QTL mapping in population originated from a bi-parental commercial cross of sugarcane

Orientadores: Anete Pereira de Souza, Monalisa Sampaio CarneiroTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A cana-de-açúcar é uma fonte renovável de energia e com potencial para expansão. A complexidade genética da cana-de-açúcar decorrente de seu alto nível de ploidia e aneuploidia, aliada à natureza quantitativa da maioria dos caracteres agronômicos tem dificultado, atualmente, a obtenção de elevados índices de ganho genético através do melhoramento convencional. O desenvolvimento de marcadores moleculares e a construção de mapas genéticos podem auxiliar na elaboração de estratégias a serem introduzidas nos programas de melhoramento de forma a aumentar a eficiência dos processos de seleção e acelerar o desenvolvimento de novas cultivares. Desta forma, a proposta desta tese foi a construção de um mapa genético em cana-de-açúcar visando a identificação de regiões genômicas que controlam características de interesse através do mapeamento de QTL (Quantitative Trait Loci). Uma população de cana-de-açúcar com 153 indivíduos oriundos do cruzamento comercial bi-parental entre as cultivares SP80-3280 e RB835486 foi utilizada para alcançar os objetivos citados. O experimento de campo foi instalado em duas localidades, Araras-SP e Ipaussu-SP, usando o delineamento de blocos aumentados incompletos com 3 repetições. As avaliações fenotípicas foram realizadas ao longo de três anos (2011, 2012 e 2013). Empregou-se a abordagem de modelos mistos para análise das características fenotípicas relacionadas com componentes de produção e resistência à ferrugem marrom. Os dados de severidade à ferrugem marrom foram analisados, como uma primeira abordagem, via modelo misto linear generalizado. As estimativas de herdabilidade das características fenotípicas foram altas, variando de 0.78 (altura de colmos) a 0.92 (diâmetro de colmos), e a análise de severidade à ferrugem marrom mostrou que 66% dos clones possuem, no mínimo, 90% de probabilidade de serem resistentes à doença. Para construção do mapa genético integrado foram utilizados marcadores moleculares do tipo microssatélites (Simple Sequence Repeats, SSR), TRAP (Target Target Region Amplification Polymorphism), além de SNPs (Single Nucleotide Polymorphims) e indels (inserções e deleções) oriundos da técnica de GBS (Genotyping-by-Sequencing). Para descoberta de marcadores baseados em GBS foram utilizadas quatro pseudo-referências: genoma de sorgo (Sorghum bicolor), genoma metil-filtrado da cana-de-açúcar, transcriptoma da cana-de-açúcar (RNAseq) e sequências do projeto SUCEST. A ploidia e dosagem de cada loco bi-alélico foi estimada através do software SUPERMASSA. Utilizando o software Onemap (v. 2.0-4) e empregando-se LOD > 9.0 e fração de recombinação 9.0 and recombination fraction <0.10, a total of 993 markers in single dose were mapped. These markers were distributed throughout 223 linkage groups, which were clustered in 18 homo(eo)logous groups. The total length of the map was 3,682.04 cM with an average marker density of 3.70 cM. Using composite interval mapping (Composite Interval Mapping, CIM) were mapped seven QTLs considering four of the 11 phenotypic traits evaluated. The results suggest the presence of a stable QTL across locations to soluble solid content (BRIX) and sucrose content of the cane (POL%C). Furthermore, QTLs to BRIX and fiber content (FIB) traits had associated markers with candidate genes, which had great potential for validation and future use for molecular breeding in sugarcane. This study is the first to report the use of GBS for large-scale variant discovery and genotyping of a population in sugarcane with posterior analysis to composite interval mappingDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecular2010/50091-4 , 2008/52197-4FAPES

Gbs-based Single Dosage Markers For Linkage And Qtl Mapping Allow Gene Mining For Yield-related Traits In Sugarcane

