Genetic variation in complex polyploids : unveiling the dynamic allelic features of sugarcane

Orientador: Anete Pereira de SouzaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A cana-de-açúcar é utilizada principalmente na produção de açúcar e etanol. As cultivares atuais são originárias de uma série de hibridizações naturais e artificiais e retrocruzamentos. Esse processo de domesticação e melhoramento resultou em um genoma altamente complexo, com elevados níveis de ploidia e aneuploidia, além de um genoma de aproximadamente 10 Gb. Sorgo, além de possuir alta sintenia com cana-de-açúcar, é o ancestral mais próximo com genoma completamente sequenciado e anotado. A presente tese teve como objetivo estudar as diferenças genéticas, a arquitetura gênica e a expressão gênica de uma região do genoma de cana-de-açúcar, assim como gerar ferramentas moleculares para este e outros estudos envolvendo o genoma desta espécie. Genes de cópia única foram buscados em transcritos de arroz, milho e sorgo e o resultado comparado com sequências expressas de cana-de-açúcar. Treze genes candidatos foram encontrados, dos quais o gene HP600 (em sorgo Sobic.003G221600) encontrava-se, além de em provável cópia única, em um QTL localizado em sorgo para Brix. Foi construída uma biblioteca de BAC para a variedade SP80-3280 e outra para a IACSP93-3046, com o objetivo de acessar o genoma de cana-de-açúcar. Foram desenvolvidos dois métodos de seleção de clones: macroarranjos (desenvolvido para ambas as bibliotecas) e mistura ordenada de clones (Pool3D ¿ desenvolvido para metade da biblioteca SP80-3280). As bibliotecas de BACs das variedades SP80-3280 e IACSP93-3046 resultaram em 221.184 e 165.888 clones, ambas com um tamanho médio de 110 kb, representando aproximadamente 2,4 e 1,8 vezes o genoma de cana-de-açúcar, respectivamente. Os treze genes foram utilizados para selecionar BACs nas duas bibliotecas utilizando macroarranjos e o Pool3D na biblioteca SP80-3280. Os BACs resultantes dos dois métodos de seleção da biblioteca da SP80-3280 para o gene HP600 foram utilizados para estudar a arquitetura gênica e transcritos do gene para estudar a expressão gênica em cana-de-açúcar. O gene da Proteína Centromérica C (CENP-C) foi encontrado ao lado do gene HP600 em cana-de-açúcar e sorgo, e ambos foram utilizados para exemplificar a expressão alélica e o comportamento genômico e genético. Os genes HP600 e CENP-C foram encontrados em dois grupos cromossômicos homeólogos. O primeiro grupo (Region01), com ploidia oito, representa a região sintênica de Sorghum bicolor, com todos os haplótipos dos dois genes expressos, porém os haplótipos do HP600 exibiram expressão diferencial. O segundo grupo de homeologos (Region02), com ploidia dez, é uma região formada a partir de diferentes genes não-colineares com sorgo contendo duplicações dos genes HP600 e CENP-C (parálogos). Essa duplicação ocorreu após a separação de sorgo e cana-de-açúcar, resultando em um pseudogene HP600 não expresso e uma versão fusionada e recombinada do CENP-C com um terceiro gene (ortólogo do Sobic.003G299500) com pelo menos dois haplótipos quiméricos expressos. O mapa genético evidenciou que marcadores em regiões duplicadas são incorporados ao mapa com distância genética enviesadas, afetando diretamente o mapeamento genético. Esta tese apresenta a complexidade envolvida na genética, genômica e expressão gênica de cana-de-açúcar e na dinâmica genômica e alélica, o que pode ser útil para a compreensão de outros genomas poliploidesAbstract: Sugarcane is mainly used in the production of sugar and ethanol. The contemporaneous cultivars originate from a series of natural and artificial hybridizations and backcrossing. The process of domestication and breeding resulted in a complex genome with high levels of ploidy, aneuploidy and approximately 10Gb genome. In addition, Sorghum has high sintenia with sugarcane and it is the closest ancestor with genome completely sequenced and annotated. The aim of this thesis was to study the genetic architecture, genomic and gene expression of a region in sugarcane, as well as generate molecular tools involving sugarcane genome. Single copy genes were searched in rice, maize and sorghum transcripts and the result compared to expressed sequences of sugarcane. Thirteen candidates¿ genes were found, which the HP600 gene (in sorghum Sobic.003G221600) was in silgle copy and also located in a QTL in sorghum for Brix. A BAC library was constructed for the sugarcane SP80-3280 variety and another for IACSP93-3046 with the objective of accessing the sugarcane genome. Two methods of clone selection were developed: macroarrays (developed for both libraries) and orderly clone mixing (Pool3D - developed for half of the SP80-3280 library). The BAC libraries construction of the varieties SP80-3280 and IACSP93-3046 resulted in 221,184 and 165,888 clones, both with an average size of 110 kb, representing approximately 2.4 x and 1.8 x the sugarcane genome, respectively. The thirteen genes were used to select BACs from both libraries using macroarrays and the Pool3D selection of the SP80-3280 library. The BACs of the SP80-3280 library resulting from the two selections for the HP600 gene were used to study the genomic architecture and transcripts of the gene to study the genic expression in sugarcane. The gene of Centromeric Protein C (CENP-C) was found side-by-side the HP600 gene in sugarcane and sorghum, and both were used to exemplify allelic expression and genomic and genetic behavior. The HP600 and CENP-C genes were found in two homeologue chromosomic groups. The first group (Region01), with ploidy eight, represents the synthenic region of Sorghum bicolor, with all the haplotypes of the two genes expressed, but the HP600 haplotypes exhibited differential expression. The second group of homoelogues (Region02), with ploidy ten, is a region formed from different non-collinear genes with sorghum containing duplications of the HP600 and CENP-C genes (paralogues). This duplication occurred after sorghum and sugarcane separation, resulting in an HP600 pseudogene and a fused and recombined version of CENP-C with a third gene (ortholog of Sobic.003G299500), with at least two expressed chimeric haplotypes. The genetic map evidenced that markers in duplicate regions are mapped in linkage groups with bias in the genetic distance, affecting the genetic mapping. This thesis presents the complexity involved in genetics, genomics and gene expression of sugarcane and in genomic and allelic dynamics, which may be useful for understanding other polyploid genomesDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecular2010/50119-6142950/2010-0FAPESPCNPQCAPE

