Instituto Nacional de Ciência e Tecnologia de Genômica

para o Melhoramento de Citros

National Science and Technology Institute of Genomic for

Citrus Breeding

FAPESP 08/57909-2

CNPq 573848/08-4

First Annual Report

Coordinator: Marcos A. Machado

Members of the Program Centro APTA Citros Sylvio Moreira, Instituto Agronômico

Embrapa Mandioca e Fruticultura Tropical

Esalq/USP

Cena/USP

Instituto Biológico

Universidade de Mogi das Cruzes

Universidade Federal de Campinas Grande

Universidade Estadual do Sudoeste da Bahia

Universidade de Santa Cruz (Ilhéus)

Unicamp

Estação Experimental de Citricultura de Bebedouro

Cordeirópolis – SP

- Abril, 2010 -

INSTITUTOS NACIONAIS DE CIÊNCIA E TECNOLOGIA – INCT

ACOMPANHAMENTO E AVALIAÇÃO

PERÍODO: de 14/5/2009 a 14/6/2010

IDENTIFICAÇÃO DO PROJETO TÍTULO: INCT de Genômica para Melhoramento de Citros PROCESSO Nº: 573848/2008-4 VIGÊNCIA: de 14/5/2009 a 13/5/2014 RECURSOS TOTAIS APROVAD0S: R$ 7.125.422,70 CUSTEIO – R$ 4.417.762,07 CAPITAL – R$ 1.852.609,91 BOLSAS – R$ 855.050,72 COORDENADOR: Marcos Antonio Machado INSTITUIÇÃO SEDE: Instituto Agronômico de Campinas INSTITUIÇÕES PARTICIPANTES DO PROJETO: (vide formulário de submissão) EQUIPE DO PROJETO: (vide formulário de submissão)

PROJETO DE PESQUISA (Anexar Relatório Parcial) HOUVE ALTERAÇÕES NOS OBJETIVOS E/OU METAS PROPOSTOS? ( x ) SIM ( ) NÃO EM CASO POSITIVO REGISTRAR AS ALTERAÇÕES OCORRIDAS: Plataforma de Informação Genômica - Objetivos 1a (Ampliar a base de dados do CitEST) decidiu-se mudar a metodologia de seqüenciamento de genoma expresso com a tecnologia Sanger original para a plataforma Illumina. As razões para isso foram: menor custo de seqüenciamento terceirizado em empresas especializadas, maior rapidez na obtenção de dados, maior eficiência na geração de resultados e maior volume de informações sobre genoma expresso. Portanto, maiores ganhos para o programa. - Objetivos 1b (Genoma completo de citros). É o projeto do Consórcio Internacional do Genoma Citros. A participação do INCT Citros, inicialmente previsto com pagamento de cobertura 2x do genoma com tecnologia Sanger, passou para 1x, em função da utilização de novas tecnologias de re-seqüenciamento (particularmente com 454), uma das atividades previstas no programa original (Objetivos 1c). Por outro lado, o serviço está sendo executado pelo AlphaHundson Institute of Biotechnology da Universidade do Arkansas ao invés do Joint Genome Institute do Departamento de Energia dos Estados Unidos. O grande volume de informação em processamento (ESTs de citros da base CitEST e de outras bases), assim como os dados a serem gerados com as novas plataformas de seqüenciamento, exigem nova estrutura de computação, em aquisição no programa. HOUVE ALTERAÇÕES NO CRONOGRAMA ORIGINAL? ( x ) SIM ( ) NÃO EM CASO POSITIVO REGISTRAR AS ALTERAÇÕES OCORRIDAS: Plataforma de Informação Genômica A estruturação da base de dados de genoma expresso e genoma completo ainda está incompleta em função das alterações apresentadas acima. Deve ser destacado que essas bases são essenciais na montagem dos sistemas de expressão previstos na Plataforma de Aplicação

Genômica. HOUVE PROBLEMAS E/OU DIFICULDADES NA EXECUÇÃO DO PROJETO?: ( ) SIM ( x ) NÃO EM CASO POSITIVO DETALHAR:

EQUIPE HOUVE ALTERAÇÃO NA COMPOSIÇÃO ORIGINAL DA EQUIPE? ( x ) SIM ( ) NÃO EM CASO POSITIVO INDIQUE O NÚMERO DE INCLUSÕES E EXCLUSÕES: Responsável p/ laboratório associado ( x ) Inclusão ( ) Exclusão Justificar:

• Centro de Biologia Molecular Estrutural CeBiME) da Associação Brasileira de Tecnologia de Luz Sincronton (ABTLus). Diretor: Kleber Gomes Franchini (ver ofício em anexo). Laboratório referencia em biologia estrutural no Brasil.

• Departamento de Ciências Biológicas, Faculdade de Ciências e Letras, Universidade Estadual Paulista (UNESP). Grupo emergente na área de genética e biotecnologia vegetal (ver ofício em anexo).

Pesquisador ( x ) Inclusão ( ) Exclusão Justificar:

• O pesquisador Celso E. Benedetti do Centro de Biologia Molecular Estrutural (CeBiME) foi convidado em função de seus trabalhos sobre genoma funcional de Xanthomonas axonopodis pv citri e transformação genética de citros (ver ofício em anexo).

• O pesquisador Dario Abel Palmieri da Faculdade de Ciências e Letras e Departamento de Ciências Biológicas foi convidado em função de seus trabalhos sobre marcadores moleculares e mapeamento genético de citros (ver ofício em anexo).

Pós Doutorando ( x ) Inclusão ( ) Exclusão Abaixo estão relacionados os Pós Doutorandos que atuam no programa com bolsas CNPq:

• Leonardo Pires Boava, Bolsa PDJ – Genoma funcional de citros sob estresse biótico (infecção por Phytophthora parasítica). Centro de Citricultura Sylvio Moreira. Supervisão: Mariângela Cristofani-Yaly

• Polyana Kelly Martins, Bolsa DTI (ex-PDJ) – Construção de vetores para transformação

genética de citros. Centro de Citricultura Sylvio Moreira. Supervião: Juliana Freitas-Astúa

• Evandro Henrique Schinor, Bolsa DTI – Avaliação da rede experimental de citros. Centro

de Citricultura Sylvio Moreira. Supervisão: Mariângela Cristofani-Yaly

• Maria Gerolina Silva Cardoso, Bolsa DTI – Avaliação de genótipos de porta-enxertos de

citros sobre estresse com alumínio. Embrapa Mandioca e Fruticultura Tropical. Supervisão: Antonio da Silva Souza, Embrapa Mandioca e Fruticultura Tropical

• Luciano Takeshi Kishi, Bolsa DTI – Bioinformática do programa. Centro de Citricultura

Sylvio Moreira. Supervisão: Marcos A. Machado

• Rosely Pereira da Silva, Bolsa PDJ – Produção de haplóides de citros. Embrapa Mandioca

e Fruticultura Tropical. Supervisão: Walter dos Santos Soares Júnior

Estudante de Doutorado ( x ) Inclusão ( ) Exclusão

Justificar: Os alunos relacionados abaixo foram aprovados em programas de pós graduação e estão desenvolvendo suas Teses dentro do INCT Citros:

• Rosangela Naomi Inui, Curso Biologia Funcional e Molecular da Unicamp, com o tema ‘Prospeção e análise da atividade de peptídeos antimicrobianos no controle de patogenos de citros’. Orientação: Marcos A. Machado Co-orientação: Juliana Freitas Astúa

• Danila Souza Oliveira Coqueiro, Curso Biologia Funcional e Molecular da Unicamp, com o

tema ‘Expressão gênica diferencial induzida por elicitores nos patossistemas huanglongbing e CVC. Orientação: Marcos A. Machado Co-orientação: Alessandra Alves de Souza

• Tahise Magalhães de Oliveira, Curso de Genética e Biologia Molecular da Universidade

Estadual de Santa Cruz (Ilhéus), com o tema ‘Transformação genetica de citros para tolerância a seca’. Orientação: Marcio Costa Co-orientação: Walter Soares Filho, Embrapa Mandioca e Fruticultura Tropical

Estudante de Mestrado ( ) Inclusão ( ) Exclusão Justificar: Os alunos relacionados abaixo foram aprovados em programas de pós graduação e estão desenvolvendo suas Dissertações dentro do INCT Citros:

• Alexandre Dias Dutra, Curso de Ciências Agrárias da Universidade Federal do Recôncavo Baiano. Bolsa CAPES. Orientação: Abelmon da Silva Gesteira Co-orientação: Walter Soares Filho, Embrapa Mandioca e Fruticultura Tropical

• Diana Matos Neves, Curso de Genética e Biologia Molecular da Universidade Estadual de

Santa Cruz (Ilhéus). Bolsa CAPES. Orientação: Abelmon da Silva Gesteira Co-Orientação: Márcio G.C. Costa

• Joadson Dutra de Souza, Curso de Genética, Biodiversidade e Conservação da

Universidade Estadual do Sudoeste da Bahia, Campus de Jequié. Bolsa CAPES. Orientação: Antonio Carlos de Oliveira Co-Orientação: Marcos A. Machado

• Luciana Faldoni, Curso de Agroecologia e Desenvolvimento Rural da UFSCar. Bolsa

CAPES. OrientaçãoL Kátia Critina Kupper Co-Orientação: Mariângela Cristofani-Yaly

Estudante de Graduação ( x ) Inclusão ( ) Exclusão Justificar: Os alunos relacionados abaixo estão desenvolvendo atividades de Iniciação Científica no programa:

• Gabriela Marteloso Carrer (Bolsa IC) – Uniararas/SP. • Joice Fernanda Garbin (Bolsa IC) – UFCG/PB. • Jose Alberto Diogo (Bolsa IC) – Unicamp/SP. • Patrícia Herrmann Corrêa – (Bolsa IC) – Uniararas/SP. • Salete Rocha (Bolsa IC) – UFCG/PB. • Fernanda Nara Mauricio - (Bolsa ITI-A) – Uniararas. • Gessica Laize Berto Gomes (Bolsa II-A) – UFCG/PB. • Janaína Andréa Mendes – (Bolsa ITI-A) – Uniararas. • Mariana Cardoso Miguel - (Bolsa ITI-A) – UFSCar. • Mariela Thim Vitorino (Bolsa IC) – UFSCar/SP.

Outros (Bolsa de Capacitação ou Desenvolvimento Tecnológico) ( x ) Inclusão ( ) Exclusão Justificar: Os bolsistas relacionados abaixo estão envolvidos com as atividades do programa:

• Samanta Marengo, bolsa de Especialização na empresa Diversity Arrays Technology, em Yarralumla, Austrália, para desenvolvimento de marcadores tipo DArt para citros. Orientação: Andrzej Kilian

• Adriano Malosso, bolsa DTI nas atividades de transformação genética de citros. Orientação: Raquel Luciana Boscariol-Camargo.

• Jacqueline Camargo Olivato, bolsa DTI nas atividades de genoma funcional de Xylella fastidiosa. Orientação: Alessandra Alves de Souza

• Silvia de Oliveira Dorta, bolsa DTI nas atividades de genoma funcional de plantas. Orientação: Helvécio Della Coletta Filho

Responsável p/ laboratório associado ( x ) Inclusão ( ) Exclusão Justificar:

• Ver acima. Pesquisador ( x ) Inclusão ( ) Exclusão Justificar:

• O pesquisador Abelmon da Silva Gesteira foi incluído no programa em função de sua contratação pela Embrapa Mandioca e Fruticultura Tropical.

Pesquisador ( ) Inclusão ( x ) Exclusão Justificar:

• O pesquisador Alexandre Morais do Amaral (Embrapa, porém sediado no Centro de Citricultura Sylvio Moreira) assumiu o Labex da Inglaterra, afastando-se, portanto, das atividades técnicas do programa.

DESCREVER OS MECANISMOS DE INTERAÇÃO UTILIZADOS ENTRE GRUPOS DE PESQUISA PARTICIPANTES DO INCT Alem da comunicação rotineira entre os membros do programa com o coordenador, foi estabelecido um sistema de gestão de projetos com coordenadores definidos por área, sendo o coordenador responsável por consolidação da informações e intercâmbios entre os grupos. Na sede do INCT Citros foi feita uma reunião geral com todos os participantes do programa em 01 e 02 de outubro de 2009 para discussão geral (ver programa em anexo). A gestão financeira é feita diretamente pelo Centro de Citricultura que concentra todas as compras e prestações de contas. Todos os grupos participantes receberam planilhas financeiras com valores aprovados pelo CNPq e FAPESP (grupos de São Paulo). A execução orçamentária é feita com base na disponibilidade financeira do programa. RELATAR EVENTUAIS DIFICULDADES ENCONTRADAS ENTRE OS GRUPOS DE PESQUISA PARTICIPANTES DA REDE E POSSÍVEIS MECANISMOS UTILIZADOS PARA SUPERAR ESTAS DIFICULDADES: Embora sem afetar a condução do programa, merece ser destacado que nem sempre a equipe vê no INCT Citros um programa conjunto. Existe a tendência de vê-lo como a reunião de projetos individuais. A expectativa é que no segundo ano essa visão se altere.

HOUVE A INCLUSÃO OU EXCLUSÃO DE INSTITUIÇÕES E EMPRESAS? ( x ) SIM ( ) NÃO EM CASO POSITIVO INDIQUE O NÚMERO: Instituição de Ensino e/ou Pesquisa ( x ) Inclusão ( ) Exclusão Justificar:

• Centro de Biologia Molecular Estrutural CeBiME) da Associação Brasileira de Tecnologia de Luz Sincronton (ABTLus). O grupo tem atuado em genoma funcional e comparativo de citros, alem de ter grande experiência em biologia molecular e estrutural, de acordo com as linhas de atuação do INCT Citros.

• Departamento de Ciências Biológicas de Faculdade de Ciências e Letras da UNESP/Assis,

por ser um grupo novo e expansão, com experiência em mapeamento genético de citros. O Dr. Dario Palmieri atuou no nosso grupo como Pós Doutorando durante o Instituto do Milênio. Grupo emergente na área de genética e biotecnologia de plantas.

RESULTADOS OBTIDOS / METAS ENUMERE E COMENTE OS RESULTADOS CIENTÍFICOS E/OU TECNOLÓGICOS OBTIDOS ATÉ O MOMENTO PARA A – PESQUISA

• Construído mapa genético com marcadores moleculares para Poncirus trifoliata e Citrus sunki para avaliação de tolerância ao huanglongbing (greening).

• A expressão de genes que codificam terpeno sintase (enzima chave na via de síntese de óleos essenciais) é controlada ao nível de desenvolvimento nos frutos de laranja doce.

• Análises de rendimento e qualidade de óleos essenciais de tangerinas estão em avaliação, bem como de expressão de genes que codificam terpeno sintases.

• Clonados genes que codificam para limoneno hidroxilases, enzimas da via de síntese de óleos essenciais, em vetores para expressão heteróloga.

• Clonados promotores específicos de genes que se expressam no floema. • Comprovada a existência de enzima de restrição no genoma de Xylella fastidiosa,

agente causador da clorose variegada dos citros. • Mutante para formação de biofilme em Xylella fastidiosa comprova o envolvimento

do processo de formação de biofilme no desenvolvimento da clorose variegada dos citros.

• Obtidas plantas autotetraploides de variedades de porta-enxertos (limão Cravo, tangerinas Cleopatra e Sunki, citrumelo Swingle, citrange Troyer e dois citrandarins) e de variedades de copa (laranjas doces, tangor Murcott, tangerinas Poncan e Clementina e Fortunella Meiwa).

• Construção de bibliotecas genômicas de Candidatus. Liberibacter americanus, agente do huanglongbing dos citros.

• Seqüenciado parcialmente o genoma de Candidatus. Liberibacter americanus com tecnologia 454 a partir de imunocaptura da bactéria em psilídeos.

• Construção do primeiro chip de DNA de citros com a base de dados do genoma seqüenciado no Centro de Citricultura Sylvio Moreira.

• Análise através de chips de DNA dos genes de laranja doce durante o processo de infecção por Ca. Liberibacter americanus.

• Ampliada a base de dados de genoma expresso de citros com seqüências de outros países. A base passou de 300 mil para 550 mil seqüências expressas.

• Construído o primeiro mapa de proteoma do fungo Alternaria alternata, agente da mancha marrom dos citros.

• Construído o primeiro mapa de proteoma do ácaro Brevipalpus phoenicis, agente da leprose dos citros.

• Avaliados a expressão dos principais genes relacionados ao florescimento da

laranja Valência através de chip de DNA (microarranjos). • Avaliados plantas transgênicas de laranja doce para tolerância ao huanglongbing e

ao cancro cítrico. • Produzidas plantas transgênicas de porta-enxertos com maior tolerância ao

estresse hídrico. • Seqüenciamento de genoma expresso do fungo Guignardia citricarpa, agente da

mancha preta dos citros. • Monitorado a flutuação populacional da bactéria Candidatus Liberibacter spp em

plantas de laranja doce em condições de campo. • Avaliado com chips de DNA os principais genes de Poncirus trifoliata e Citrus sunki

associados a infecção por Phytophthora parasítica. • Publicado o primeiro estudo sobre mapeamento de QTL (Quantitative Trait Loci)

para resistência à leprose dos citros. • Marcadores moleculares associados ao gene Ctv no mapa de Poncirus trifoliata

foram encontrados no mapa de citrumelo Swingle • Mapeados QTL (Quantitative Trait Loci) para resistência a gomose de

Phytophthora no mapa de ligação de citrumelo Swingle. • Foram encontrados marcadores moleculares microssatélites específicos para

algumas variedades de tangerinas e híbridos. • Seleção de 37 citrandarins (híbridos de Citrus sunki vs Poncirus trifoliata) como

potenciais porta-enxertos com resistência a tristeza, gomose e morte súbita do citros, sendo que dentre eles alguns apresentam elevada tolerância ao estresse hídrico.

• Seleção de oito híbridos de limão Cravo vs citrumelo Swingle e onze híbridos de limão Cravo vs Poncirus trifoliata resistentes à gomose de Phytophthora e à tristeza dos citros.

• Seleção de nove variedades de tangerinas resistentes à leprose. • Obtidas novas populações de híbridos de tangerina para seleção de plantas

resistentes a mancha marrom de alternaria. • Selecionados os porta-enxertos nanicantes: citrandarins Clementina x trifoliata,

Cleópatra x Swingle 715 e 1.614, Cleópatra x Rubidoux e Cleópatra x Cristhian. Eles induziram a formação de laranjeiras Valencia com altura inferior a 3,5.

• Comprovado que limão cravo Limeira, Ipanema e Pennivesiculata assim como o limão Volkameriano Catania-2 e os limões rugosos FM, Schaub, do Cabo, 58329 e 58328 são tolerantes a morte súbita dos citros.

• Demonstrado que os citrumelos Swingle, W-2, Swingle, F.80-7, F.80-5, F.80-8, F.80-6, F.81-18, F.80-3 e 4481 são tolerantes a morte súbita dos citros e ao declínio dos citros.

• Comprovada a primeira incompatibilidade copa com porta-enxerto em laranja Valência enxertada sobre trangpur (Limão Cravo x Citrange Carrizo) 1524.

• Selecionados dez híbridos de tangor Murcott vs laranja Pêra e tangerina Cravo vs laranja Pêra com resistência a mancha marrom de alternaria e com qualidade da fruta com grande potencial para o mercado de fruta fresca.

• Feito o levantamento de tolerância à mancha marrom de alternaria e ao cancro cítrico no germoplasma de citros.