Genetic diversity analysis among pigeonpea genotypes adapted to South American regions based on microsatellite markers

The pigeonpea [Cajanus cajan (L) Millspaugh] is one of the most important perennial legume crops utilized in the food, fodder, soil conservation, crop-livestock integrated systems, reclaiming of degraded pastures and symbiotic nitrogen fixation. Microsatellite markers were used to estimate the genetic diversity of 77 pigeonpea genotypes selected from the germplasm collections at Embrapa Cattle-Southeast and, to evaluate their transferability to Phaseolus vulgaris and Vigna unguiculata species. The number of alleles per locus ranged from 2 to12, with an average of 5.1 alleles. The PIC values ranged from 0.11 to 0.80 (average 0.49) and the D values from 0.23 to 0.91 (average 0.58). The averages of observed and expected heterozygosity were 0.25 and 0.47, respectively, showing a deficit in heterozygosity. A model-based Bayesian approach implemented in the software STRUCTURE was used to assign genotypes into clusters. A dendrogram was constructed based on the modified Roger's genetic distances using a neighbor-joining method (NJ). A total of four clusters were assembled by STRUCTURE and a strong tendency of correspondence between the Bayesian clusters in the NJ tree was observed. The genetic distance ranged from 0.09 to 0.62 (average 0.37), showing a low genetic diversity in the pigeonpea genotypes. Transferability of pigeonpea-specific microsatellites revealed a cross-amplification and the presence of polymorphic alleles in P. vulgaris and V. unguiculata

Identificação de cultivares de batata por marcadores moleculares

The objective of this work was to evaluate a set of microsatellite markers for varietal identification and characterization of the most widespread potato cultivars in Brazil. The DNA from 14 potato cultivars was genotyped using microsatellite markers and the alleles were scored in silver-stained polyacrylamide gel. Twenty-four microsatellite markers were evaluated, and only one locus was monomorphic. Based on band patterns, a set of two microsatellites that were able to identify and differentiate all examined cultivars was obtained.O objetivo deste trabalho foi avaliar um conjunto de marcadores microssatélites para identificação e caracterização varietal das cultivares de batata mais amplamente utilizadas no Brasil. O DNA das 14 variedades de batata foi genotipado com marcadores microssatélites, e os alelos foram visualizados em gel de poliacrilamida corado com prata. Vinte e quatro marcadores foram avaliados e apenas um loco foi monomórfico. Com base no padrão de bandas, foi obtido um conjunto com dois microssatélites capazes de identificar e diferenciar todas as cultivares analisadas.11011