• Comprovada a existência de resistência à mancha preta dos citros em laranja doce de maturação tardia.

• Concluída a caracterização nutracêutica e agronômica de frutos de laranjas sanguíneas.

• Produzido clones de Yuzu para a indústria de óleo essenciais. • Estabelecido o Sistema Protegido do Banco Ativo de Germoplasma de Citros com

recursos IAC/APTA/SAA. • Construído a primeira plataforma de micro arranjos de DNA para estudos de expressão

gênica global de laranja doce, tangerina e Poncirus trifoliata. • Duplicado o banco de genoma expresso de citros (ESts) em colaboração com grupos no

exterior. • Primeiros relatos da expressão diferencial de genes em laranja doce afetada por

Candidatus Liberibacter americanus, uma das bactérias causadoras do huanglongbing dos

citros. • Identificação de híbridos de porta-enxertos de citros com tolerância à gomose de

Phytophthora e à tristeza (Programa Embrapa). • Identificação de híbridos de porta-enxertos com maior tolerância à seca (Programa

Embrapa). • Identificação de híbridos com potencial para plantas ornamentais.

B – FORMAÇÃO DE RECURSOS HUMANOS

• Incorporados 06 (seis) pós doutorando ao Programa. • Incorporados 03 (três) alunos de Doutorado e 04 (quatro) de Mestrado ao Programa. • Quatro bolsistas DTI (nível de graduação) estão atuando no Programa. • 10 novos alunos de IC passaram a participar do Programa.

C – TRANSFERÊNCIA DE CONHECIMENTO E TECNOLOGIA

• Participação em eventos técnico-científicos. • Parceria com a empresa Fischer Agropecuária SA na montagem de experimentos para

avaliação de potenciais porta-enxertos e copa de citros em suas fazendas. As potenciais variedades deverão ser utilizadas pela empresa, respeitando-se os direitos previstos na Lei de Proteção de Cultivares.

• Iniciada parceria com a empresa Cambuhy Agrícola (Matão) para a montagem de experimentos de avaliação de variedades copa originadas do programa de melhoramento do Centro de Citricultura.

D – EDUCAÇÃO E DIVULGAÇÃO DA CIÊNCIA Nada a acrescentar. ENUMERE OS IMPACTO(S) CAUSADO(S) PELAS AÇÕES E RESULTADOS DO PROJETO PARA A AMPLIAÇÃO, MELHORIA E CONSOLIDAÇÃO DA COMPETÊNCIA TÉCNICO-CIENTÍFICA NACIONAL PARA A – PESQUISA Embora a melhor maneira de medir impacto de pesquisa seja através de publicações referenciadas. Essas estão relacionadas no relatório de atividades. No entanto, o INCT Citros tem também o potencial de efetivamente reunir todos os pesquisadores brasileiros que trabalham com citros, particularmente na área de melhoramento. Com o avanço do huanglongbing (HLB, greening) o Centro iniciou a instalação do Sistema Protegido do Banco Ativo de Germoplasma de Citros sob telado com proteção contra insetos vetores de doenças. Ao BAG Citros foram incorporados mais de 500 novos híbridos resultantes do programa de Melhoramento do Centro. Portanto, o Centro de Citricultura, como uma das unidades do INCT Citros, continuará a ser um fornecedor de material básico com certificação genética e fitossanitária. O Centro continuou sendo referencia no fornecimento de material básico de propagação (sementes de porta-enxertos e borbulhas de variedades copa), muito embora forneça essencialmente para multiplicação de novas borbulheiras no setor privado. Nos últimos anos reduziu significativamente o volume de borbulhas e sementes fornecidas, uma vez que o setor privado cada vez atende às suas próprias necessidades. - Métodos de diagnóstico de doenças de citros – ajustes os métodos para diagnóstico de Xylella fastidiosa, Ca. Liberibacter spp e Xanthomonas axonopodis pv citri utilizando-se de PCR com iniciadores fluorescentes, o que permite rapidez, precisão e economia nos testes. A metodologia está sendo transferida para a Clínica Fitopatológica de Citros. - Variedades livres de patogenos – Com a rotina de limpeza clonal por microenxertia, variedades de clones novos e velhos do BAG Citros foram recuperadas e foram incluídas no programa de proteção em ambiente protegido. Merece destaque a limpeza e liberação para testes de variedade Yuzu, de interesse para a indústria de essenciais. - Registro de variedades - Foram solicitados registros de 11 novos cultivares no RNC/MAPA somando-se aos mais de 60 previamente registrados. - Plantas geneticamente modificadas – Dentre vários eventos de transformação genética, algumas

plantas mostraram-se tolerantes ao cancro cítrico. Liberação controlada no ambiente está sendo solicitada junto à CTNBio. - Novos genes e promotores – A partir das informações sobre genoma foram identificados novos genes e promotores dentro do grupo dos citros, permitindo a continuidade dos trabalhos de obtenção de variedades geneticamente modificadas com genes do próprio grupo. B – FORMAÇÃO DE RECURSOS HUMANOS Praticamente todos os participantes de iniciação científica, treinamento técnico, pós graduação e pós doutorado encontram-se inseridos em atividades do Programa e deverão ser capacitados em áreas como biologia molecular, bioinformática, genética e melhoramento de citros. Todos são potenciais pesquisadores que poderão estar envolvidos com pesquisa de citros no futuro próximo, portanto, com efeito multiplicador. C – TRANSFERÊNCIA DE CONHECIMENTO E TECNOLOGIA Parte considerável de participam do INCT Citros, particularmente no Centro de Citricultura em Cordeirópolis, estão envolvidos em eventos de transferência representado por eventos no Centro destinados a produtores, extencionistas, viveiristas, etc. São eventos de caráter técnico destinados a usuários diretos da informações tecnológicas (agronômicas). No período desse relatório foram organizados e efetivados os seguintes eventos: - 2o. Dia do Huanglongbing, no dia 13 de março de 2009, com sete palestras e mais de 300 participantes. - 3o Dia do Huanglongbing, no dia 12 de março de 2010, com oito palestras e cerca de 350 participantes. - 10o. Dia do Limão: no dia 26 de março de 2009, com três palestras e cerca de 60 participantes. - 11o. Dia do Limão: no dia 25 de março de 2010, com debates sobre mercado e exportação e 55 participantes. - 4o. Dia do Porta-Enxerto, em 30 de abril de 2009, com três palestras e 30 participantes. - 5o. Dia do Porta-Enxerto, em 07 de maio de 2009, com três palestras e 25 participantes. -XII Dia da Tangerina, concomitantemente com o 1º Encontro de Citricultura na Região Sudoeste de São Paulo e o VIII Dia de Campo de Tangerina, em Capão Bonito (SP), no dia 18 de junho de 2009, com sete palestras e 80 participantes. - 31a Semana da Citricultura, de 01 a 05 de junho de 2009, com apresentação de 41 palestras e cerca de 6 mil participantes. - 32a Semana da Citricultura, de 07 a 11 de junho de 2010, com apresentação de 43 palestras e aproximadamente 8 mil participantes. - 35a Expocitros, junto com a Semana da Citricultura, com participação de 54 empresas do setor citrícola. - 36a Expocitros, junto com a Semana da Citricultura, com participação de 45 empresas do setor citrícola. - 15o. Dia do Viveirista em 11 de agosto, com quatro palestras e 250 participantes. - 9o. Dia da Laranja em 21 de outubro, em sistema de entrevista tipo Roda Viva com 150 participantes. D – EDUCAÇÃO E DIVULGAÇÃO DA CIÊNCIA O Centro de Citricultura, coordenador do INCT Citros, possui publicação anual da revista Laranja, considerada técnico-científica, com grande penetração no setor de produção. A partir de 2010 a revista passa a se chamar Citrus Research & Technology, com o objetivo de fomentar a melhoria na qualidade de publicações e atender a sistemas de indexação exigidos pela Capes. (ver citrusrt.centrodecitricultura.br). O Centro ainda mantém um curso anual de citricultura destinado a alunos em final de curso e recém formados, com abrangência em todas as áreas da citricultura. O curso tem carga de 80 horas. O Curso de Doenças de Citros e seu manejo, regularmente ministrado uma vez ao ano, envolve aspectos aplicados no controle de doenças de citros e destina-se a todos os profissionais do setor.

PARA FINS DE DIVULGAÇÃO, RELACIONAR RESULTADOS OBTIDOS QUE MEREÇAM DESTAQUE PARA O DESENVOLVIMENTO CIENTÍFICO, TECNOLÓGICO E/OU SOCIAL:

• Mutante para formação de biofilme em Xylella fastidiosa comprova o envolvimento do processo de formação de biofilme no desenvolvimento da clorose variegada dos citros.

• Seqüenciado parcialmente o genoma de Candidatus. Liberibacter americanus com tecnologia 454 a partir de imunocaptura da bactéria em psilídeos.

• Construção do primeiro chip de DNA de citros com a base de dados do genoma seqüenciado no Centro de Citricultura Sylvio Moreira.

• Ampliada a base de dados de genoma expresso de citros com seqüências de outros países. A base passou de 300 mil para 550 mil seqüências expressas. (Consórcio Internacional do Genoma Citros)

• Construído o primeiro mapa de proteoma do fungo Alternaria alternata, agente da mancha marrom dos citros.

• Construído o primeiro mapa de proteoma do ácaro Brevipalpus phoenicis, agente da leprose dos citros.

• Avaliados plantas transgênicas de laranja doce para tolerância ao huanglongbing e ao cancro cítrico.

• Publicado o primeiro estudo sobre mapeamento de QTL (Quantitative Trait Loci) para resistência à leprose dos citros.

• Mapeados QTL (Quantitative Trait Loci) para resistência a gomose de Phytophthora no mapa de ligação de citrumelo Swingle.

• Seleção de 37 citrandarins (híbridos de Citrus sunki vs Poncirus trifoliata) como potenciais porta-enxertos com resistência a tristeza, gomose e morte súbita do citros, sendo que dentre eles alguns apresentam elevada tolerância ao estresse hídrico.

• Seleção de oito híbridos de limão Cravo vs citrumelo Swingle e onze híbridos de limão Cravo vs Poncirus trifoliata resistentes à gomose de Phytophthora e à tristeza dos citros.

• Seleção de nove variedades de tangerinas resistentes à leprose. • Obtidas novas populações de híbridos de tangerina para seleção de plantas

resistentes a mancha marrom de alternaria. • Selecionados os porta-enxertos nanicantes: citrandarins Clementina x trifoliata,

Cleópatra x Swingle 715 e 1.614, Cleópatra x Rubidoux e Cleópatra x Cristhian. Eles induziram a formação de laranjeiras Valencia com altura inferior a 3,5.

• Comprovado que limão cravo Limeira, Ipanema e Pennivesiculata assim como o limão Volkameriano Catania-2 e os limões rugosos FM, Schaub, do Cabo, 58329 e 58328 são tolerantes a morte súbita dos citros.

• Demonstrado que os citrumelos Swingle, W-2, Swingle, F.80-7, F.80-5, F.80-8, F.80-6, F.81-18, F.80-3 e 4481 são tolerantes a morte súbita dos citros e ao declínio dos citros.

• Selecionados dez híbridos de tangor Murcott vs laranja Pêra e tangerina Cravo vs laranja Pêra com resistência a mancha marrom de alternaria e com qualidade da fruta com grande potencial para o mercado de fruta fresca.

• Comprovada a existência de resistência à mancha preta dos citros em laranja doce de maturação tardia.

• Estabelecido o Sistema Protegido do Banco Ativo de Germoplasma de Citros com recursos IAC/APTA/SAA.

RESULTADOS EM NÚMEROS

A – INDICADORES DE PESQUISA NÚMEROS DA PRODUÇÃO TÉCNICO-CIENTÍFICA E ARTÍSTICA NO PERÍODO

(anexar referências):

TIPO QUANTIDADE LIVROS 0

CAPÍTULOS DE LIVROS 0 ARTIGOS PUBLICADOS EM PERIÓDICOS NACIONAIS 09 ARTIGOS PUBLICADOS EM PERIÓDICOS INTERNACIONAIS 08 TRABALHOS APRESENTADOS EM CONGRESSOS NACIONAIS

12

TRABALHOS APESENTADOS EM CONGRESSOS INTERNACIONAIS

07

SOFTWARE 0 PATENTE

PRODUTOS 0 PROCESSOS 0

PRODUÇÃO ARTÍSTICA (ESPECIFICAR) 0 OUTROS (ESPECIFICAR): 0

B – INDICADORES DA FORMAÇÃO DE RECURSOS HUMANOS NÚMEROS DA FORMAÇÃO DE RECURSOS HUMANOS NO PERÍODO

TIPO QUANTIDADE

ENCERRADOS: INICIAÇÃO CIENTÍFICA 5 MESTRE 1 DOUTOR 0 PÓS-DOUTOR 0 OUTROS (ESPECIFICAR): 0

EM ANDAMENTO:

INICIAÇÃO CIENTÍFICA 19 MESTRE 11 DOUTOR 09 PÓS-DOUTOR 07 OUTROS (ESPECIFICAR): BOLSAS DTI (APERFEIÇOAMENTO) 04

C – INDICADORES DE TRANSFERÊNCIA DE CONHECIMENTO E TECNOLOGIA NÚMEROS DA PRODUÇÃO NO PERÍODO

(especificar e anexar referências):

TIPO QUANTIDADE Dias Tematicos 05

Semana da Citricultura 01

D – INDICADORES DE EDUCAÇÃO E DIVULGAÇÃO DA CIÊNCIA

NÚMEROS DA PRODUÇÃO NO PERÍODO (especificar e anexar referências):

TIPO QUANTIDADE

Revista Laranja 01

Curso de Citricultura 01

Curso de Doenças de Citros 01

INFORMAÇÕES ADICIONAIS

DESCREVER OUTRAS FORMAS DE DISPONIBILIZAÇÃO PÚBLICA DOS RESULTADOS DO PROJETO: Vídeo institucional do INCT Citros. Página do Programa: http://www.centrodecitricultura.br/inct_citros.php

DESCREVER AS MELHORIAS IMPLANTADAS NAS INSTALAÇÕES FÍSICAS DA SEDE E DOS LABORATÓRIOS ASSOCIADOS AO INSTITUTO, COMO ADPTAÇÕES FÍSICAS, EQUIPAMENTOS, ETC. Embora o programa INCT Citros esteja contratado a pouco mais de um ano, foi somente a partir de novembro de 2009 que o recurso do CNPq começou a ser utilizado. Houve dificuldades no uso do Cartão de Pesquisa, posteriormente trocado por depósito em conta corrente. O único processo de importação encaminhado ao CNPq em dezembro de 2009, até a presente data (junho de 2010) ainda não se concretizou. Para complicar, o CNPq afirma que não mais irá importar nesses projetos, repassando a função às instituições sedes do INCT. Um complicador quando a instituição não tem capacitação para isso e o projeto está em nome do coordenador. Os grupos fora do Estado de São Paulo dependem desse serviço para suas atividades no INCT. De qualquer modo, foram adquiridos equipamentos com recursos da FAPESP para o Centro de Citricultura (citometro, termociclador para RT-qPCR e servidor para análise de dados de seqüenciamento). Para a Esalq foi adquirida estufa climatizada para manutenção de plantas transgênicas. HOUVE ATIVIDADES DE INTEGRAÇÃO COM OUTROS INCT’S: ( X ) SIM ( ) NÃO EM CASO POSITIVO DETALHAR: INCT de Controle Biorracional de Insetos pragas, da Universidade Federal de São Carlos, em trabalhos sobre doenças de citros, como mancha marrom de alternaria, mancha preta (Guignardia citricarpa) e clorose variegada dos citros (CVC), em parceria com a Profa. Dra. Maria de Fátima G.F. da Silva.

CONSIDERAÇÕES FINAIS COMENTAR OUTROS ASPECTOS RELEVANTES DO DESENVOLVIMENTO GERAL DO PROJETO De modo geral, o programa está sendo conduzido a contento, mas poderia ter avançado mais na sua primeira parte (Plataforma de Informação Genômica) em função de desencontros dentro do Consórcio Internacional e da saída do Join Genome Institute como centro de seqüenciamento do genoma com tecnologia Sanger. Essa atividade está sendo conduzida no AlphaHudson Institute of Biotechnology da Universidade do Alabama. Por outro lado também, houve um aumento expressivo na base de dados de genoma expresso exigindo capacidade de processamento que não havia no grupo, pois a estrutura usada no Instituto do Milênio (programa anterior ao INCT) encontrava-se desatualizada e sem capacidade de processamento para aquele volume de dados. QUAL O PAPEL DO INCT PARA A FORMAÇÃO DA REDE DE PESQUISA? Como colocado acima o INCT tem sido uma oportunidade do grupo entender que o trabalho em rede nem sempre é a soma de projetos individuais. Efetivamente existe o trabalho em rede em alguns frentes do Programa, como na transgenia, onde o grupo tem boa afinidade. Nas atividades de genoma esse trabalho fica restrito ao grupo do Centro de Citricultura, o único de tem envolvimento maior com esses resultados. Nas atividades de melhoramento os grupos caminham paralelos, muito provavelmente em função da natureza dessa atividade (experimentos de campo de longa duração, áreas e focos distintos, material genético diverso). Como parte considerável dessas atividades também estão no Centro de Citricultura, o INCT está se tornando o suporte essencial ao programa de melhoramento. AVALIE A INTERLOCUÇÃO DO INCT COM O CNPq E DEMAIS FINANCIADORES DO PROGRAMA: De modo geral, sempre muito boa. A equipe do CNPq responsável pelo INCT (Ana Lucia, Elpígio, Alcebíades) sempre foram solícitos e colaboradores. A equipe da importação também. O impasse, sobre se o CNPq continuará ou não a importar, persiste. Ofício encaminhado à Presidência ainda

não foi respondido. Na FAPESP o contato principal é com a Diretoria Administrativa nos processos de compra e prestação de contas.

Anexar relatório de resultados parciais, de no máximo 50 páginas, contendo:

1. Comitê Gestor – reuniões realizadas e decisões Em anexo.

2. Atividades de cooperação entre os grupos de participantes do INCT. Descritas no relatório técnico.

3. Atividades de cooperação entre INCT’s e com outras instituições (empresas, ongs, instituições governamentais, etc). Descritas no relatório técnico.

4. Principais resultados técnico-científicos Relatório técnico (em inglês)

5. Eventos nacionais e internacionais: apresentação de trabalhos, organização de cursos, seminários; palestras; mesas redondas. Descritas no relatório técnico.

6. Atividades de formação e capacitação de recursos humanos Descritas no relatório técnico.

7. Perspectivas e futuros desdobramentos. No relatório técnico.

LOCAL E DATA: Cordeirópolis, 07 de junho de 2010.

Marcos A. Machado

Coordenador

Instituto Nacional de Ciência e Tecnologia de

Genômica para o Melhoramento de Citros

National Science and Technology Institute of Genomic for Citrus Breeding

FAPESP 08/57909-2 CNPq 573848/08-4

First Annual Report

Coordinator: Marcos A. Machado

Members of the Program Centro APTA Citros Sylvio Moreira, Instituto Agronômico

Embrapa Mandioca e Fruticultura Tropical Esalq/USP Cena/USP

Instituto Biológico Universidade de Mogi das Cruzes

Universidade Federal de Campinas Grande Universidade Estadual do Sudoeste da Bahia

Universidade de Santa Cruz (Ilhéus) Unicamp

Estação Experimental de Citricultura de Bebedouro

Cordeirópolis – SP April 2010

Instituto Nacional de Ciência e Tecnologia de Genômica para o Melhoramento de

Citros

National Science and Technology Institute of Genomic for Citrus Breeding

FAPESP 08/57909-2 CNPq 573848/08-4

Program Summary

The citrus industry is one of the leading activities of the agribusiness in

Brazil, with the states of Sao Paulo, Sergipe and Bahia as the main citrus

producers. Brazil is by far the main producer and exporter of frozen

concentrated (FCOJ) and not from concentrated (NFC) orange juice in the

world. The low Brazilian yield (approximately 2 boxes 40.8 kg/tree/year) is

highly associated to the incidence of pests and diseases (resulting in a

substantial influence over the production costs), the production in non-irrigated

areas, and the narrow genetic basis that has been used.

It is estimated that more than 60 % of the costs for citrus production in

Brazil are associated with control of pests and diseases. Diseases like citrus

variegated chlorosis, leprosis, black and brown spot, sudden death, citrus

canker, gummosis, tristeza and, recently, huanglongbing (HLB, former

greening). Indeed, in recent years, the citrus industry has witnessed substantial

structural changes in its structures due to diseases like HLB. Most of the

models for the management of the disease vectors, including chemical control,

have failed due to their high economic and environmental costs, which enhance

even more the importance of studies on genetic breeding as a powerful and

durable strategy to control diseases. The Centro de Citricultura Sylvio Moreira

(CCSM) – IAC and Embrapa have been investigating citrus breeding for a long

time as well as producing and evaluating new genetic materials by using

biotechnological tools to accelerate genetic gains. As the leading member of the

Millennium Institute of CNPq /MCT (2002-2005), CCSM constructed an

extensive database on expressed citrus genome (CitEST), which comprises

tools for studying genetic breeding and comparative and functional genomics of

citrus and some of its pathogens. Besides producing the largest collection of

genomic data worldwide, the project enlarged profoundly the number of new

citrus hybrids under field evaluation, molecular markers for genetic mapping

and discovery of new genes discovery with potential association to disease

resistance.

This current program reflects the continuation of the program “Millennium

Institute”, not only bringing together the main groups that investigate citrus in

Brazil, but also focusing on subjects related to genetic breeding and functional

genomics of the citrus group, looking forward on the integration of three

platforms:

Platform of Genomic Information, which focuses on studies regarding

comparative and functional genomics to enlarge the basis of information,

including the complete genome associated with the International Consortium on

Citrus Genome and the database on expressed citrus genome, studies on gene

expression (microarrays) of citrus and some of its pathogens, support to

bioinformatics and genetic transformation to produce modified plants based on

data from the citrus genome.

Platform for Genomic Application, which the focus is the plant-pathogen

interaction in limiting pathosystems faced by the citrus industry and the plant-

environment interaction (water stress). Such studies will allow enlarging the

knowledge useful in other activities of the Program.

Platform of Apllied Genetics, which represents the advanced interface of

breeding from which previously generated data (markers, maps, genes,

promoters), as well as genetic material (hybrids of controlled crosses and

events of transformation already available) will be validated under field

conditions, to permit a reliable approach to the strategy of marker-assisted

breeding. From this platform, new materials will be released for the Brazilian

citrus industry.

The multi-disciplinar and multi-institutional network of the Program includes,

besides the CCSM as the coordinator member, Embrapa Mandioca e

Fruticultura and its network for citrus breeding, Embrapa Recursos Genéticos e

Biotecnologia, Escola Superior de Agricultura Luiz de Queiróz (ESALQ/USP)

and Centro de Energia Nuclear na Agricultura (Cena), Universidade Estadual

de Campinas (Unicamp), Instituto Biológico, Universidade Federal de Campina

Grande, Universidade Estadual do Sudoeste da Bahia (Vitória da Conquista),

Universidade de Mogi das Cruzes (UMC), and University of Florida.

Report Summary

The CitEST (Database of expressed citrus genome) was doubled and

shared with other citrus databases around the world. Thus the NSTI Citrus has

an expressive coverage of the expressed genome, which will support the

activities of annotation and anchorage of the complete citrus genome. The new

database needs to be processed and assembled as soon as the hardware

platforms are working. The current computer facilities in our laboratory are not

able to process this amount of data. Although originally planned to be conduced

in our facilities, the sequencing of new EST libraries with the objective to

increase the number of unigenes of other citrus species will be carried out

abroad at lower costs and higher efficiency. We also decided to use new

technologies for sequencing since such data will be useful for both EST

assembly and re-sequencing activities included in the Program. More important

than perform the sequencing activities inside the group is to acquire and to

process the generated data.

To accomplish this goal, we contacted Sequencing companies that

perform pyrosequencing, since it had been proposed as a third party service.

Using the Genome Sequencer FLX Instrument, powered by GS FLX Titanium

series reagents, we will be able to generate more than 1,000,000 individual

reads with improved Q20 read length of 400 bases per 10-hour instrument run.

Therefore, one run can generate 400 MB, which is more than the estimated

genome size. To get the job done we will need 4 runs per species and that is

the object of the discussion with the Sequencing companies.

The proposal to sequence the full genome of citrus within the

International Citrus Genome Consortium (ICGC) has faced problems of lack of

funds in US and Brazil, and lack of leadership especially in the European

groups. With the advance of new sequencing technologies, the ICGC decided

to include a set of such data and decrease the amount of sequences with

Sanger technology. Instead of the originally proposed 10x coverage, it was

decided to run 6x coverage with Sanger technology and 4x with 454 technology.

Thus Brazil, USA and Spain are in charge of 6x coverage (2x coverage each),

whereas France shall be responsible for 4x coverage, and Italy, 1x additional

coverage. Joint Genome Institute of DOE/US started to sequence 2x (American

part) but will not assume the sequencing of our part since they are moving to

the new sequencing technologies. AlphaHudson Institute of Biotechnology of

the University of Alabama will do the service for the NSTI Citrus.

Although the database did not reach the main program goals yet, it has

been used for several proposals in the NSTI Citrus, including search for new

genes, molecular markers inside the ESTs, and the built of microarray platforms

for studies on gene expression.

Slides were designed using UniGene selected from the CitEST database,

assembled from the ESTs submitted to NCBI. The sequences were used to

construct genome-wide 60-mer oligonucleotide cDNA microarrays by Roche

NimbleGen Systems using a multi-step approach to select probes with optimal

predicted hybridization characteristics. Slides were composed of 31,535

sequences of sweet orange (Citrus sinensis), plus probes for six additional

sequences, three probes per target, four copies on array, totalizing 378,420

probes of C. sinensis. Mixed slides (sweet orange, mandarin and Poncirus)

were also built. Additional arrays of mandarin and Poncirus trifoliata are also

planned as the database of unigenes are concluded. The DNA array technology

has been used as an approach for global gene expression of citrus and its

pathogens. Results on such approach are presented in the report, pointing out

the plant pathogen interaction in the pathosystems gummosis, leprosis, and

huanglongbing. Studies on interactions with Xylella fastidiosa and Xanthomonas

axonopodis pv. citri are underway.

Although the technology of DNA arrays has been the main proposed

approach for analysis of global gene expression, there is a tendency to replace

this transcriptome analysis using high throughput technology for sequencing.

Transcriptome by sequencing combines global gene expression with genome

studies and increase the efficiency in obtaining information in different citrus

pathosystems. However, for specific studies on gene expression the technology

of real time PCR has been more useful, and is well established in the routine of

several activities.

Expressive advance was done on the characterization of new candidate

genes for genetic transformation, all of them from the CitEST database. The

technology of transformation of juvenile tissue was well established with a

production of several potential events (transformed plants). The plants have

been challenged for biotic stresses to test for resistance or susceptibility to the

pathogens. Field experiments are planned after authorization of CTNBio.

In conclusion, both the platform of genomic information and of application

in the NSTI Citrus has extended the database of information on the genomes of

citrus and its pathogens, but has also pointed out emphasis in the use of new

approaches integrating with the breeding platform.

Next steps and priorities in these platforms will be:

• to increase the database of expressed genome and its application

(annotation, molecular markers, new candidate genes);

• to conclude the complete genome of citrus in the ICGC;

• to conclude the experiments of global gene expression both by

transcriptome (microarrays and sequencing) or proteome, and

• to produce new events of genetic transformation for disease

resistance.

The main challenge of the program is undoubtedly the integration of

information on genome to genetics at the breeding level. The proposed

approaches include the identification of molecular markers based on ESTs,

genetic mapping, genotyping of new population of hybrids, analysis of

expression QTLs, and production of genetically modified plants. An expressive

indicator of the program is the number o new hybrids under field evaluation both

as rootstock and scion potential varieties. Five populations of hybrids are being

used for genetic mapping with several classes of molecular markers including

DArts in a collaborative program with Australia.

With the increase of the citrus expressed genome database and the

availability of a complete genetic map with molecular markers, new approaches

could be launched. The possibility to map genes and to associate them in a

physical map of BAC libraries opens new perspectives in the associative

genetics. The Program should be prepared for this jump.

It should be pointed out that several field experiments have been

conduced with support of the industry, which maintain the orchards.

Next steps and priorities in these platforms will be:

• to increase the number of molecular markers in the available

genetic maps;

• to saturate the genetic maps with all classes of molecular markers;

• to start a physical map using BAC libraries of three species;

• to evaluate more hybrids of scion and rootstocks in the field;

• to test genetically transformed varieties under field conditions.

All scientific and technical activities of the NSTI Citrus include

undergraduate and graduate students from several graduate programs.

Progress Report

1. Platform of Genomic Information Platforms of genomic information are considered as the combined data both

from the citrus plant genome and its pathogens, which will play a role during the

program activities. The platforms have concentrated on studies regarding

comparative and functional genomics, the completion of the Citrus genome

sequencing (International Citrus Genomics Consortium) and the enrichment of

the expressed citrus genome databases, investigations on gene expression

(microarrays) of citrus plants as well as some of their pathogens, support to

genetic transformation based on data from the expressed citrus genome and

strengthening the bioinformatics to support the development of the tools that will

be needed during the development of the program.

Objective 1a. Increase CitEST database.

Goal 1a1: Construct and sequence cDNA libraries of sweet orange, mandarin,

trifoliate orange, Rangpur lime, and Sunki mandarin up to 30,000 unigenes per

species (approximately 120 thousand ESTs/species, in addition to the existing

CitEST database).

Indicators: Number of sequenced libraries

As described in the proposal, CitEST was composed of more than 280

thousands sequences, which resulted in more than 91 thousands unigenes.

From that, Citrus sinensis, C. reticulata and Poncirus trifoliata accounted for

most of the sequences, 32,121, 18,873, and 12,873 unigenes, respectively. For

increasing the CitEST database, we sequenced ESTs of 17 different new

libraries from different species as listed below.

Common name Species Library

name Number of library

Number of ESTs

Sweet orange Citrus sinensis CS 6 12,767 Mandarin Citrus reticulata CR 2 10,368 Poncirus Poncirus trifoliata PT 3 17,368

Sour orange Citrus aurantium CA 1 960 Tahiti lime Citrus aurantifolia CG 1 960 Mexican lime Citrus latifolia LT 1 960 Rangur lime Citrus limonia CL 2 5,278 Sweet lime Citrus limettiodes CM 1 480 Sunki mandarin Citrus sunki TS 1 1,536 Swingle citrumelo Citrumelo swingle CW 1 10,552

Total 19 3417,869 The goal of this part of the Program was to construct and sequence cDNA

libraries from sweet orange (C. sinensis), mandarin (C. reticulata), P. trifoliata,

C. sunki, and Rangpur lime (C. limonia). To these species we added others that

were interesting to our group but still we will accomplish the proposed goal for

each of the initially proposed species. However, due to the new technologies

that are fully available for DNA sequencing, we are planning to change the

method for increasing the CitEST database. Instead of the capillary Sanger

sequencing we are planning to use the Next-generation platform from Ilumina,

which is able to generate around 1.5 Gb per run. To make a rough comparison

with the Sanger technology, our reads are close to 750 bp maximum, which

means that it is proposed to sequence 90 Mb per species and, therefore, just

one run of the Genome Analyser II (Illumina) is enough to generate more bases

sequenced than the proposed for all the species together.

Because of that, we made contact with different companies that have this

service and chose Macrogen as service provider. We discussed the technical

conditions for sequencing our materials and we are now setting up the

bureaucratic aspects of the contract.

The material to be used for these experiments will be obtained from

experiments that were already set up for this project, like the evaluation of citrus

sudden death in the field, or material of plants infected with both bacteria of

huanglongbing.

The next step toward the accomplishment of this goal is to set the

agreement with Macrogen in relation to bureaucracy and prepare total RNA

from all the plant material to send for sequencing.

Goal 1a2: Process and analyze ESTs (approximately 310 thousand) database

and unigenes from all of the species of the Program and make available

annotation and search services.

Indicators: Number of assembled unigenes (singlets + high quality contigs).

We downloaded the data sequence from the University of California -

Riverside (214,224 ESTs) that comprises libraries prepared from different

laboratories in the world and includes different species. These sequences were

used for assembly with the CitEST set in our computational setup but we

completely failed in getting it done because our processing capacity was much

lower than the required for the task. On the other hand, small sets of ESTs were

successfully assembled, confirming the need for a more powerful setup to work

with large sets of sequences. The computational setup was included in the

original proposal.

Species CitEST Harvest

CitEST +

Harvest

New Assembled Unigenes

CS 112,420 89,182 201,602 np

CR 48,627 87,016 135,643 np PT 34,920 20,981 5,5901 np

CA 6,797 960 7,757 5,294 CG 7,377 0 7,377 3,989

LT 6,380 0 6,380 3,754 CL 6,666 0 6,666 1,407

CM 2,077 0 2,077 np TS 2,320 0 2,320 1,378

CW 6,908 8,758 15,666 6,799 TC 0 2,679 2,679 1,987

CF 0 1,728 1,728 1,153 CD 0 468 468 327

CY 0 1,635 1,635 728 ST 0 817 817 703

Total 234,492 2456,979 1682,1201 np: not clustered

Because of that, we contacted several suppliers for increasing this

capacity by acquring new equipments. We talked to IBM, Apple and Silicon

Graphics, deciding for the former (see Goal 1.c.1 as well). A cluster was

acquired and is now in process of import. So the next step is to get the machine

working and do all the necessary assemblies.

Goal 1a3: Generate and release SSR markers based on ESTs.

Indicators: Number and validation of SSR markers (primers, amplification

conditions etc).

Coordinators: Mariângela Cristofani-Yaly, Marco Aurélio Takita and Luciano T.

Kishi

We implemented a service in our server to identify SSR markers in the

EST databases. For that we used Misa (pgrc.ipk-gatersleben.de/misa/) and

obtained the following results with the very first CitEST set of unigenes.

Number CS CR PT CA CG LT CL CM TS

Sequences 32.121 18.873 12.873 5.607 4.327 4.883 5.945 4.540 2.652 Identified SSRs

26.588 10.830 10.794 3.495 2.779 3.184 8.642 7.028 4.768

SSR containing sequences

14.416 6.866 6.066 2.253 1.785 2.178 4.026 2.861 1.842

sequences containing more than 1 SSR

5.988 2.254 2.377 750 573 659 1.962 1.759 1.107

Number of SSRs present in compound formation

7.544 2.389

2.994 699 565 591 3.216 3.023 2.083

The validation of some of these SSRs was done as mentioned in

Objective 3a. The main problem was the bioinformatics resource available for

the Program so far. As mentioned before, the server we have is not able to

assemble some sets of ESTs available in CitEST right now, mainly the most

comprehensive ones. As soon as the new machines arrive (the equipments are

expected to arrive within the next four months) we will do the assembly to

analyze the whole EST datasets.

Objective 1b. Participate in the effort of sequencing, assembling, and

annotating a model citrus genome with a local database available for the

Program in collaboration with the International Citrus Genomics Consortium

(ICGC).

Goal 1b1: Assemble bioinformatics database for the complete citrus genome;

Indicators: Percentage of partial and final coverage of the genome throughout

the sequencing and assembling processes.

Goal 1b2: Assemble tools that allow the comparison between complete versus

expressed genomes;

Indicators: Available structured database and tools in Brazil.

Coordinators: Marcos A. Machado, Juliana Freitas-Astúa, Marco Aurélio Takita

and Luciano T. Kishi

The below text reproduces the statements and agreements among the

members of the International Citrus Genomic Consortium (ICGC) in the meeting

at Joint Genome Institute/DOE, Walnut Creek, CA, 2007 and 2008.

‘The Steering Committee of the ICGC met to discuss plans and changes to our

previously described goals and strategies, as outlined in an earlier White Paper and in

the so-called Valencia Declaration of April 2003. The outcome conclusions following

two days of deliberations were accepted and approved unanimously by the members of

the Steering Committee. These conclusions represent the essence of the ICGC plan

for international collaboration in sequencing a citrus genome, and they are listed below.

1. The long-term goal of the international consortium is a freely available, high quality

reference genome sequence for Citrus. Immediate short-term and mid-term goals

established by the ICGC are the selection of a target genome for sequencing by mid-

summer 2007, and the securing of funding commitments by late-fall 2007.’

Actually the sequencing started by mid-summer 2009 funded by Florida Citrus

Production Adviser Council (FCPRAC). A 2x cover was carried out by JGI using

cosmid libraries and Sanger quality. The Brazilian part (1x) will be sequenced

by HudsonAlpha Institute for Biotechnology at University of Alabama. The

contract between the Coordinator (Marcos A. Machado) and HudsonAlpha was

already signed, and payment will be done by FAPESP.

‘2. Based on various experiences from sequencing other heterozygous diploid plants

and the known allelic diversity of citrus, the highest quality reference sequence would

be provided by a haploid citrus genome.’

A haploid Clementine from Institute Valenciano de Investigaciones Agrícolas

(IVIA), Spain, was chosen as model species.

‘3. Requirements for the target sequencing genome:

• Haploid (or di- or tri-haploid) tree that may be freely distributed internationally;

• The candidate tree would preferably be free from infection with pathogens,

exhibit robust growth (as a partial guarantee against a defective genome), and

be relatively easy to maintain;

• The candidate tree chromosome number should be verified.

• The genome should be evaluated to assure that it is free large deletions or

other defects, as well as confirmed to be mono-allelic using molecular assays

including SSR marker assays, microarrays, and short-read sequencing

methods.’

Allmost all these activities were concluded inside the ICGC. The Brazilian group

was in charge to check the homozigozyty of the material using SSR markers.

‘5. Requirements for whole genome sequencing partnership:

• Target of 8-10x total coverage (3 billion bp, US$3M estimated cost)

• To begin ~fall 2007

• Rapid deposition of data with free access to all partners

• Sequencing partnership, pending funding approval, across Brazil, France,

Spain Italy and USA;

• Use common libraries, participants contribute reads through exchange

• Plan in place for the retention (multiple locations), maintenance and distribution

of clone resources (BACs and fosmids.)‘

With the possibility to use new technologies for sequencing the original proposal

was changed in the following way:

- 6x coverage with Sanger quality (2x for US, 2x for France, 1x for Brazil and 1x

for Italy) and 4x 454 quality (2x for Spain, 1x for Brazil and 1x for US).

- 1x Sanger quality of Brazilian group will be concluded by HudsonAlpha

Institute (University of Alabama), and will be paid by FAPESP (0.5x) and

Funarbe (0.5x) (Embrapa).