Genetic diversity analysis among pigeonpea genotypes adapted to South American regions based on microsatellite markers

The pigeonpea [Cajanus cajan (L) Millspaugh] is one of the most important perennial legume crops utilized in the food, fodder, soil conservation, crop-livestock integrated systems, reclaiming of degraded pastures and symbiotic nitrogen fixation. Microsatellite markers were used to estimate the genetic diversity of 77 pigeonpea genotypes selected from the germplasm collections at Embrapa Cattle-Southeast and, to evaluate their transferability to Phaseolus vulgaris and Vigna unguiculata species. The number of alleles per locus ranged from 2 to12, with an average of 5.1 alleles. The PIC values ranged from 0.11 to 0.80 (average 0.49) and the D values from 0.23 to 0.91 (average 0.58). The averages of observed and expected heterozygosity were 0.25 and 0.47, respectively, showing a deficit in heterozygosity. A model-based Bayesian approach implemented in the software STRUCTURE was used to assign genotypes into clusters. A dendrogram was constructed based on the modified Roger's genetic distances using a neighbor-joining method (NJ). A total of four clusters were assembled by STRUCTURE and a strong tendency of correspondence between the Bayesian clusters in the NJ tree was observed. The genetic distance ranged from 0.09 to 0.62 (average 0.37), showing a low genetic diversity in the pigeonpea genotypes. Transferability of pigeonpea-specific microsatellites revealed a cross-amplification and the presence of polymorphic alleles in P. vulgaris and V. unguiculata.431439Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Potato cultivar identification using molecular markers

O objetivo deste trabalho foi avaliar um conjunto de marcadores microssatélites para identificação e caracterização varietal das cultivares de batata mais amplamente utilizadas no Brasil. O DNA das 14 variedades de batata foi genotipado com marcadores microssatélites, e os alelos foram visualizados em gel de poliacrilamida corado com prata. Vinte e quatro marcadores foram avaliados e apenas um loco foi monomórfico. Com base no padrão de bandas, foi obtido um conjunto com dois microssatélites capazes de identificar e diferenciar todas as cultivares analisadas.The objective of this work was to evaluate a set of microsatellite markers for varietal identification and characterization of the most widespread potato cultivars in Brazil. The DNA from 14 potato cultivars was genotyped using microsatellite markers and the alleles were scored in silver-stained polyacrylamide gel. Twenty-four microsatellite markers were evaluated, and only one locus was monomorphic. Based on band patterns, a set of two microsatellites that were able to identify and differentiate all examined cultivars was obtained

Adaptação da técnica de fluorescência para fins de genotipagem com novos marcadores microssatélite em feijoeiro

The objectives of this work were to adapt the fluorescent labeling (PCR) technique using M13 universal primer for genotyping purposes, and to present a new set of microsatellite markers for common bean (Phaseolus vulgaris L.). A large population (380 common bean lines) was used for microsatellite genotyping. PCR fluorescent labeling method showed to be very efficient for multiplex analysis, providing lower costs and saving time, thus increasing the quality of genotyping analysis. A new set of 50 microsatellites developed from an enriched library derived from cultivar IAC-UNA was presented. This study provides better tools for assisting common bean breeding programs.Os objetivos deste trabalho foram adaptar a técnica de marcação fluorescente de produtos da (PCR) com uso do iniciador universal M13, para aplicação em genotipagem, e apresentar novos marcadores microssatélite para o feijoeiro (Phaseolus vulgaris L.). Uma população de grande tamanho amostral (380 linhagens) foi utilizada para genotipagem dos microssatélites. O método de PCR marcado por fluorescência demonstrou ser muito eficiente para a análise "multiplex" e proporcionou a redução de custos e ganho de tempo, aumentando a qualidade de análise da genotipagem. Foram apresentados 50 novos locos de microssatélites, desenvolvidos a partir de biblioteca enriquecida a partir da cultivar IAC-UNA. Este estudo fornece ferramentas melhores para assistir aos programas de melhoramento do feijoeiro

Gene Duplication in the Sugarcane Genome: A Case Study of Allele Interactions and Evolutionary Patterns in Two Genic Regions