6. Other components identified as essential components of genome sequencing

efforts:

• A BAC library from the selected genome source, and BAC-end sequences

(greater than 7x, at least two enzymes)

• Physical map of the selected genome, to aid genome sequence assembly

• Probably no new large-scale EST sequencing is needed but complete

sequences from full length cDNAs would be desirable

• Organized effort to characterize variation across Citrus and related Rutaceae

(species and varieties)

• Integration of existing genetic maps to have highest density combined map

• Informatics

o Assembly to be performed at the various participating sequencing

centers, and released to the ICGC partners

o Analysis of variation (e.g., sweet orange shotgun vs. genome)

o “Structural annotation” (automated pipelines exist for gene prediction)

o Distribution (various capabilities for “genome browsers”)

o Curation’

BAC libraries of the haploid genome are available at Amplicon Express

(www.amplicon-express.com) and available to be purchased. We are ordering

not only libraries of haploid Clementine but also libraries of sweet orange

(Pineapple) and Poncirus trifoliata (Rubidoux).

Several activities above are not direct responsability of our group but can be

included in the Program as soon as they are concluded.

Objective 1c. Establish sequencing and assembling databanks of other citrus

species from the pyrosequencing effort.

Goal 1c1: 4x genome coverage (~380 Mb/species) of Ponkan mandarin,

Poncirus trifoliata and Rangpur lime with pyrosequencing sequences.

Indicators: Percentage of partial coverage of the genomes of these species.

To accomplish the goal of this part of the Program we made contact with

the Sequencing companies to verify the costs for pyrosequencing since it was

proposed as a third party service. For that, using the Genome Sequencer FLX

Instrument, powered by GS FLX Titanium series reagents, we will be able to

generate more than 1,000,000 individual reads with improved Q20 read length

of 400 bases per 10-hour instrument run. Therefore, one run can generate 400

MB, which is more than the estimated genome size. To get the job done we will

need 4 runs per species and that is the object of the discussion with the

Sequencing companies.

So far Macrogen was the one that offered us the best deal. We are now

discussing the technical conditions for sequencing our materials and setting up

the bureaucratic aspects of the contract. The material to be used for these

experiments will be obtained from plants grown in greenhouses.

We also evaluated the performance of two systems in order to have a

better idea about the computational resources needed for the task. We used

two different softwares, Mira (www.chevreux.org/projects_mira.html) and

Newbler (454 Life Sciences) to assemble two sets of 454 reads as listed below.

Reads Base pairs Assembling

time Software OS Server

610178 183.994.141 7 hours MIRA linux x 86_64 8 core 48 GRAM

1140276 388.321.828 > 30 days MIRA linux x 86_64 2 core 16 GRAM

1140276 388.321.828 > 40 days NEWBLER linux x 86_64 2 core 16 GRAM

The results show that for 1x coverage a 2-core 16 GRAM machine is not

enough to process the information reasonably fast. An 8-core 48 GRAM

machine performed much better than the other machine, so we considered that

the minimum for running the project. We discussed with the IT companies what

we need and what we could get for the resources we have and decided to buy a

system from Silicon Graphics with one head node of 2 6-core processors, 48

GRAM, two compute nodes of 4 6-core processors, 48 GRAM, and 1 compute

node of 2 6-core processor, 144 GRAM. So each node will have at least 12 core

and 48 GRAM and, therefore, we expected a much better performance.

As mentioned before, the machines are already in process of import. As

soon as they arrive, we will rerun the samples as well as test some other

multiprocessor softwares. To do the assembly of genomes from other species it

is necessary to have the complete genome of Citrus clementina available to

guide the assembly of other species.

Goal 1c2: Assemble tools for analyzing pyrosequencing sequences.

Indicators: Assembled databanks and available tools.

Goal 1c3: Assemble tools that allow the comparison between complete

genome (Sanger quality) versus partial genomes obtained from

pyrosequencing.

Indicators: Available tools.

The goals 1c2 and 1c3 depend on the results produced in 1b and 1c1. Objective 1d. Assemble and analyze citrus high-density microarrays for gene

expression and gene expression QTLs studies;

Goal 1d1: Assemble and analyze sweet orange high-density microarrays (380

thousand spots) for large-scale expression.

Indicators: Number of assembled and analyzed slides (approximately 36 slides

for each bioassay) for gene expression studies on the plant-pathogen and plant-

water stress interactions.

Slides were designed using UniGene selected from the CitEST database,

assembled from the ESTs submitted to NCBI (GenBank accession numbers

EY649559 to EY842485). The sequences were used to construct genome-wide

60-mer oligonucleotide cDNA microarrays by Roche NimbleGen Systems using

a multi-step approach to select probes with optimal predicted hybridization

characteristics. One of the slides was composed of 31,535 sequences of C.

sinensis, plus probes for six additional sequences three probes per target, four

copies on array, totalizing 378,420 probes of C. sinensis. According to the

experiment schedule (see goals 2a 2h) and upon request new sets of arrays

can be ordered.

Goal 1d2: Assemble and analyze high density microarrays (380 thousand

spots) for large scale gene expression studies in mandarin.

Indicators: Number of assembled and analyzed slides (approximately 36 slides

for each bioassay) for gene expression studies on the plant-pathogen and plant-

water stress interactions (see Objectives 1a to 1f and 1h).

Slides of a total of 62,876 UniGenes (31,583 of C. sinensis, 18,712 of C.

reticulata and 12,581 of P. trifoliata) selected from the CitEST database,

assembled from the ESTs submitted to NCBI (GenBank accession numbers

EY649559 to EY842485) were used to construct genome-wide oligonucleotide

cDNA microarrays by Roche NimbleGen Systems using a multi-step approach

to select probes with optimal predicted hybridization characteristics. Three

probes were selected per UniGene, comprising a probe set, and each probe set

is represented on the final array by two replicates. All probes were designed as

perfect match oligonucleotides. The slide was composed of 377,256 probes.

Goal 1d3: Assemble and analyze high density microarrays (380 thousand

spots) of Poncirus trifoliata for large scale expression.

Indicators: Number of assembled and analyzed slides (approximately 36 slides

for each bioassay) for gene expression studies on the plant-pathogen and plant-

water stress interactions (see Objectives 1f, 1g and 1i).

The available number of unigenes of Poncirus trifoliata both of CitEST

and public is not enough to build such array yet. As soon as the number of

unigenes increases they will be used in the arrays.

Goal 1d4: Assemble and analyze high density microarrays (380 thousand

spots) of sweet orange and Ponkan mandarin for expression QTL studies.

Indicators: Number of assembled and analyzed slides (approximately 72 slides

for each bioassay) for expression QTL studies (see Objectives 3f).

The available number of unigenes of Ponkan mandarin is also not

enough to build feasible arrays. As soon as the number of unigenes increases

they will be used in the arrays.

Goal 1d5: Develop proteome maps of sweet orange and mandarin during the

process of Alternaria infection.

Indicators: Number of maps consistently produced and number of identified

spots (see Objective 1g).

Two dimensional gel electrophoresis (2DE) and tandem mass

spectrometry (MS/MS) were used to evaluate the interaction of citrus and

Alternaria alternata in a proteomic level. Young leaves (2-3 cm length) of

susceptible Murcott tangor and tolerant Pera sweet orange were inoculated with

a conidial suspension (106 spores/mL) of A. alternata. In order to normalize the

patterns of translated proteins before the inoculation, plants were maintained for

two weeks in a plant growth chamber at 27 °C, 12h photoperiod (light/dark), and

70% relative humidity. Healthy plants were used as experimental control.

Twelve hours after inoculation, leaves were collected, ground in liquid nitrogen,

and the proteins were extracted with phenol according to Hurkman and Tanaka

(1986). Isoelectric focusing was performed in polyacrylamide IPGstrips (pH 3-

10NL) using 750�g of proteins. For each genotype, spots differentially detected

between healthy and inoculated plants were excised and digested in-gel by

trypsin. MS/MS analysis are been conducted in a Waters-Micromass Q-TOF-

Ultima API (ESI-MS/MS) coupled to a Waters CapLC system.

2DE gels were successfully obtained for healthy and inoculated plants of

each genotype (Figure 1). When treatments were compared, initial data

exhibited 191 and 76 differentially detected spots (p�0.05) in Murcott tangor and

Pera sweet orange, respectively. All the spots were excised from 2DE gels for

further MS/MS analysis. In October 2009, the Mass spectrometer showed

technical problems and could not be used until March 2010. In order to improve

analysis and reduce the amount of peptides to be sequenced, only those spots

showing p�0.01 were selected, corresponding to 56 spots from Murcott and 39

from Pera. During March 2010 9 digested proteins from Murcott and 21 from

Pera were sequenced (Table 1).

Amino acid sequences were automatically deduced using ProteinLynx

v.2.2.5 software (Waters) and Mascot server 2.3 (Matrix Science) coupled to

CitEST (http://biotecnologia.centrodecitricultura.br/), NCBI (http://www.ncbi.nlm.

nih.gov) and UniPROT/Swiss-Prot (http://www.expasy.ch/sprot) databanks. In

order to confirm automated deduction, sequences are been manually checked

(de novo sequencing) using PepSeq (Waters) and mMass v.3.0

(www.mmass.org) softwares. As soon as the mass spectrometer starts to work,

MS/MS analysis will be performed in order to conclude all the proteomic-related

experiments.

Figure 1. Coomassie Brilliant Blue G250 stained 2DE gels (pH 3-10NL) of healthy (A)

and 12 hours inoculated (B) plants of Murcott tangor (1) and Pera sweet orange (2).

A1 A2

B1 B2

pH 3-10 NL pH 3-10 NL

pH 3-10 NL pH 3-10 NL

Table 1. Citrus trypsin-digested proteins submitted to MS/MS analysis

Objective 1e. Assemble microarrays of the complete genome of citrus pathogens for

the functional genomics studies.

Goal 1e1: Microarray assay with the complete genome (~2,400 ORFs) of

Xylella fastidiosa.

Indicators: Number of assembled and analyzed slides (approximately 12 slides

for each bioassay) for the studies of biofilm formation during the process of

colonization of the xylem vessels.

DNA microarray chips were designed with thirteen 60-mer oligonucleotide

probes per gene, according to the available X. fastidiosa genomic sequences.

Probes were synthesized by Roche-NimbleGen, Inc. (Madison, WI, USA) in situ

by photolithography on glass slides using a computer-generated randomized

pattern on the array. All gene probe set were repeated five times in each chip

and we used three biological replicates per treatment. The raw data were

normalized, briefly, a Robust Multichip Average (RMA) convolution model was

applied for background correction, and the corrected probe intensities were then

normalized using a quantile-based normalization procedure. Differentially

expressed genes were identified, using the ArrayStar software, by Student's t

test (P.< 0.05) and the multiple test correction of raw p-values was performed

using the False Discovery Rate (FDR). We also used a cutoff of 2.0 fold change

in expression.

Goal 1e2: Microarray assay with the complete genome (of unkwnown size at

this point) of Candidatus Liberibacter americanus and Ca. L. asiaticus (if

bacterial DNA is available after cultivation).

Indicators: Number of assembled and analyzed slides (approximately 12 slides

for each bioassay) for the studies of infection in sweet orange.

The available database on genome of Ca. Liberibacter spp. does not

allow the construction of a robust array yet. As soon as more sequences are

submitted, allowing a minimal assembling of the genome, arrays will be

performed.

Objective 1f. Establish a routine analysis of large-scale gene expression using

RT-qPCR for gene validation.

Goal 1f1: Validate by RT-qPCR differentially expressed (up- or down-regulated)

genes in response to the interaction assays (see Objectives 1a to 1j, except for

1h).

Indicators: Number of genes validated for each assay.

To validate differentially expressed genes (up- or down-regulated) in

response to biotic stress [(Xylella fastidiosa, Phytophthora parasitica, and Citrus

leprosis virus C (CiLV-C)], relative quantification was made using the ABI

PRISM 7500 Sequence Detector System (Applied Biosystems). The primers

were designed using the ‘PrimerExpress’ (Applied Biosystems) program. All the

amplicons were sequenced using the automatic sequencer ABI 3730 to confirm

the amplification of the genes assessed. The primers amplification efficiency

were tested by serial dilution (at least 5) of cDNA samples and value of slope

assessed by E =10(-1/slope)-1. Selected primers were those with efficiency values

between 0.9 and 1. The reactions were done in triplicate using negative control

(without cDNA) to detect contaminants. Each reaction was composed by 2 μL of

cDNA, 1 μM by each primer (forward and reverse) and 12.5 μL of “SYBR green

PCR master mix” (Applied Biosystems); the final volume was adjusted for 25

μL. The reactions were incubated in 50ºC for 2 minutes, 10 minutes in 95ºC and

40 cycles of 15 seconds in 95ºC and 1 minute in 60ºC. The amplifications were

checked in ABI PRISM 7500. After amplifications, the reactions were submitted

to dissociation curve to identify contaminants in the test.

A short experiment to select the best normalizer genes was performed.

The �-tubulin is frequently used for these types of studies without a robust

comparison with other candidate genes. For this experiment, the expression

levels of �-tubulin, ubiquitin, ciclofilin, CEF2 and actin were tested with all the

samples of the experiments with CiLV-C inoculation. Their expression was

analyzed with the software GenEx, using the algorithm GeNorm. We concluded

that it is advisable to use two or three normalizers to provide a robust

comparison.

Gene expression of X.fastidiosa in biofilm condition - Some genes were

selected for validation by RT-qPCR, identified as probably related to persistent

cells. As endogenous control, genes with differential expressions in microarray

experiments are being tested.

Under tetracycline inhibitory conditions, 160 genes were differentially

expressed in DNA microarray experiments (114 genes repressed and 46

induced). From those, 10 were selected for validation by RT-qPCR. Under

copper inhibitory conditions, from the 868 differentially expressed genes in DNA

microarray experiments (407 repressed and 461 induced), 9 were selected for

validation by RT-qPCR.

The RT-qPCR assays are being conducted. The evaluation of possible

endogenous controls indicated that the genes XF1353 and XF0656 are good

candidates, since they were not differentially expressed from the control

(without antimicrobial compounds) in microarray experiments and RT-qPCR.

The expression of other genes, differentially expressed in microarray

experiments, is being tested.

Gene expression of Poncirus trifoliata, Citrus sunki and their hybrids during

infection of Phytophthora parasitica - In this study, genes differentially

expressed between hybrids of Poncirus trifoliata and Citrus sunki (resistant and

susceptible, respectively) may provide key candidates to identify transcripts

involved in disease resistance. We investigated gene expression in pools of four

resistant and four susceptible hybrids in comparison to their parents 48 hours

after P. parasitica inoculation using RT-qPCR. Our analysis searched

upregulated genes (fold > 2 and p-value � 0.05) in the resistant genotypes

relative to the susceptible, found in previous microarray study. Nine genes were

selected due to the biological interest based on their function according to

CitEST database; among them, genes that encode enzymes participating in

defense-related metabolic pathways such as the biosynthesis of

phenylpropanoids and antimicrobial compounds such as phytoalexins, flavonoid

biosynthesis and resistance genes such as CC-NBS-LRR and TIR-NBS-LRR.

Gene expression of Pera sweet orange, Murcott tangor and their hybrids during

infection of Xylella fastidiosa - A total of 163 unigenes in several functional

categories were detected as differentially expressed (Fold> 2) between the

pools in relation to resistant pools susceptible in microarray study. The

functional classification of induced transcripts revealed genes with defensive

function that candidates may be involved in resistance to CVC. Thirteen genes

were selected due to the biological interest based on their function according to

CitEST database, among them, genes that encode enzymes participating in

defense-related. The RT-qPCR assays are being tested for validation.

Gene expression of Pêra sweet orange and Murcott tangor in response to

infestation with Brevipalpus phoenicis non-viruliferous or viruliferous to CiLV -

This study compared the response of Pera sweet orange and Murcott tangor

(resistant and susceptible, respectively) to CiLV-C. Two-way ANOVA (p-value �

0.1 and fold-change � 2) analysis resulted in 80 differentially expressed genes,

68 of them upregulated and 12 downregulated. Among them, 11 genes were

chosen for validation by RT-qPCR based on their similarity with genes involved

in defense response to pathogens and fold-change. The RT-qPCR assays are

being tested for validation.

Gene expression in response to infection with Citrus leprosis virus C (CiLV-C) -

Hybrids of Pêra sweet orange and Murcott tangor were infested with

Brevipalpus phoenicis viruliferous for CiLV-C. These plants had been evaluated

for 6 years in prior field experiments order to find the most susceptible and the

most resistant individuals. A pool of seven resistant or seven susceptible plants

was collected in four biological repetitions for microarray analysis. The

comparison of the resistant pools to the susceptible ones showed 466 genes

differentially expressed. From these, 289 were upregulated and 177 were

downregulated. For validation, four genes upregulated and three downregulated

were chosen based on known genes for defense response to pathogens. In

addition to them, ten other genes without a clear function in the roll of plant

defense were chosen to try to identify genes not usually described.

Objective 1g. Increase the genetically modified citrus program.

Goal 1g1: Produce new sweet orange transformation events with constructions

derived from the Program database.

Indicators: Number of new constructions and events of transformation.

New constructions for candidate genes.

Defensins - Analysis of the citrus ESTs has shown the occurrence of at least

two genes encoding defensins similar to those described to other plant species

that present antifungal and/or antibacterial activities.

Primers were designed for the coding regions of these defensins that

resulted in the amplification of products of 815 bp (CsDef1) and 213 bp

(CsDef2) from Valencia sweet orange genomic DNA. The genes were cloned in

pGEM-T Easy vector (Promega) and submitted to DNA sequencing. CsDef1

has shown an intron interrupting the coding region while the same was not

observed for CsDef2. The defensin encoded by CsDef1 has 82 aa including 8

cysteins that are conserved among plant defensins and has shown 43.9%

sequence identity to defensin PDF1.4 from Arabidopsis thaliana. Amino acids

sequence analysis with the Psort program indicated the presence of a 26 aa

non-cleavable signal peptide and a putative localization in the endoplasmic

membrane. The defensin encoded by CsDef2 has 76 aa including the 8

conserved cysteins and has shown 59.7 % identity to A. thaliana PDF2.1. Psort

analysis indicated a 19 aa cleavable signal peptide and a high probability of

secretion to the outside of the cell.

The next step of vector construction is to isolate the cassettes

constructed and to transfer them to the binary vector pCambia2201

(www.cambia.org.au). After this, citrus plant transformation will be performed.

Citrus phloem protein 2 (PP2) promoter - The citrus phloem protein 2 (PP2)

promoter is capable to drive vascular tissue-specific expression of reporter

genes in citrus transgenic lines. The gene was characterized in the citrus

shotgun library and the upstream region was identified. Thus two regions (PP2-

2 and PP2-3) of full promoter were isolated from leaves of Valencia sweet

orange (Citrus sinensis L. Osbeck) and cloned. These regions were digested

with Eco RI and Bgl II and cloned in the pCAMBIA 3301 vector. Citrus

transformation is in progress.

Npr1 from Citrus - Initially, using the Gene Projects environment of CitEST, we

selected 51 sequences either by npr1 keyword search or tBlast N. These

sequences were clusterized in 11 contigs and 6 singlets. The analysis of the

contigs allowed us to identify 12 clones carrying plasmids with sequences of

npr1 from Citrus. These clones used for plasmid extraction and DNA

sequencing. E. coli DH5� strain carrying plasmids from different CitEST

libraries were recovered from the -80oC freezers. The bacterial cells were grown

in 2 mL of LB medium, supplemented with ampicillin (100 μg/mL), at 37oC for 16

hours under rotation (250 rpm). Plasmidial DNA extraction was done using

alkaline lysis protocol.

The sequences were assembled in Seqman and 5 different forms of Npr1

were identified. To verify the absence of problems in the DNA sequence present

in each of the clone, new primers were designed using the contigs generated in

the assembly as templates. This was done because the fragment was large and

sequencing from the 5’ and 3’ ends was not enough to cover the entire

sequence. Twenty new primers were designed in Oligo Explorer 1.2

(http://www.genelink.com/tools/gl-oe.asp) and used for sequencing the DNAs.

P29 (putative coat protein) and P32 (movement protein) of Citrus leprosis virus

C (CiLV-C) - Genes coding for the viral proteins P29 and P32 were selected to

construct cassettes to induce silencing. This strategy is normally used to avoid

the virus multiplication in plants. The pKANNIBAL vector was used during the

cloning. Specific primers were designed for gene cloning in sense and

antisense using the program ‘Primer3’

After isolation of viral genes from the RT-PCR using cDNA of sweet

orange plants infected with CiLV-C, the genomic fragments (789 bp for P29 and

891 bp for P32) were purified, sub-cloned into vector pGEM-T (Promega) and

sequenced to confirm the inserts. The presence of the inserts was confirmed by

specific primers for the 35S promoter as well as primers for the genes of

interest.

As a next step, the constructions pKANNIBAL+sense gene (for the two

candidate genes) will be digested with specific enzymes for cloning of antisense

gene according to methods previously described.

Goal 1g2: Produce new transformation events of Rangpur lime, Sunki mandarin

and Swingle citrumelo (rootstocks) with constructions derived from the Program

database.

Indicators: Number of new constructions and events of transformation.

New constructions for candidate genes.

NAC4 and WRKY17 - The full-length cDNA sequences of the genes NAC4 and

WRKY17 were identified, cloned, and characterized from leaves of Citrus

reshni. These genes were excised from the pGEM-T vector, by means of

digestion with SalI and NotI, and subcloned in sense orientation at the same

restriction sites of the pUC118/CaMV35S plasmid. This plasmid contains the

CaMV 35S promoter. The cassetes have been now excised of

pUC118/CaMV35S, by digestion with PstI, and will be subcloned at the same

site in the pCAMBIA 3301 vector. This vector contains the chimeric genes bar

and gusA.

- Actions for the continuation and fulfillment of the goal in the next period.

In the next period, these constructions will be used to transform citrus

rootstocks.

P5CS - The coding region of p5cs gene (involved in the proline synthesis) from

Citrus sinensis was amplified by PCR, using Pfu DNA Polymerase with specific

primers. The gene amplified was cloned into pCR2.1 vector, using TOPO TA

Cloning kit (Invitrogen). After this, the coding region was isolated with restriction

enzymes BamHI and EcoRI and subcloned in p35SPT (pGreen vectors),

previously digested with the same enzymes. The expression cassette (P35S-

p5cs-T35S) was isolated by digestion with EcoRV and cloned in pCambia

2305.1 in the SmaI restriction site. This vector was transferred to Agrobacterium

tumefaciens EHA105 and used for plant transformation.

Protocol for plant transformation - The vectors containing the desired

construction were introduced into EHA105 lines of A. tumefaciens, using the

freeze-thaw method. The verification of transformed colonies was done by PCR

using specific primers for the gene. Epicotyl segments of citrus (0.8 to 1 cm)

were obtained from seedlings germinated in vitro. Seeds were treated with

commercial bleaching solution diluted 1:3 with distillated water, for 20 minutes,

and placed for germination and elongation in the dark (4 weeks), at 27oC.

Epicotyl segment were inoculated with A. tumefaciens for 20 minutes. The

explants were incubated for 3 days at 24oC, and then transferred to EME

selection and regeneration medium, supplemented with BAP, cefotaxime and

kanamycin (100 mg L-1) or Finale® at 27oC, 16-h of light. Sprouts from

regenerated plants were analyzed by GUS hystochemical analysis and by PCR

with specific primers to detect the studied gene. Positive plants were

micrografted on selected rootstock for later acclimation in greenhouses.

Transformation of Rangpur lime and Sunki mandarin - Seeds of ‘Cravo Santa

Cruz’ Rangpur lime (Citrus limonia), ‘Sunki Maravilha’ mandarin (C. sunki), and

‘TSKxSW 314’ hybrid were collected in the active citrus germplasm bank

collection of Embrapa Cassava and Tropical Fruit (Cruz das Almas, BA). Seeds

were germinated in plastic dibble tubes (115 cm3 and 180 cm3) containing a mix

of Plantmax (Eucatex Agro.) substrate and coconut fiber, at 1:1 proportion,

under greenhouse conditions. Four months after germination, the plants were

transplanted to 15 L pots containing a mix of soil, sand, and Plantmax

substrate, at 2:1:1 proportion. After four months, plants of nucellar origin were

selected based on their uniformity and splitted in two groups: (i) a group of 4

plants of each genotype that were daily irrigated and (ii) a group of 11 plants of

each genotype that were drought stressed. All the pots were closed with

aluminum foil in order to allow the water loss only by transpiration. The water

content of the pots has been measured by time domain reflectometry (TDR)

probes, the stomatal resistance by a porometer, and the water potential by a

Scholander pump. Samples of leaf and root tissues have been collected for

further molecular analyses. At this point, three harvests of tissues were done

already. The RNA was extracted using the RNAqueous kit (Applied

Biosystems).

Objective 1h. Develop, improve, and implement bioinformatic tools in support

of the database.

Goal 1h1: Support the Program.

Indicators: Pipelines, services and interfaces in use.

Our citrus genome database, with 448,716 reads (CitEST, Harvest and

International Citrus Genome Consortium), requires constant updates to the

analysis of new sequences, including ESTs libraries on the following:

- Submission System of electropherograms;

- Pipeline to trim and assemble ESTs;

- System Project Management (GP);

- System for quantitative analysis of data from CitEST – in silico

hybridization; - Scripts to generate data in HTML format of the results of hybridization in

silico and to categorize and compare against a number of public databanks;

- Support the work of expression by microarray, where it was necessary to

develop a database (MySQL) for storing microarray data and a web interface

(CGI-PERL) to make the results available, integrating with the Unipaper data

base;

- Evaluation and development of scripts for new methodologies for

categorizing genes and systems for categorization via Gene Onthology;

- Understanding and study of the KEGG metabolic maps for

implementation of unigenes in studies of metabolic pathways.

Difficulties found were related to low processing power and delay to

process the blasts and assembling programs with data of pyrosequencing.

2. Plataform of Genomic Application Objective 2a. Transcriptome and gene expression profile to study citrus-

Candidatus Liberibacter spp. interactions

Goal 2a1: Characterize and compare at least two profiles (= bioassays) of

global gene expression of sweet orange infected with Candidatus L. spp.

Indicators: Number of differentially expressed genes putatively associated with

susceptibility to HLB.

To obtain healthy and infected plants, approximately 60 plants of sweet

orange (C. sinensis) originated from seeds and grafted onto Rangpur lime were

used. After reaching around 30 cm of vegetative growth, 25 plants were

inoculated with Ca. Liberibacter americanus (CLam), and other 25 with Ca.

Liberibacter asiaticus (CLas) by grafting of two buds infected from symptomatic

plants. All the plants were maintained under greenhouse conditions. Once

confirmed the presence of the bacteria in all of the inoculated plants, they were

pruned to induce sprouting and uniform vegetative growth.

The presence of the bacteria occurred approximately forty days after

pruning, with the appearance of the first shoots, having reached vegetative

growth of about 15 cm and tissues maturation of the first samples of infected

and healthy plants. The leaves and twigs tissues were collected from five citrus

plants positives for CLam; five plants positive for CLas, and three healthy

plants, with their developmental stage as uniform as possible. Plant tissues

were ground in liquid N2 and a small amount of different tissues of each plant

were evaluated to confirm the presence of the bacteria both by conventional

PCR and qPCR.

Once confirmed the presence of the bacteria in the tissues, RNAs from

these samples were extracted using the RNeasy Mini Kit (Qiagen) and sent to

NimbleGen for hybridization on microarrays containing about 32,121 species-

specific unigenes of citrus (C. sinensis). CLas was first detected about 90 days

after inoculation (d.a.i) and the first symptoms appeared around 120-150 d.a.i in

approximately 95% of the infected plants. CLam was first detected 150 d.a.i and

the first symptoms appeared approximately 220 d.a.i in 30% of the inoculated

plants. Only three plants infected by CLas were confirmed by conventional

PCR. All other test plants were positive for CLam and CLas by qPCR. After 40

days of pruning it was possible to identify lack of uniformity in vegetative growth.

Moreover, plants infected with CLas exhibited leaf symptoms similar to nutrient

deficiency and, in plants infected with CLam, the leaves tended to curl down.

The RNA extracted from juvenile tissues (leaves and branches exemplified in

Fig.3) was of high quality and concentration (Fig. 4), varying from 790 to 4,496

ng/ul, ratio 260/280 varying from 1.53 to 2.12 and ratio 260/230 varying from

1.20 to 2.27. Preliminary results obtained by our group, using two biological

replicates, reveal 634 sweet orange genes differentially expressed after

inoculation with CLam compared to the healthy plants. From those, 419 were

induced and 215 repressed by CLam inoculation. Figure 5 A and B shows the

classification of the differentially expressed genes found according to MIPS

categorization.

Figure 3. Sweet orange Plant 1 (Citrus sinensis L. Osbeck) infected with CLam. Leafs and branchs (juvenile tissues) used in microarray analysis

Leaves

Branches

Figure 4. RNA extracted of Sweet orange Plant 1 (Citrus sinensis L. Osbeck) infected with CLam. Leafs and branchs tissues used in microarray analysis.

Figure 5. Categorization of differentially expressed genes in sweet orange

(Citrus sinensis L. Osbeck) infected with Ca. Liberibacter americanus according

to MIPS database category of Arabdopsis thaliana.

Objective 2b. Transcriptome and gene expression profile to study citrus-Xylella

fastidiosa interactions.

Goal 2b1: Characterize and compare at least three profiles (= bioassays) of

global gene expression of sweet orange and mandarin infected with Xylella

fastidiosa.

Indicators: Number of differentially expressed genes putatively associated with

susceptibility and resistance to CVC.

The experiments were conducted with plants of sweet orange and

mandarin grafted on Rangpur lime and kept in a protected environment. For

inoculation, 10�l of X. fastidiosa suspension were deposited by needle.

Total genomic DNA (plant + bacteria) was extracted from inoculated

tissue after 1, 7, 14 and 21 days. These samples were used for the monitoring

of bacterial population through qPCR. The analyses were carried out with an

ABI PRISM 7500 Sequence Detector System. Each sample was tested in

triplicate with three biological replicates. The standard curve was produced

using known concentrations of genomic DNA of X. fastidiosa obtained from 10-

fold serial dilutions from 1x103 a 1x108 CFU per mL. This showed a linear range

with a correlation coefficient of 0.999. All the results reported from qPCR were

based on the number of cells of X. fastidiosa in each sample, which was

estimated from the standard curve. Negative (no template DNA) and positive

(DNA from X. fastidiosa) controls were included in all experiments to exclude or

detect any possible contamination.

It was found that sweet orange plants infected with X. fastidiosa showed

a gradual increase of population over time, which was expected due to the

susceptibility of this variety to CVC (Fig. 6). However, the population of bacteria

in tangerine remained stable at all time course evaluated (Fig. 6). This is

consistent with previous results where X. fastidiosa can stay for a certain period

of time in tolerant plants, but then the population decreased by a total absence.

Figure 6. Population mean of X. fastidiosa estimated by qPCR for analysis of

leaf samples taken from sweet orange and tangerine on various days after

inoculation. Bars represent errors of the averages of three biological replicates.

Originally the experiments were proposed to characterize and compare at

least three global gene expression profiles of sweet orange and tangerine

infected with X. fastidiosa. However, our group decided firstly investigate the

changes that occur in the early stage of response to infection that possible

involves resistance genes and signaling pathway. To determine which time

point could represent the time of expression of genes related with resistance

and signaling network, we did a pilot experiment with the four time points after

inoculation (1, 7, 14 and 21 days) in tangerine resistance plants through the

expression of some genes previously found in Citrus EST Project (CitEST).

Among the selected genes we chose those involved in early and delay stages

of plant response to pathogens (Table 2).

Table 2: ORFs from CitEST database possibly associated with resistance to X.

fastidiosa in mandarin.

ORF Putative Function

NBS-LRR type disease resistance protein

Resistance to plant pathogens

Apetala2/ethylene responsive factor

Cell signaling

pad4 Involved in the synthesis of AS and response to pathogens

npr1 Inductor of PR proteins

pr1 Inductor resistance and response to pathogen

The same plant material collected from the experiment to follow up the

population of X. fastidiosa was used to do the pilot experiment. The same

material will also be used for microarray analysis. The total RNAs were

extracted with Trizol and treated with RNase-Free DNase Set. The samples

were done using a pool of three biological replicates for each time of evaluation

in plants infected or not (controls). For cDNA synthesis we used 500 ng/ �l of

total RNA. The evaluations of gene expression were performed on the ABI

PRISM 7500 using relative quantification analysis. The detection of PCR

products was measured by monitoring the increase in fluorescence emitted by

SYBR green marker. The endogenous control (EC) used was �-tubulin. For all

reactions the qPCR was performed in a dissociation curve to check for

nonspecific amplification resulting from contamination.

The results showed that all genes were induced after one day of

inoculation, but not apetala, which showed no expression at any time of

evaluation (Fig. 7). Based on these results we chose one day after inoculation

to evaluate the change of global gene expression by microarray. We expected

mainly identify the network of signalization genes in mandarin that might trigger

other genes and as a consequence reflecting in its resistance.

Figure 7. Relative quantification of the possible genes associated with

resistance in Poncan mandarin. cDNAs pool consisting of three biological

replicates for each time measured from plants challenged with X. fastidiosa, or

not. The samples were used for the quantification in the ABI PRISM 7500

Sequence Detector System (Applied Biosystems). The measures were

normalized using the threshold cycles (Cts) obtained for the amplifications of

the endogenous control run in the same plate. The values represent the fold

increase in gene expression compared with values obtained for cDNA from

plants without pathogen (calibrator). The results are averages of samples tested

in triplicate. Bars represent standard errors of the means.

Objective 2c. Transcriptome and gene expression profile to study citrus-

Xanthomonas axonopodis pv citri interactions.

Goal 2c1: Characterize and compare at least two profiles (= bioassays) of

global gene expression of sweet orange and mandarin infected with

Xanthomonas axonopodis pv citri.

Indicators: Number of differentially expressed genes putatively associated with

susceptibility and tolerance to citrus canker.

The experiments will be conduced in Spring 2011. Concentration of

bacteria, methods for inoculation into the tissue, and time of infections were

previously determinated.

Objective 2d. Transcriptome and gene expression profile to study citrus-Citrus

leprosis virus C (CiLV-C) interactions.

Goal 2d1: Characterize and compare at least two profiles (= bioassays) of

global gene expression of sweet orange, tangor, and lime infected with Citrus

leprosis virus C (CiLV-C).

Indicators: Number of differentially expressed genes putatively associated with

susceptibility, resistance, and tolerance to leprosis.

Plants of the susceptible Pera sweet orange (C. sinensis) and the

tolerant Murcott tangor (Citrus sinensis x C. reticulata) were infested with

Brevipalpus phoenicis mite vector non-viruliferous or viruliferous for CiLV-C.

Samples were collected 48 hours post infestation for whole transcriptome

analysis. Total RNA from these samples were extracted using the RNeasy Mini

Kit (Qiagen) according to manufacturer´s instructions. The integrity, quality and

concentration of RNA was analyzed using denaturing gel and NanoDrop 8000

Spectrophotometer-Thermo Scientific equipment according to manufacturer’s

instructions and sent to Roche NimbleGen for hybridization on 385K density

microarrays containing 32,121, 18,873 and 12,873 unigenes from Pêra sweet

orange, Ponkan mandarin (C. reticulata) and Poncirus trifoliata, respectively.

These unigenes were obtained from the Citrus EST database (CitEST) of

Centro de Citricultura Sylvio Moreira/IAC.

Using a two-way ANOVA test (p-value � 0.1 and fold-change � 2), 80

differentially expressed genes were identified (Fig. 8). The majority of the up-

regulated genes were related to cellular communication/signal transduction

mechanism and transcription; the few genes related to cell rescue, defense and

virulence will be validated by RT-qPCR.

Figure 8. Categorization of differentially expressed upregulated genes in

Murcott tangor inoculated with CiLV-C according to MIPS database category of

Arabdopsis thaliana.

Because of the significant amount of work, as well as their economic

importance, we decided to compare the response of only two citrus genotypes

to leprosis: sweet orange (susceptible) and Murcott tangor (resistant/ tolerant).

On the other hand, another experiment was conducted aiming to find

differentially expressed genes in plants responding to Citrus leprosis virus C

(CiLV-C) infection. In this study, pools of seven highly tolerant and seven highly

susceptible hybrids from Murcott tangor and Pêra sweet orange were used for

comparison of response to the disease by microarray analysis. In order to

define susceptible and tolerant hybrids, they were infested by viruliferous mites

in 2002 and the symptoms were evaluated during six years. Symptomless

leaves of each hybrid were collected for RNA extraction. The experiment was

designed in ten experimental blocks, the leaves were collected from four blocks,

each being a biological repetition and each repetition containing seven

Upregulated

individuals. The same microarray chip containing 32,121; 18,873 and 12,873

unigenes from Pêra sweet orange, Ponkan mandarin and Poncirus trifoliata,

respectively, was used. The microarrays were performed by Roche Nimblegen

and Bayesian moderated T-test yielded 466 diferentially expressed genes (p.val

� 0.05 and fold-change � 2). The differentially expressed genes and their

classification according to MIPS is showed in Figure 9. With exception of genes

encoding unknown proteins and with classification not clear yet, the majority

was related to metabolism, cell rescue, defense and virulence, and protein fate.

Figure 9. Categorization of differentially expressed genes in bulks of resistant

compared to susceptible hybrids according to MIPS database category of

Arabdopsis thaliana.

Goal 2d2: Characterize and compare at least two profiles (= bioassays) of

global gene expression of sweet orange, Murcott tangor, and lime infested with

the false spider mite Brevipalpus phoenicis (vector of CiLV-C).

Indicators: Number of differentially expressed genes putatively associated with

response to the false spider mite.

The experiments will be conduced in Winter 2010. Number of mites, time

of virus transmission and incubation period were previously determined.

Objective 2e. Transcriptome and gene expression profile to study citrus-

CTV/CSD interactions.

Up-regulated Down-regulated

Goal 2e1: Characterize and compare at least two profiles (= bioassays) of

global gene expression of Sunki mandarin and Poncirus trifoliata infected with

CTV.

Indicators: Number of differentially expressed genes putatively associated with

susceptibility and resistance to tristeza.

The experiments will be conduced in Winter 2010. Rangpur lime will be

included in the evaluation.

Goal 2e2: Characterize and compare at least two profiles (= bioassays) of

global gene expression of Rangpur lime and Sunki mandarin infected with CTV

in plants exhibiting citrus sudden death (CSD) symptoms.