Sugarcane (Saccharum spp.) is highly polyploid and aneuploid. Modern cultivars are derived from hybridization between S. officinarum and S. spontaneum. This combination results in a genome exhibiting variable ploidy among different loci, a huge genome size (~10 Gb) and a high content of repetitive regions. An approach using genomic, transcriptomic, and genetic mapping can improve our knowledge of the behavior of genetics in sugarcane. The hypothetical HP600 and Centromere Protein C (CENP-C) genes from sugarcane were used to elucidate the allelic expression and genomic and genetic behaviors of this complex polyploid. The physically linked side-by-side genes HP600 and CENP-C were found in two different homeologous chromosome groups with ploidies of eight and ten. The first region (Region01) was a Sorghum bicolor ortholog region with all haplotypes of HP600 and CENP-C expressed, but HP600 exhibited an unbalanced haplotype expression. The second region (Region02) was a scrambled sugarcane sequence formed from different noncollinear genes containing partial duplications of HP600 and CENP-C (paralogs). This duplication resulted in a non-expressed HP600 pseudogene and a recombined fusion version of CENP-C and the orthologous gene Sobic.003G299500 with at least two chimeric gene haplotypes expressed. It was also determined that it occurred before Saccharum genus formation and after the separation of sorghum and sugarcane. A linkage map was constructed using markers from nonduplicated Region01 and for the duplication (Region01 and Region02). We compare the physical and linkage maps, demonstrating the possibility of mapping markers located in duplicated regions with markers in nonduplicated region. Our results contribute directly to the improvement of linkage mapping in complex polyploids and improve the integration of physical and genetic data for sugarcane breeding programs. Thus, we describe the complexity involved in sugarcane genetics and genomics and allelic dynamics, which can be useful for understanding complex polyploid genomes

Genetic diversity analysis among pigeonpea genotypes adapted to South American regions based on microsatellite markers

The pigeonpea [Cajanus cajan (L) Millspaugh] is one of the most important perennial legume crops utilized in the food, fodder, soil conservation, crop-livestock integrated systems, reclaiming of degraded pastures and symbiotic nitrogen fixation. Microsatellite markers were used to estimate the genetic diversity of 77 pigeonpea genotypes selected from the germplasm collections at Embrapa Cattle-Southeast and, to evaluate their transferability to Phaseolus vulgaris and Vigna unguiculata species. The number of alleles per locus ranged from 2 to12, with an average of 5.1 alleles. The PIC values ranged from 0.11 to 0.80 (average 0.49) and the D values from 0.23 to 0.91 (average 0.58). The averages of observed and expected heterozygosity were 0.25 and 0.47, respectively, showing a deficit in heterozygosity. A model-based Bayesian approach implemented in the software STRUCTURE was used to assign genotypes into clusters. A dendrogram was constructed based on the modified Roger's genetic distances using a neighbor-joining method (NJ). A total of four clusters were assembled by STRUCTURE and a strong tendency of correspondence between the Bayesian clusters in the NJ tree was observed. The genetic distance ranged from 0.09 to 0.62 (average 0.37), showing a low genetic diversity in the pigeonpea genotypes. Transferability of pigeonpea-specific microsatellites revealed a cross-amplification and the presence of polymorphic alleles in P. vulgaris and V. unguiculata

Potato cultivar identification using molecular markers Identificação de cultivares de batata por marcadores moleculares

The objective of this work was to evaluate a set of microsatellite markers for varietal identification and characterization of the most widespread potato cultivars in Brazil. The DNA from 14 potato cultivars was genotyped using microsatellite markers and the alleles were scored in silver-stained polyacrylamide gel. Twenty-four microsatellite markers were evaluated, and only one locus was monomorphic. Based on band patterns, a set of two microsatellites that were able to identify and differentiate all examined cultivars was obtained.O objetivo deste trabalho foi avaliar um conjunto de marcadores microssatélites para identificação e caracterização varietal das cultivares de batata mais amplamente utilizadas no Brasil. O DNA das 14 variedades de batata foi genotipado com marcadores microssatélites, e os alelos foram visualizados em gel de poliacrilamida corado com prata. Vinte e quatro marcadores foram avaliados e apenas um loco foi monomórfico. Com base no padrão de bandas, foi obtido um conjunto com dois microssatélites capazes de identificar e diferenciar todas as cultivares analisadas