Indicators: Number of differentially expressed genes putatively associated with

susceptibility and resistance to CSD.

Infected samples will be collected in the field in Spring 2011. Global gene

expression will be evaluated not only for Sunki mandarin, but also for Rangpur

lime and Swingle citrumelo, instead of Poncirus trifoliata.

Objective 2f. Transcriptome and gene expression profile to study citrus-

Phytophthora spp. interactions.

Goal 2f1: Characterize and compare at least two profiles (= bioassays) of

global gene expression of Sunki mandarin and Poncirus trifoliata infected with

Phytophthora parasitica.

Indicators: Number of differentially expressed genes putatively associated with

susceptibility and tolerance to citrus gummosis.

A microarray containing 62,876 UniGene of P. trifoliata, Citrus sinensis

and C. reticulata selected from the CitEST database and prepared by

NimbleGen Systems was used for analyzing global gene expression 48 hours

after infection with P. parasitica. RNA was extracted from resistant and

susceptible hybrids (four each), and then the four samples were pooled into

resistant and susceptible samples.

A total of 8,522 UniGene transcripts distributed in several functional

categories (Figure 10) were detected as upregulated (fold change > 2) in P.

trifoliata relative to C. sunki, while 4,337 transcripts were detected as

upregulated in the resistant bulk samples relative to C. sunki, and 1,168

transcripts were upregulated in resistant hybrids relative to susceptible ones.

These analyses identified 102 upregulated (Figure 11) UniGene transcripts (p-

value � 0.05) in all three comparative pair analyses, and nine of the top genes

with described functions retrieved from CitEST were selected for validation by

real-time RT-PCR (Figure 12). We encountered several previously reported

defense-related genes, such as one encoding CC-NBS-LRR and TIR-NBS-LRR

resistance protein, upregulated in the resistant genotypes compared to the

susceptible genotypes. NBS–LRR-mediated resistance has been identified

against numerous types of biotrophic or hemibiotrophic pathogens, including

fungi, oomycetes, viruses and bacteria, and these types of R genes have been

identified across a wide range of plants.

Our results showed that the libraries were useful for identifying genes

involved in the Phytophthora-citrus compatible interaction. On the other hand,

the use of a mixed-species DNA microarray was feasible because there was

enough complementarity among the libraries used to build the arrays. Pooled

samples can also be beneficial to find outgroup-specific expression profiles

despite large interindividual variation.

The genes that we have identified as upregulated across the resistant

genotypes will be valuable for ongoing work in eQTL mapping.

Figure 10. Functional categories according to MIPS classification scheme of

significantly upregulated UniGene transcripts (fold change > 2 and p-value �

0.05) common to the three pairwise comparative analyses (Rub vs. Sunki, Pool

R vs. Sunki, and Pool R vs. Pool S) obtained using BLASTX searches against

the GenBank database.

Figure 11. Venn diagrams showing the differentially upregulated UniGene

transcripts common to the resistant genotypes based on three different

comparisons between resistant (Pool R) and susceptible (Pool S) F1 hybrids

and their parents, Citrus sunki (Sunki) and Poncirus trifoliata Rubidoux, (Rub),

respectively susceptible and resistant to P. parasitica, 48 hours after

inoculation. Summary of UniGene selection results by two methods: (A) Venn

diagram summarizing the number of UniGene transcripts identified using 2-fold

cut-offs without a p-value restriction; and (B) Venn diagram summarizing the

number of genes identified using 2-fold cut-off and p-value � 0.05.

Figure 12. Validation of microarray data by quantitative real-time RT-PCR.

Real-time RT-PCR fold-changes are shown for nine genes upregulated in all

microarray pairwise comparisons (Rub vs. Sunki, Pool R vs. Sunki, and Pool R

vs. Pool S) 48 hours after P. parasitica inoculation, and wer compared with fold-

changes obtained by microarray analysis. Real-time RT-PCR data were

normalized to the two most stable candidate endogenous control genes (UBQ

and CYP). Bars represent standard deviations of the means of three technical

replicates and three biological replicates. *, p-value � 0.05; **, p-value � 0.01.

Objective 2g. Transcriptome and gene expression profile to study citrus-

Alternaria alternata interactions.

Goal 2g1: Characterize ans compare at least two profiles of global gene

expression of sweet orange and mandarin during infection by Alternaria

alternata patotype tangerine.

Indicators: Number of differentially expressed genes putatively associated with

susceptibility and tolerance to alternaria.

Instead of analyzing gene expression using microarrays, the

transcriptome of the interaction between citrus and Alternaria will be carried out

by transcripts sequencing using the Illumina platform. The experiments are

planned for the next year.

Objective 2h. Citrus transcriptome and gene expression during water stress.

Goal 2h1: Characterize and compare at least two profiles (= bioassays) of

global gene expression of Sunki mandarin, Rangpur lime, and Poncirus trifoliata

under water stress conditions.

Indicators: Number of differentially expressed genes putatively associated with

the response to hydric deficit.

Instead of analyzing gene expression using microarrays, the

transcriptome of the interaction between the rootstostocks under water stress

conditions will be carried out by transcripts sequencing using the Illumina

platform. The experiments are planned for the next year.

Objective 2i. Xylella fastidiosa transcriptome and gene expression during

infection.

Goal 2j1: Characterize and compare conditions for the biofilm formation by

Xylella fastidiosa.

Indicators: Number of differentially expressed genes putatively associated with

biofilm formation and pathogenicity.

Microarray analysis was peformed to identify genes associated with

programmed cell-death in Xylella fastidiosa biofilm. To accomplish that,

inhibitory concentration of copper and tetracycline were added when the biofilm

reached mature phase. The samples from three biological repetition where sent

to NimbleGen to microarray hybridization. Each oligonucleotide, which

represents one gene from X. fastidiosa genome, was spotted five times on the

chip. Each spot has 5 repetitions. The nomalization data was done using the

“quantile” methodology by NimbleScan software. The statistic analysis of the

normalized data was done using DNAStar software. The values of differentially

expressed genes were obtained using fold change more then 2 and T-Student

(95% of confidence) test.

3. Plataform of Applied Genetics

Objective 3a. Assess promising new citrus rootstock and scion hybrids under

biotic and abiotic stresses.

Goal 3a1: Assess rootstock trials for resistance to gummosis and tolerance to

CSD and tristeza.

Indicators: Number of assessed rootstocks; number of rootstocks with higher

or lower tolerance to CSD; number of rootstocks with higher or lower tolerance

or resistance to gummosis; number of rootstocks with higher susceptibility to

tristeza; number of rootstocks with potential to be recommended in new

experimental areas; number of rootstocks recommended as cultivars; number of

rootstocks registered in the “Serviço Nacional de Proteção de Cultivares” (or

National Plant Variety Protection Service).

Rootstock and scion breeding program of Instituto Agronômico

At this stage of the work, the agronomic characteristics of rootstock

hybrids under field and greenhouse conditions, with emphasis on disease

resistance, are being evaluated. In Table 3 there were described the

populations, traits and localities where the experiments were conducted.

Table 3. Description of the experiments in field and greenhouse, local,

pathosystem in evaluation and total number of plants evaluated.

Experiments in field

Hybrids Pathosystem Place Number

of hybrids

Nr of plants

Date

Sunki mandarin x P. trifoliata

Tristeza Gummosis

Cordeirópolis

270 1104 2003

Sunki mandarin x P. trifoliata

Tristeza Gummosis Sudden death

Colômbia

111 P/T 444 2004

111 V/T 444

165

V/T/LC 660

Experiments in greenhouse

Rangpur Lime x Swingle

Tristeza Gummosis

Cordeirópolis 120

480 2006

citrumelo Sudden death Colina 120 720 2006 Rangpur Lime x T. trifoliata

Sudden death Colina

65 390 2006

Sunki mandarin vs P. trifoliata

Sudden death Colina

198 594 2006

The results of the evaluation for resistance to CTV (Citrus tristeza virus),

CSD (Citrus Sudden Death) and Phytophthora gummosis provided hybrids with

level of resistance/tolerance similar to the resistant parental. Resistant/tolerant

hybrids to CTV and Phytophthora gummosis were obtained from the controlled

crosses: Citrus sunki x Poncirus trifoliata, C. limonia x P. trifoliata and C. limonia

x Swingle citrumelo. Some of them showed tolerance to hydric stress. These

hybrids have been included in breeding programs for citrus rootstocks.

The citrandarins (hybrids of Sunki mandarin x Poncirus trifoliata) were

evaluated for agronomic traits conferred by the rootstock to the scion variety

(Pera sweet orange) as water stress tolerance, plant height, incompatibility, fruit

quality and production. For tolerance to water stress, highly susceptible plants

and plants with tolerance comparable to Rangpur were found. In the present

study, only three hybrids displayed necrosis in the region of grafting, a typical

symptom of incompatibility between scion and rootstock. As for plant height, we

observed plants ranging from 0.99 to 2.37 m, indicating that rootstocks have

influence on their height. Thirty seven citrandarins were pre-selected with

potential for rootstocks varieties. Evaluations of the agronomic characteristics

and quality of the juice were performed in order to verify the importance of

rootstock on the development and ripening point of the scion variety, since

some hybrids induced early ripening of the scion (with the ratio above 16), late

ripening (with ratio below of 12) and hybrid plants with fruit ripening ideal for the

juice industry, with the ratio ranging between 14 and 16.

Evaluation of hybrids for scion varieties

The severity of ABS (Alternaria Brown Spot) was quantified by visual

assessment of the symptoms (scale of notes from 0 to 4). The hybrids were

evaluated in Bebedouro-SP, Botucatu-SP and Cordeirópolis-SP. The severity of

the CBS (Citrus Black Spot) was measured in 20 fruits of each treatment. We

used a scale consisting of six levels, 0 when there is no damage on the surface

of the fruit, and notes 1 to 6, corresponding respectively to the percentages of

areas damaged by 0.8%, 1.6%, 3.1%, 6.2%, 12.5% and 25%. The hybrids were

also evaluated in Cordeirópolis-SP.

For the evaluation of CVC, leaves of the hybrids were collected for total

DNA extraction and subsequent PCR diagnosis, quantifying the percentage of

infection from 0 to 100%. The hybrids were evaluated in Colina-SP.

A histogram of the distribution of mean severity of ABS for the hybrids in

each experiment is shown in Figure 13. It is observed that the highest disease

severity was detected in Botucatu.

Figure 13. Histogram distribution of alternaria brown spot severity in the

population of hybrids of tangor Murcott x Pera sweet orange (2009).

For CVC, during the evaluation performed in May 2009, 84 hybrids were

found PCR positive. The infection rate ranged from 25 to 100% among the

hybrids (Figure 14).

Figure 14: Histogram distribution of percentage of plants positive for CVC in the

population of hybrids of tangor Murcott x Pera sweet orange (2009).

Most hybrids showed notes of severity of CBS between 0 and 1 (0.8%

fruit area with lesions) and only five had notes between 1 and 3 (3.1%) (Figure

15).

Figure 15. Histogram distribution of citrus black spot mean severity in the

population of hybrids of tangor Murcott x Pera sweet orange (2009).

Rootstock breeding projects of Embrapa

Gummosis of Phytophthora - Nucelar seedlings from 25 genotypes were

cultivated in soil substrate containing Pinus spp. bark. After completing two

years of age, a portion of 5 mm in diameter of the bark was taken out by using a

cork borer at 15 cm from the bottom. Afterwards, a disk containing culture

media with Phytophthora parasitica var. nicotianae mycelium was inserted into

the lesion. Then, a section of the bark detached from the trunk was placed over

the mycelium disk. The inoculation spot was covered with wet cotton and

protected using tape. After the inoculation the seedlings were maintained at

25ºC and photoperiod of 12 hours of light. The experiment was installed in

complete random design with 25 genotypes and 10 replicates. Each

experimental plot was represented by one plant.

The genotypes Poncirus trifoliata, ‘Beneke’ selection, ‘Swingle’ citrumelo,

common ‘Sour Orange’, ‘Sunki’ mandarin, ‘Tropical’ selection, as well as the

hybrids: HTR - 010, HTR - 051, HTR - 069, HTR - 127, LRF x (TR x LCR) - 004,

LRF x (TR x LCR) - 005, LVK x LVA - 009, TSKC x CTSW - 018, TSKC x

CTSW - 019, TSKC x CTSW - 033, TSKC x CTSW - 041, TSKC x CTSW - 064,

TSKC x TRFD - 007 and TSKC x TRBK - 010 presented high tolerance to

Phytophthora foot rot. The genotypes, Rangpur’ lime, ‘Santa Cruz’ selection,

‘Sunki’ mandarin, ‘Maravilha CNMPF 02’ selection, as well as the hybrids LCR x

LRF - 034, TSKC x CTTR - 002, TSKFL x CTARG - 002 and TSKFL x CTARG -

028 presented intermediate susceptibility. The common ‘Sunki’ mandarin

presented high susceptibility to the pathogen.

Citrus sudden death – Three experiments were installed from March 2007 to

July 2009 in areas with high incidence of CSD located in the county of

Colômbia, north of the State of São Paulo. The experimental design was in

random blocks with three replicates and variable number of plants per plot

(three to five). Disease evaluation, of possible viral etiology, will be carried out

mainly by observing the presence of yellowish color in the internal tissues of the

bark of rootstocks in the region of the functional phloem in contrast to the

tissues of the bark of the scions which are lighter.

Tristeza - 20 cm stems from 40 hybrid nucelar seedlings selected from the

CGBP were collected from the different quadrants of the scions and, after

removing the bark, evaluated regarding stem pitting symptoms using a grade

scale. The indirect ELISA test was carried out in order to detect the presence of

the virus in the tissues of the hybrids evaluated.

The TSKFL x CTARG - 029, TSKFL x CTSW - 004, TSKC x CTSW - 022,

TSKC x TRFD - 007, TCLN x TRDP - 015, HTR - 206 and TSKC x CTTR - 029

hybrids behaved as resistant; The TSKFL x CTARG - 023, TSKC x CTARG -

015, TSKC x CTSW - 017, TSKC x CTSW - 058, TSKC x TRFD - 003, TSKC x

(TR x LCR) - 017, TSKC x CTQT1439 - 026, TSKFL x CTTR - 004, TSKFL x

CTTR - 021, TSKC x LHA - 001, HTR - 208 hybrids, behaved as very tolerant;

the TSKC x CTARG - 020, TSKC x CTARG - 069, TSKC x CTARG - 081,

TSKFL x CTSW - 009, TSKC x CTSW - 055, TSKC x CTSW - 060, TSKC x (TR

x LCR) - 018, TSKC x (TR x LCR) - 020, TSKC x CTQT1439 - 014, TSKC x

TRDP - 026, TCLC x CTSW - 005, TSKC x CTQT - 010, LVKC x CTSW - 009

and HTR - 207 hybrids behaved as tolerant; and the TSKC x CTARG - 079,

TSKC x CTSW - 025, TSKC x CTSW - 036, TSKC x (TR x LCR) - 032, TSKC x

CTQT - 003, TSKC x LHA - 007, TSKC x LHA - 004, TSKC x LVKCT2 - 001 and

TSKFL x CWEB - 004 hybrids behaved as non tolerant.

Goal 3a2: Assess rootstock trials in areas with hydric stress.

Indicators: Number of assessed rootstocks; number of rootstocks with higher

or lower tolerance to hydric stress; number of rootstocks with potential to be

recommended in new experimental areas; number of rootstocks recommended

as cultivars; number of rootstocks registered in the National Plant Variety

Protection Service.

During the period of intense water stress, evaluations based on the

presence or lack of leaf curling, characteristic of plants under water stress,

considering a grade criteria, were carried out.

The crosses which showed greater potential of drought tolerant hybrids

were: ‘Rangpur Santa Cruz’ lime (Citrus limonia) (LCRSTC) x common ‘Sour’

orange (C. aurantium) (LAZC), ‘Rangpur CNPMF-005’ lime (LCR CNPMF-05) x

‘Smooth Flat Seville Sour’ orange (LAZSFS), LCRSTC x LAZSFS, LCRSTC x

[common ‘Sunki’ (C. sunki) mandarin hybrid x P. trifoliata ‘English’ - 256) (TSKC

x TRENG - 256) and LCRSTC x (TSKC x TRENG - 264).

A competition assay with 50 rootstocks selected from the CGBP in

combination with ‘Valencia’ sweet orange scions was installed in the county of

Colômbia, SP. The experimental design was in random blocks with three

replicates with variable number of plants per plot (three to five). Visual

evaluations such as leaf curling characteristics and bud, flower and fruit

formation were carried out using the following grade criteria: 0 (presence) and 1

(absence).

The TSKC x (TR x LCR) - 001, TSKC x (TR x LCR) - 059, TSKC x (TR x

LCR) - 073, TSKC x CTSW - 033, TSKC x CTSW - 041, TSKC x CTQT1434 -

010, TSKC x CTQT1439 - 004, TSKC x CTQT1439 - 026, TSKC x CTTR - 002,

TSKC x CTARG - 001, TSKC x LHA - 006, TSKC x LHA - 011, LVK x LCR -

010, TSKC x CTRK - 001, LCR x TR - 001, HTR - 053 and HTR - 116 hybrids,

as well as selections of the ‘Rangpur’ lime and ‘Tropical Sunki’ mandarin,

provided the least drought sensitivity to the scions of the ‘Valencia’ sweet

orange (no leaf curling occurred) and quick reaction to the presence of water

(previous rain at the time of the evaluations), according to the observations

regarding bud formation.

Objective 3b. Evaluate fruit quality from promising new hybrids between

oranges and mandarins or tangors as scion varieties;

Goal 3b1: Assess a number of new promising hybrids for scion varieties

regarding fruit quality in ongoing trials.

Indicators: Number of new potential varieties assessed; number of maturation

curves performed and evaluated; number of new scion varieties with potential to

be recommended in new experimental areas; number of new scion varieties

recommended as cultivars; number of scion varieties registered in the National

Plant Variety Protection Service.

Evaluations were carried out on physical-chemical characteristics of fruits

in September and October. For the analysis, 10 fruits were collected in the outer

portion of the canopy in the whole length of the perimeter of the plant. We

evaluated height and diameter of the fruit, color, total mass, juice yield, soluble

solids, acidity of the juice and soluble solids / acidity (ratio).

For the evaluation of fruit production, they were harvested and weighed

separately at each plant. We evaluated the total weight of fruit per plant, total

fruit number per plant and number of fruit per bag for collection of 27.2 kg,

which represents a measurement inversely proportional to the size of the fruit.

Some hybrids were selected showing a production of more than 1.52 boxes per

tree (average yield of Valencia / Natal oranges with 5 years without irrigation in

the region of evaluation) and juice percentage above 55%.

It was observed that some hybrids showed good characteristics for

consumption as fresh fruit and that they were more resistant to Alternaria brown

spot (Figure 16). The assessments of fruit quality will be continued in the

following years.

Figure 16: Plant and fruit of the hybrid between Murcott tangor x Pêra sweet

orange (TM x LP 281), resistant to Alternaria brown spot. A) detached leaves

subjected to in vitro inoculation of the fungus showing the absence of symptoms

and (B) the parental Murcott, highly susceptible to the fungus.

Objective 3c. Validate RGAs and SSR markers based on CitEST and map

them within the existing linkage maps;

Goal 3c1: Validate at least 500 new SSR markers and 30 new RGAs derived

from citrus ESTs.

Indicators: Number of SSR markers validated by PCR and inserted in the

following maps: Sunki mandarin (C. sunki) x Poncirus trifoliata cv Rubidoux,

Swingle citrumelo (C. paradisi x P. trifoliata) x Rangpur lime (C. limonia),

Murcott tangor (C. sinensis x C. reticulata) x Pêra sweet orange (C. sinensis),

Cravo mandarin (C. reticulata) x Pêra seet orange (C. sinensis).

Microsatellites were investigated in the unigene sequences from Citrus

spp. and Poncirus trifoliate (CitEST project). So far, 134 primer sequences

flanking SSR motifs were successfully designed and synthesized. In this stage

of work were assessed the SSR loci to build a consensus map through the

integration of four linkage maps: Pêra sweet orange (Citrus sinensis), Murcott

tangor (C. sinensis x C. reticulata), Cravo mandarin (C. reticulata) and

Pummelo (C. grandis).

The Isolation of DNA of 94 hybrids of each population: Pêra sweet

orange vs. Cravo mandarin (PE x CM), Pêra sweet orange vs. Murcott tangor

(PE x MU), and Pummelo vs Cravo mandarin (PU x CM) was performed as by

Murray and Thompson (1980). All hybrids and parents were genotyped with

SSR markers - 134 EST-SSRs and 171 Genomic-SSR.

From the total SSR markers (134 EST-SSRs and 171 Genomic-SSR),

35 pairs of primers SSR proceeding from sequences of ESTs were selected on

the basis of the polymorphism between the genitors and segregation in the

hybrid plants and 40 SSR pairs of primers were selected from the genomic

libraries. From the 75 SSR pairs of primers, 25 pairs of primers produced

bands that segregated in an informative way in all the progenies, that is, both

parents were heterozygous and at least three different alellos were segregating,

so that four different genotypes could be identified in the population.

Goal 3c2: Map the molecular markers within the linkage maps already available

in the Program.

Indicators: Number of markers that can be inserted in the maps.

The total SSR evaluated 25 of them showed to be completely informative

(4 alleles) and they could be used as anchors to combine the different maps. In

a linkage analysis congregating the selected RAPD, SSR, AFLP and TRAPS

markers (Tab. 4), 336 out of 619 molecular markers grouped in ten linkage

groups totalizing 1,053 cM (Fig. 16). Almost 54% of the total number of the

markers could be integrated in one map. Only the linkage groups with markers

of at least two parents were considered to compose the consensus map.

Additionally to SSR markers, dominant markers as AFLP and RAPD that

segregated in a ratio of 3:1 functioned as anchors to combine the different

maps.

Table 4: Number of molecular markers of the Integrated Map from each

individual map

Species Number of molecular markers Total

AFLP RAPD SSR TRAPs

Pêra sweet orange 68 11 07 6 92

Murcott tangor 60 16 16 12 104

Cravo mandarin 22 47 09 - 78

Pummelo 14 23 25 - 62 Total 164 97 57 18 336

New genetic maps were obtained for the populations of hybrids (94

plants) Rangpur x Swingle Swingle and Rangpur lime x P. trifoliata (50 plants)

and location of regions associated with HLB, SDC, resistance to Phytophthora

and CTV.

Moreover, two populations of F1 hybrids derived from intraspecific

crosses between the Pera-de-Abril sweet orange (with monoembryonic seeds)

and Tobias sweet orange (plants with short juvenile cycle), obtained in 2006,

are being mapped using Traps and SSR molecular markers. Approximately

30% of the plants of both populations, with two years old, had already flowered

in the field. Screening for molecular data of 25 pair of Traps primers and 12 pair

of SSR primers have been generated with all plants of both populations and,

after that, the linkage map will be done.

Figure 16. Consensus map of Citrus.

Goal 3c3: Genotype the molecular markers aiming the construction of

new linkage maps for the populations of intraspecific hybrids of sweet orange,

Rangpur lime x Poncirus trifoliata cv. Rubidoux and Clementine mandarin (C.

reticulata) x Murcott tangor.

Indicators: Number of markers that can be inserted in the maps.

Microsatellites were investigated in the unigene sequences from Citrus

spp. and Poncirus trifoliate (CitEST project). So far, 134 primer sequences

flanking SSR motifs were successfully designed and synthesized. In this stage

of work were assessed the SSR loci to build a consensus map through the

integration of four linkage maps: Pêra sweet orange (Citrus sinensis), Murcott

tangor (C. sinensis x C. reticulata), Cravo mandarin (C. reticulata) and

Pummelo (C. grandis).

R7-1565 0 AFLP1 1 AFLP78 2 N5-1249 8 C7-1779 12 B4-1650 14 AT7-620 Q6-738 16 AFLP584 AFLP155 17 AB14-767 AFLP411 19 B5-939 20 AT14-2115 AFLP637 21 AFLP351 26 AFLP341 27 AFLP743 AFLP79 31 AFLP350 32 AFLP720 33 AFLP222 34 AFLP332 AFLP220 38 AFLP589 39 AFLP719 41 AFLP403 AFLP55 AFLP631 42 AFLP270 AFLP223 AFLP714 43 AFLP119 44 AFLP218 AFLP152 46 AFLP156 48 AFLP335 AFLP154 49 AFLP376 50 AFLP221 52 AFLP319 53 AFLP145 54 AFLP227 57 AFLP352 58 AFLP414 AFLP118 64 AFLP501 AFLP225 68

Pt160 0 AFLP70 27 AFLP215 37 G10-1416 45 A9-1189 54 AFLP464 55 E19-1282 59 AFLP213 60 Q10-747 62 AFLP675 65 AFLP549 72 AFLP1549 AFLP211 AFLP214 74 AFLP504 76 AFLP785 78 AFLP512 81 AFLP545 83 AFLP522 84 AFLP309 AFLP636 86 AFLP515 89 AFLP514 92 AFLP524 95 AFLP5003 98 AFLP526 AFLP525 102 AFLP503 103 AFLP187 AFLP202 104 AFLP782 116 AFLP5001 135

AFLP207 1 AFLP168 AFLP160 AFLP67 AFLP833 14 AFLP831 16 AFLP167 AFLP303 20 AFLP162 22 AFLP296 24 AFLP272 25 AFLP306 27 AFLP248 30 AFLP147 32 AFLP244 35 AFLP243 AFLP295 36 AFLP273 AFLP302 AFLP240 39 AFLP242 AFLP291 40 AFLP241 42 AFLP290 AFLP238 44 AFLP234 46 AFLP285 49 AFLP276 50 AFLP237 52 AFLP301 AFLP279 54 AFLP281 57 AFLP280 AFLP283 61 AFLP286 67 AFLP275 71

AFLP52 AFLP805 0 AFLP46 8 AFLP37 10 AFLP468 17 AFLP51 24 AFLP50 25 AFLP482 26 AFLP48 27 AFLP513 29 AFLP356 31 AFLP475 AFLP53 AFLP581 33 AFLP236 AFLP44 AFLP791 38 AFLP731 43 AFLP474 45 AFLP789 49 AFLP134 AFLP473 51 AFLP653 56 AFLP5 57 AFLP457 AFLP1480 58 AFLP480 60 AFLP10 61 AFLP790 62 AFLP550 64 AFLP8 AFLP486 66 AFLP266 67 AFLP7 76 AFLP17 78 AFLP809 80

A10-2015 0 AT7b 1 AV5-1626 2 AT5-484 3 AFLP488 6 AFLP383 7 G17-1101 8 B13-1832 9 AFLP393 11 AFLP391 12 AFLP392 15 N14-1386 18 AFLP390 19 E20-576 27 AFLP532 AFLP397 29 AFLP481 33 AFLP530 AFLP781 38 AFLP396 AFLP188 41 AFLP538 45 AFLP534 51 AFLP193 54 H15-335 AFLP537 58 AFLP540 60 P6-1339 61 AFLP665 65 G17-459 68 G18-1871 69 B2-1156 70 AFLP762 B11-2037 71 AFLP324 H16-705 73 B10-1986 77 H16-1349 83 AT13d 85 AFLP399 86 H1-1922 90 H19-1107 98 AFLP355 103 AFLP415 AV12-1987 105 AB8-1544 115 Pm99 117 AT7a 121 E17a 153

A7-1261 A7-1201 0

G19-1261 62 A19-2743 65 Q8-657 68 N15-1535 N9-533 B3-1778 P20-635 Q5-2961 70 A8-580 71 A7-643 Y11-1654 72 Q17-1822 73 AT13-986 75 AV12-1495 76 AB18-1357 80 AFLP110 118

A19-2743 0 Q8-657 6 N15-1535 7 P20-635 N9-533 Q5-2961 B3-1778 8 AV12-1495 AT13-986 A7-643 9 A8-580 Q17-1822 10 Y11-1654 11 G19-1261 17 AB18-1357 18

A4-717 0 U3-1107 29 B3-1698 33 AB5-1308 37 N15-597 54 G8-623 D15-1838 56 AB6-655 58 P9-1435 C6-652 60 E16-2267 I11-434 62 A8-341 63 C3-762 64 P11-1408 66 AFLP228 69 AB6-1268 71 A8-2011 72 B19-1571 B19-1730 73 H19-1673 83 G17-841 88 AT13-1432 95

Pc49 129 Pc55 149 Pc53 160

A8-769 0 H15-1014 7 U8-783 E16-1967 AT18-1053 N14-295 9 N15-612 11 C6-1076 35 H19-1584 68 G17-1807 71 E16-805 N8-1362 73 M7-1426 77 M13-1189 80 M7-438 81 AB4-1226 M11-379 82 P1-1020 C6-866 A9-628 A8-497 AV8-822 P9-1692 83 R19-1027 84 P10-2261 86 G19-1203 88 B12-813 89 AV12-1840 90

A10-2015 0 AV5-1626 8 AT5-484 11 G17-1101 19 B13-1832 20 N14-1386 33 E20-576 42

Pc119 71 H15-335 74 P6-1339 77 G17-459 84 B2-1156 85 G18-1871 H16-705 86 B11-2037 88 B10-1986 96 H16-1349 99 AT13d H1-1922 106 H19-1107 114 AV12-1987 AFLP415 121 AB8-1544 131 AT7a 138

The Isolation of DNA of 94 hybrids of each population: Pêra sweet

orange vs. Cravo mandarin (PE x CM), Pêra sweet orange vs. Murcott tangor

(PE x MU), and Pummelo vs Cravo mandarin (PU x CM) was performed as by

Murray and Thompson (1980). All hybrids and parents were genotyped with

SSR markers - 134 EST-SSRs and 171 Genomic-SSR.

The SSR markers were added to the preexisting maps built with RAPD,

AFLP and TRAPs markers. The genetic maps were obtained using the JoinMap

software v.3.0, through the simultaneous analysis of markers that had 1:1,

1:2:1, 3:1 and 1:1:1:1 Mendelian segregations.

Objective 3d. Include phenotypic data from assessments of promising new

hybrids within the respective linkage maps;

Goal 3d1: Validate the data on resistance or tolerance to CVC, leprosis, and

Alternaria brown spot on the assessed scion hybrids.

Indicators: Number of features inserted in the maps.

New evaluations for CVC, leprosis and ABS were conducted in field

experiments but the characteristis were not included in the map in this first

stage yet. Since several traits seem to be quantitative more evaluation should

be considered before to include them in the maps.

Goal 3d2: Validate the data on resistance or tolerance to tristeza, gummosis,

and water deficit on the assessed rootstock hybrids.

Indicators: Number of features inserted in the maps.

Detection and mapping of QTLs in the populations were analyzed with

the program MapQTL v. 4.0 (Van Ooijer et al., 2002), using parametric tests

Interval Mapping (IM) and "Multiple QTL Mapping (MQM).

QTL mapping for resistance to CTV in the linkage map of Swingle citrumelo

For the QTL location to CTV resistance, the phenotypic data measured

(absorbance in ELISA) was analyzed separately and the QTL was localized in

the linkage maps using the program MAPQTL v.4.0. The QTL was located

between SSR marker 109 and P1R1-420, the latter being a marker linked to

genes involved in the synthesis of lignin. This QTL may be considered of a large

effect since it explains 95.1% of the phenotypic variance.

CTV gene had been mapped in a previous work, in Poncirus trifoliate,

using a mapping population between P. trifoliata vs Sunki mandarin. Since

Swingle citrumelo is a hybrid of Citrus paradisi vs P. trifoliata, we used the

markers found in the linkage group of P. trifoliata gene associated with CTV

(Figure 18). Thus, one can see that the markers associated with the CTV gene in

the P. trifoliata map are also associated with the gene in map of C. Swingle. It

can be stated that the CTV resistance gene of P. trifoliata was inherited by C.

Swingle.

Figure 18. (A) Linkage map of Swingle citrumelo with markers near the CTV

gene of P. trifoliata, showing the location of a QTL for CTV resistance (B) a

group of P. trifoliata map of CTV with the gene (C) linkage group of Citrumelo

Swingle showing the location of a QTL for resistance to CTV, without the

markers found in P. trifoliata.

Mapping QTLs for resistance to Phytophthora in the linkage map of Swingle

citrumelo

The average length of lesions in hybrids and parents was between 5.6

and 25.23 mm. Parents Rangpur lime and Citrumelo Swingle had an average of

24.05 and 6.08 mm, respectively. In a linkage analysis with RAPD, SSR and

TRAP, 77 of 154 markers were grouped into eight linkage groups of Citrumelo

Swingle and 33 markers were grouped into 7 linkage groups of Rangpur lime.

Forty-four markers (26.78%) did not bind to any linkage group. The phenotypic

data were analyzed for localization of QTL through the program MAPQTL v.4.0.

The analysis by the method Multiple QTL Mapping (MQM) has detected the

presence of two QTLs for resistance to Phytophthora in linkage groups 3 and 6

(LG 3 and 6) of Swingle citrumelo. The permutation tests (1000 replicates)

detected the critical LOD scores critics that confirmed the presence of QTL (p

<0.05) that explained 50.8% and 37.7% of the phenotypic variation.

Figure 19. Swingle citrumelo linkage map with 86 markers (41 RAPD, 29 SSR

and 16 TRAP) in 8 linkage groups and QTL associated with resistance to

Phytophthora. Five RAPD and 4 SSR markers that were linked to CTV in the

Poncirus trifoliata map were incorporated into the linkage group 6.

Figura 20. P. trifoliata linkage map with 202 markers (135 RAPD, 33 AFLP, 10

SSR and 24 TRAP) in 10 linkage groups; TRAP markers are highlighted in red

and SSR merkers in blue; QTLs are highlighted in yellow.

Objective 3e Assess expression QTLs (eQTLs) for disease resistance.

Goal 3e1: Validate through RT-qPCR at least 20 differentially expressed genes

in the parental lines and in pools or groups of 7 to 10 contrasting hybrids

F2P3_320

CAT/ACA_105

F2P6_520 0

N13_1400 1

N01_850 1

R14_800 2

M12_410 2

AV02_11 3

N14_100 4

M104_239 5

N01_1030 5

N08_310 6AV05_1200 7N14_400 7

H04_110 8N15_120 8N06_1500 9N15_2000 9Q05_500 9AV08_1800 100 M06_1250 101 Q07_250 103 CAG/ACG_70 107 AT07_750 111 A10_200 113 A10_1200 118 M02_2110 124 CTC/ACT_304 127 A10_300 129 A10_800 139

CCSM168_176 85

AV11_13 0

AV12_410 13 CAT/ACA279 14 AT07_3000 17

CTC/AAG_188 31 CTC/AAG_199 35

AV11_2000 45

CCSM75_232 54

CCSM170_ 254 60

R01_1000 73

AF3_153 0 AV05_250 3 CTG/ACA_121 5 A18_1800 7 F2P2_4900 CAC/AAC_252 Q06_1020

12

E20_920 Q12_490 13 AV01_180 21 CCSM11_150 26 R01_710 28 C07_1400 29 M06_1100 30 U11_1550 34 UO3_1550 37 CCSM13_234 38 U05_750 46 CAC/ACT_407 51 CTC/AGC_199 57 F1P5_780 AV08_900

58

M16_300 59 CTG/ACA_347 70 N01_1550 82 U05_740 83

M20_1030 95

A10_410 0

C14_1300 2 C14_650 5 N16_500 11 N16_510 12 C05_1410 22 A08_1622 28 H04_1400 30 C11_400 35 CAC/ACC_255 37 C15_110 38 C15_410 39 AV02_250 AV02_3050 41 M01_860 43 C10_170 47 F1P1_740 F2P1_850 49 C06_110 50 F4P4_150 51 N06_1510 55 AV08_1750 57 E15_1000 58 AT03_1000 66 F1P3_490 67 CTA/ACA316 F2P4_580 75

R01_1100 F2P1_650 93

N08_750 0

R09_710 6 CAC/ACT_215 10 R20_800 15 A04_100 16 M06_2300 19 M06_2000 20 Q06_1600 22 Q05_510 25 AT07_1700 29 M13_750 34 F2P6_370 38 F2P6_340 42 Q05_408 45 M12_1100 48 AT18_500 51

F2P2_4.300

M01_202

A01_180 0

CTC/ACA_197 5

F4P4_400 10

AV03_750 22

C06_450 28

R07_1610 33 AT14_600 37

CCSM111_180 42

C05_750 48

M02_1650 65 F4P5_680 66 M20_730 68

A04_470 81

AT14_1550 93

C14_1500 105

CAA/ACA_179 115

CAC/AAC_243 121

CAA/ACG_255

CCSM40_180

E07_100

AV11_250 0

AV02_2100 2

P05_820 2CAA/ACC_202 3CAA/ACT_193 3

H04_1200 4

A18_3100 4F4P1_380 5

F2P6_800 5

Q07_430 6CTC/ACT_294 7A10_750 7F4P1_200 7F2P3_390 7CCSM09_156 8 8CTG/AAC_147 9AV12_330 9CCSM156_310 102 CAC/ACC_344 103

CCSM12_280 112 CAA/ACC_215 115

E07_800 0 N08_2500 6 AV11_15 11 AT07_400 15 E16_510 16 M05_310 20 N15_1050

F4P5_960

24 CAC/AAC_294 28

29 N12_1550 31 E20_510 32 N15_1610 33 Ct36 W18_480 38 AV12_470 43 M01_140 46 A04_480 51 G02-540 57 CAT/ACA_107 65

C05-1500 0

R20_1100 1

C19_380 2

Z_680 3

CAT/ACT_187 3U05_740 4AV02_1100 4

H04_1200 4W13_1650 4

AV11_1300 5

AT07_3000 6C06_1100 6

F1P5_710 7

N08_1300 0 CAC/ACG_144 3

AV11_15 11

AT07_800 15 W13_580 16

CTG/AAG_293 23 CTC/ACA_270 M05_420 26

30 F1P2_680 CAC/ACC_316 34

F2P3_510 39 F1P3_2100 41

M03_140 48

A04_780 55

CTG/AAC_194 60

CAC/ACT_283 67

6 7 8 9 1

regarding resistance to CVC, gummosis, CTV, Alternaria brown spot and

leprosis, and tolerance to water deficit, based on the microarray experiments;

Indicators: Number of differentially expressed genes validated.

Initially, the biological experiments of gene expression were established,

some genes were identified as induced in resistant hybrids and are being

validated by RT-qPCR. The step of quantification of transcripts in the hybrids

and mapping of eQTL will be held in the sequence of work.

Goal 3e2: Assess, through RT-qPCR, the level of expression of validated

genes (goal 3e1) in 94 hybrids from each map population (Sunki mandarin x

Poncirus trifoliata, Swingle citrumelo x Rangpur lime, Murcott tangor x Pêra

sweet orange, Cravo mandarin x Pêra sweet orange.

Indicators: Number of differentially expressed genes validated and mapped.

The activities depend on the results of the previous goal (3e1).

Goal 3e3: Map within the linkage groups of the eight genetic maps already

established, the genomic regions associated with disease resistance (CVC,

gummosis, CTV, Alternaria brown spot, and leprosis), and hydric stress

tolerance, based on phenotypic analyses (QTLs) (goals 3d1 and 3d2) and

expression QTLs (eQTLs) (goal 3e2).

Indicators: Number of mapped QTLs in each genetic map and correlation

between phenotypic analyses (QTLs) and expression (eQTLs)

The activities depend on the results of the previous goals (3d1, 3d2, 3e1

and 3e2).

Objective 3f. License genetically modified (GM) citrus plants previously

obtained in the Program for field trials.

Goal 3f1: Establish at least one experimental field area for evaluation of GM

plants (previously obtained in the Program) after challenging with each of the

pathogens studied (the causal agents of CVC, leprosis, HLB, and tristeza).

Indicators: Number of experimental areas and challenged plants.

A proposal for controlled experiments in the field is being prepared to

submit to CTNBio.

Objective 3g. Protect new cultivars (hybrids) after confirmation of their value of

culture and use.

Goal 3g1: Register and protect at least five new cultivars of scion and

rootstocks.

Indicators: Number of protected cultivars.

Among the new hybrids several individuals have been identified as

candidates to be new varieties both of rootstocks and scions. All of them have

been challenged under field conditions in four to seven years of experiments

(see Objetives 3a).

Objective 3h. Develop a database and web interface for managing field trials.

Goal 3h1: Establish a pipeline for management of the data obtained from the

field trials.

Indicators: Pipeline established.

This activity is planned for the next year.

Objective 3i: Prepare patent applications for processes and uses of products

developed based on the platforms of the Program.

Goal 3i1: Apply for patents on the use of novel genes and promotors.

Indicators: Number of patent applications and granted.

Several genes and promotors of the CitEST database are candidates to

be patented, but they need better characterization regarding vector construtions

and testing in heterologe systems.

Refereed Journal Articles

1. Baptista, JC, MA Machado, RA Homem, PS Torres, AA Vojnov & AM

Amaral 2010. Mutation in the xpsD gene of Xanthomonas axonopodis pv.

citri affects cellulose degradation and virulence. Genetics and Molecular

Biology, 33 (1): 46-153.

2. Basílio-Palmieri, AC, AM Amaral, RA Homem & MA Machado. 2009.

Differential expression of pathogenicity and virulence-related genes of

Xanthomonas axonopodis pv citri under copper stress. Genetics and

Molecular Biology

3. Bassanezi RB, LH Montesino, ES Stuchi. 2009. Effects of huanglongbing

on fruit quality of sweet orange cultivars in Brazil. European Journal of

Plant Pathology 125:565-572.

4. Bastianel M, VM Novelli, EK Kitajima, KS Kubo, RB Bassanezi, MA

Machado and J Freitas-Astúa. 2010. Citrus Leprosis: Centennial of an

Unusual Mite Virus Pathosystem. Plant Disease, v. 94, p. 284-292, 2010.

5. Bastianel, M, M Cristofani-Yaly, AC Oliveira, J Freitas-Astúa, AAF

Garcia, MDV Resende, V Rodrigues & MA Machado. 2009. Quantitative

trait loci analysis of citrus leprosis resistance in an interspecific

backcross family of (Citrus reticulata Blanco � C. sinensis L. Osbeck) �

C. sinensis L. Osb. Euphytica. DOI:10.1007/s10681-009-9950-3.

6. Cantuarias-Avilés T, FAA Mourão Filho, ES Stuchi, SR Silva, E

Espinoza-Nuñez. 2010. Tree performance and fruit yield and quality of

`Okitsu’ Satsuma mandarin grafted on 12 rootstocks. Scientia

Horticulturae 123: 318-322.

7. Cavalcante IHL, ABG Martins, ES Stuchi, MCC Campos. 2009. Fruit

maturation as a parameter for selection of sweet orange cultivars in

Brazil.. International Journal of Food, Agriculture and Environment

(Online) 7:132-135.

8. Coletta-Filho, HD, EF Carlos, KCS Alves, MAR Pereira, LC Fender, LL

Lotto, RL Boscariol-Camargo, AA Souza & MA Machado. 2009. In

planta multiplication and graft transmission of ‘Candidatus Liberibacter

asiaticus’ revealed by Real-Time PCR. European Journal of Plant

Pathology. DOI 10.1007/s10658-009-9523-2

9. Eiras M. SR Silva, ES Stuchi, MLPN Targon, SA Carvalho. 2009.

Viróides em citros. Tropical Plant Pathology 34:275-296.

10. Kubo, SK, J Freitas-Astúa, MA Machado & EW Kitajima. 2009. Orchid

fleck symptoms may be caused naturally by two different viruses

transmitted by Brevipalpus. J. Gen. Plant Pathol 75 (3): 250-255. DOI

10.1007/s10327-009-0167-z.

11. Kubo, SK, RM Stuart, J Freitas-Astúa, R Antonioli-Luizon, EC Locali-

Frabis, HD Coletta Filho, MA Machado & EW Kitajima. 2009. Evaluation

of the genetic variability of orchid fleck vírus by single-strande

conformational polymorphism analysis and nucleotide sequencing of a

fragment from the nucleocapsid gene. Arch. Virology 154 (6): 1009-1014.

DOI 10.1007/s00705-009-0395-8.

12. Peroni, LA, M Lorencini, JRR Reis, MA Machado & DR Stach-Machado.

2009. Differential diagnosis of Brazilian strains of Citrus tristeza virus by

epitope mappingo of coat protein using monoclonal antibodies. Virus

Research. doi:10.1016/j.virusres.2009.05.014

13. Silva SR, JC Oliveira, ES Stuchi, ET Reiff. 2009. Qualidade e maturação

de tangerinas e seus híbridos em São Paulo. Revista Brasileira de

Fruticultura 31: 977-986.

14. Souza MC, ES Stuchi, A Góes. 2009. Evaluation of tangerine hybrid

resistance to Alternaria alternata. Scientia Horticulturae 123: 1-4.

15. Stuchi ES, ABG Martins, RR Lemo, T. Cantuarias-Avilés. 2009. Fruit

quality of 'Tahiti' lime (Citrus latifolia Tanaka) grafted on twelve

rootstocks. Revista Brasileira de Fruticultura. , v.31, p.454 - 460, 2009.

16. Tomasetto F, ES Stuchi, ABG Martins. 2009. Avaliação de cinco

seleções de laranjeira 'Valência' sobre dois porta-enxertos. Revista

Brasileira de Fruticultura 31: 480-486.

17. Weiler, RL, EC Brugnara, M Bastianel, MA Machado, MT Schifino-

Wittmann & SF Schwarz. 2009. Carcterização molecular de um progênie

de tangerineira Clementina Fina e Montenegrina. Scientia Agrária

(UFPR) 10(6): 429-435.

Abstracts

1. Cristofani-Yaly M, Bastianel M, Novelli VM, Machado MA. Agronomic

Characterization, Selection and Genetic Mapping of New Hybrids of

Citrus Scion and Rootstocks Varieties. 2nd International Citrus

Biotechnology Symposium, Catania, Italy, november 30 – december 2,

2009.

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8. Marengo, S, M Cristofani-Yaly, JA Diogo & MA Machado. 2009.

Mapeamento genético de tangerina Sunki e Poncirus trifoliata para

resistência ao Huanglongbing (Greening) dos citros. In: 55 Congresso

Brasileiro de Genética, 2009, Águas de Lindóia. Congresso Brasileiro de

Genética, 2009.

9. Conceição, JPS, A Souza, KA Souza & W Soares Filho. 2009.

Obtenção e cultivo de calos nucelares de citros para isolamento de

protoplastos. In: Congresso Brasileiro de Floricultura e Plantas

Ornamentais e Congresso Brasileiro de Cultura de Tecicos de Plantas,

Aracaju. Anais... CD-ROM.

10. Pissinato, AGV, MA Coelho Filho, AS Gesteira, MGC Costa, W Soares

Filho. 2009. Protocolo experimental para estudos de déficit hídrico em

genótipos de citros passíveis de utilização como porta-enxertos. In:

Jornada Científica da Embrapa Mandioca e Fruticultura, 3., 2009, Cruz

das Almas. Anais... CD Rom.

11. Rocha, JS, W. Soares Filho. 2009. Desenvolvimento de variedades-copa

híbridas de citros: plantas ornamentais. In: Jornada Científica da

Embrapa Mandioca e Fruticultura, 3., 2009, Cruz das Almas. Anais... CD

Rom.

12. Santana, LG, W. Soares Filho, 2009. Programa de melhoramento

genético de citros da Embrapa Mandioca e Fruticultura Tropical:

obtenção de porta-enxertos híbridos. In: Jornada Científica da Embrapa

Mandioca e Fruticultura, 3., 2009, Cruz das Almas. Anais... CD Rom.

13. Soares Filho, W, OS Passos, FV Souza, J. Rocha, LG Santana, D. D.

S.; SANTOS, M. G. Recursos genéticos de citros: obtenção de plantas

híbridas ornamentais. In: Simpósio de Recursos Genéticos para América

Latina y Caribe, 7, Chile. Anais...v. 1, p. 483-484.

14. Soares Filho W, U Souza, CRC Oliveira, MG Santos, CAS Ledo, LGL

Santana, JS Rocha, AGV Pissinato, JSS Silva, AS Souza, OS Passos.

2009. Poliembrionia e potencial de obtenção de híbridos em citros. In: In:

3 Congresso Brasileiro de Melhoramento de Plantas, 2009,

Guarapari/ES. Anais do 3 Congresso Brasileiro de Melhoramento de

Plantas, 2009.

15. Rodrigues AS, CJ Barbosa, EES Santos, W. Soares Filho, J.F. Astua.

2009. Tolerância de porta-enxertos híbridos de citros ao Citrus tristeza

virus e Bahia Bark Scaling disease. Tropical Plant Pathology 34.

Resumo 935.

16. Astua JF, ES Stuchi. 2009. Worldwide status of leprosis, its mite vector, and a

casa study: sampling, diagnostic and management of the disease in Brazil In:

International Workshop on Citrus Quarantine Pests, 2009, Villahermosa

Tabasco, México. (CD-ROM).

17. Stuchi ES, H Bremer Neto, E Spinoza-Nuñez, T Cantuarias-Aviles, FAA Mourão

Filho. 2009. Drought tolerance of rootstocks and clones of ‘Tahiti’ lime. In:

International Citrus Biotechnology Symposium, Abstracts p.89.

18. Stuchi ES, H Bremer Neto, E Spinoza-Nuñez, T Cantuarias-Aviles, FAA Mourão

Filho, D Milori. 2009. Integration of physiological methods to estimate water

deficit in scions, clones, rootstocks and interstocks of ‘ Tahiti’ acid lime. In:

International Citrus Biotechnology Symposium. Abstracts p.86.

19. Stuchi ES, T Cantuarias-Aviles, FP Gonçalves. 2009. Current situation,

management and economic impact of citrus variegated chlorosis in Brazil In:

International Workshop on Citrus Quarantine Pests, 2009, Villahermosa

Tabasco, México. (CD-ROM).

Submitted manuscripts

1. Marengo S, M Cristofani-Yaly, HD Coletta-Filho, JA Diogo, MA

Machado. Genetic mapping of resistance of Poncirus trifoliata to

huanglongbing (HLB). Phytopathology.

2. Faria LM, M Cristofani-Yaly, L Faldoni, B Bastianel, MA Machado.

Genetic mapping and detection of QTLs in Rangpur lime and Swingle

citrumelo. Euphytica.

3. Cristofani-Yaly M, VM Novelli, M Bastianel, MA Machado. Transferability

and level of heterozygosity of microsatellites markers in Citrus species".

Plant Molecular Biology Reporter.

4. Boava LP, M Cristofani-Yalym MS Mafra, K Kubo, LT Kishi, MA Takita, M

Ribeiro-Alves, MA Machado. Global gene expression of Poncirus

trifoliata, Citrus sunki and their hybrids under infection of Phytophthora

parasitica. 2010. BMC Genomics.

5. Santos J, MA Machado, DA Botrel, WH Pfenning. Efeito fitotóxico e

caracterização de Fusarium solani associados a plantas de citros com

sintomas de morte súbita. Tropical Plant Pathology.

6. Amaral AM, SA Carvalho, VA Silva, MA Machado. Host range reaction of

genotypes of citrus species and varieties to xanthomonas citri subsp. citri

under greenhouse conditions. Journal of Plant Pathology.

7. Santos, FA, AA Souza, HD Coletta Filho, MLPN Targon, LA Peroni, DR

Stach-Machado, SA Carvalho, MA Machado. Evidence of synergism

between subisolates of Citrus tristeza virus in Mexican lime. Archives of

Virology.

8. Winck, FV, AM Amaral, JC Baptista, D Martins, S Marangoni, JC

Novello, MA Machado. Protein profile in mutants of hrpF from

Xanthomonas axonopodis pv. Citri. Genetics and Molecular Biology.

Requested patents

Nothing to declare.

Book chapters

Nothing to declare.

Books

Nothing to declare.

Concluded Dissertations

Marengo, S. Mapeamento genético de tangerina Sunki e Poncirus trifoliata para

resistência ao Huanglongbing (Greening) dos citros. Dissertação

apresentada para obtenção do título de Mestre em Agricultura Tropical e

Subtropical Área de Concentração em Genética, Melhoramento Vegetal e

Biotecnologia. Campinas, 2009.

Concluded Thesis

Nothing to declare.

Cordeirópolis, 07 de junho de 2010.

Marcos A. Machado Coordenador

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1a. Reunião Anual

Data: 01 e 02/09/2009 Centro de Citricultura Sylvio Moreira

Agenda

Dia 01/10, Quinta-feira

09:00 – Recepção, apresentação da agenda e apresentação do grupo 09:15 - Apresentação geral do programa – Marcos 09:45 – Intervalo e Foto do Grupo Apresentação dos grupos 10:00 – Genoma citros, Takita e Luciano Kishi 10:30 – Genoma funcional, Takita, Eliane, Juliana, Alexandre 11:00 - Transformação: Raquel, Polyana, Alexandre, Rodrigo 11:30 – Transformação na UESC: Márcio 12:00 – Almoço 13:30 – Construção de vetores e transformação no IB/ESALQ e CENA: Ricardo 14:00 – Mapeamento genético, Mariângela 14:30 - Melhoramento IAC, Marcos 15:00 - Melhoramento Embrapa, Walter 15:30 - Intervalo 16:00 – Discussão geral 17:00 – Encerramento Dia 02/10, Sexta-feira 08:30 – Vários assuntos - Constituição do Comitê Gestor - Constituição do Comitê Externo de Avaliação - Acordo de confidencialidade - Revisão de metas anuais - (Entrevistas para o vídeo) 10:30 – Intervalo

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- Sistemática de compras; financeiro, recibos, notas, material permanente e equipamentos, consumo, transporte, etc. - Bolsas CNPq e CAPES: projetos, relatórios, implementação - Relatórios dos grupos: cronograma e conteúdo 12:00 – Almoço 13:30 – (cont.) - Cronograma de reuniões do Comitê Gestor: trimestral - Cronograma de reuniões do INCT: semestral ou anual - Cronograma de avaliações externas: anual - Folder e vídeo - (Entrevistas para o vídeo) 15:00 – Intervalo Reuniões Coordenação e Grupos 16:30 – Encerramento Ata da Reunião Foi a primeira reunião com quase todos os participantes do INCT Citros. O primeiro dia foi dedicado à apresentação dos vários subprojetos componentes do programa, útil não só para normalizar as informações e atividades, mas também para deixar bem definido a responsabilidade de cada líder. A programação apresentada acima faz parte do programa aprovado no edital do INCT. Foi feita uma reunião adicional com o grupo de transformação genética e com o grupo de transformação para discussão de assuntos específicos. No grupo de transformação acordou-se para a divisão de trabalho na busca de genes, construção de vetores e protocolos de transformação. No grupo de melhoramento (rede experimental de campo) foram apresentados os experimentos em andamento, que resultarão em atividades do programa, mas deixando definido a atuação institucional (Embrapa e Instituto Agronômico). O segundo dia da reunião foi dedicado à discussão de questões gerenciais do programa. O Comitê Gestor foi nomeado, constituindo-se dos pesquisadores Marcos A. Machado (Centro de Citricultura, coordenador), Alexandre Morais do Amaral (Embrapa Cenargen), Juliana Freitas-Astúa (Embrapa Mandioca e Fruticultura), Marco Aurélio Takita (Centro de Citricultura) e Francisco Mourão Filho (Esalq/USP). Portanto, representante das principais unidades executoras do programa. O grupo é de fácil capacidade de reunião e deverá se reunir na solução de impasses. Sempre que necessário estarão resolvendo pendências através de e-mail.

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Cada grupo recebeu individualmente suas planilhas de orçamento, definidas em função do que foi solicitado na elaboração do programa ao CNPq. A execução orçamentária ficou evidentemente dependente da disponibilidade financeira. Os procedimentos para compra foram estabelecidos e se concentram no Centro de Citricultura, sendo vetada aos grupos a transferência direta de recursos, uma vez que a modalidade de contratação junto ao CNPq foi a de Auxílio Individual, em nome do Coordenador. Assim, os grupos devem enviar a propostas de compras ao coordenador, obedecidos os critérios estabelecidos pelo CNPq e FAPESP (para os grupos do Estado de São Paulo), que autorizará o gasto de acordo com a disponibilidade financeira. O mesmo se aplica a processos de importação que deverão ser executados pela FAPESP e pelo CNPq. Foi apresentada a sistemática de implementação de bolsas do CNPq e da CAPES, sendo todas encaminhadas ao coordenador que as implementa no sistema Carlos Chagas do CNPq ou encaminha documentação à CAPES. A todos foi destacada a exigência de projeto vinculado ao programa, que deve ser sempre enviada ao coordenador. Sem essa documentação nenhuma bolsa será implementada. Aos grupos foi também informado os prazos para relatório anual e a necessidade de atendimento das metas estabelecidas. O primeiro relatório anual ficou previsto para o final de abril (data da contratação do programa pela FAPESP). Discutiu-se também a constituição de um comitê externo de avaliação a se reunir na reunião anual de 2010. Esse comitê tem a função de fazer uma análise critica do programa e sugerir direcionamentos e integração, além de se constituir de pesquisadores que podem contribuir com o grupo, por serem de áreas afins. Os seguintes nomes foram sugeridos: Fred Gmitter, da Universidade da Flórida, MIkeal Roose da Universidade da Califórnia (Riverside), Dario Grattapaglia (Embrapa Cenargen). Ao coordenador cabe a função de fazer contato e convidá-los a participar da reunião prevista para setembro de 2010. Durante a reunião foi feito o vídeo institucional encaminhado ao CNPq e FAPESP.

Cordeirópolis, 02 de Outubro de 2009.

Marcos A. Machado Coordenador

Equipe

SECRETARIA DEAGRICULTURA E ABASTECIMENTO