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Sugarcane (Saccharum spp.) is predominantly an autopolyploid plant with a variable ploidy level, frequent aneuploidy and a large genome that hampers investigation of its organization. Genetic architecture studies are important for identifying genomic regions associated with traits of interest. However, due to the genetic complexity of sugarcane, the practical applications of genomic tools have been notably delayed in this crop, in contrast to other crops that have already advanced to marker-assisted selection (MAS) and genomic selection. High-throughput next-generation sequencing (NGS) technologies have opened new opportunities for discovering molecular markers, especially single nucleotide polymorphisms (SNPs) and insertion-deletion (indels), at the genome-wide level. The objectives of this study were to (i) establish a pipeline for identifying variants from genotyping-by-sequencing (GBS) data in sugarcane, (ii) construct an integrated genetic map with GBS-based markers plus target region amplification polymorphisms and microsatellites, (iii) detect QTLs related to yield component traits, and (iv) perform annotation of the sequences that originated the associated markers with mapped QTLs to search putative candidate genes. Results: We used four pseudo-references to align the GBS reads. Depending on the reference, from 3,433 to 15,906 high-quality markers were discovered, and half of them segregated as single-dose markers (SDMs) on average. In addition to 7,049 non-redundant SDMs from GBS, 629 gel-based markers were used in a subsequent linkage analysis. Of 7,678 SDMs, 993 were mapped. These markers were distributed throughout 223 linkage groups, which were clustered in 18 homo(eo)logous groups (HGs), with a cumulative map length of 3,682.04 cM and an average marker density of 3.70 cM. We performed QTL mapping of four traits and found seven QTLs. Our results suggest the presence of a stable QTL across locations. Furthermore, QTLs to soluble solid content (BRIX) and fiber content (FIB) traits had markers linked to putative candidate genes. Conclusions: This study is the first to report the use of GBS for large-scale variant discovery and genotyping of a mapping population in sugarcane, providing several insights regarding the use of NGS data in a polyploid, non-model species. The use of GBS generated a large number of markers and still enabled ploidy and allelic dosage estimation. Moreover, we were able to identify seven QTLs, two of which had great potential for validation and future use for molecular breeding in sugarcane.18FINEP (Finaciadora de Estudos e Projetos)FAPESP (Fundacao de Amparo a Pesquisa de Sao Paulo) [08/52197-4]INCT-Bioetanol (Instituto Nacional de Ciencia e Tecnologia do Bioetanol) [FAPESP 08/57908-6]INCT-Bioetanol (CNPq, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) [574002/2008-1]FAPESP [10/50091-4, 12/25236-4, 10/50549-0, 10/50031-1, 12/11109-0]CNPqCAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Molecular diversity and genetic structure of Saccharum complex accessions.

Sugarcane is an important crop for food and energy security, providing sucrose and bioethanol from sugar content and bioelectricity from lignocellulosic bagasse. In order to evaluate the diversity and genetic structure of the Brazilian Panel of Sugarcane Genotypes (BPSG), a core collection composed by 254 accessions of the Saccharum complex, eight TRAP markers anchored in sucrose and lignin metabolism genes were evaluated. A total of 584 polymorphic fragments were identified and used to investigate the genetic structure of BPSG through analysis of molecular variance (AMOVA), principal components analysis (PCA), a Bayesian method using STRUCTURE software, genetic dissimilarity and phylogenetic tree. AMOVA showed a moderate genetic differentiation between ancestors and improved accessions, 0.14, and the molecular variance was higher within populations than among populations, with values of 86%, 95% and 97% when constrasting improved with ancestors, foreign with ancestors and improved with foreign, respectively. The PCA approach suggests clustering in according with evolutionary and Brazilian breeding sugarcane history, since improved accessions from older generations were positioned closer to ancestors than improved accessions from recent generations. This result was also confirmed by STRUCTURE analysis and phylogenetic tree. The Bayesian method was able to separate ancestors of the improved accessions while the phylogenetic tree showed clusters considering the family relatedness within three major clades; the first being composed mainly by ancestors and the other two mainly by improved accessions. This work can contribute to better management of the crosses considering functional regions of the sugarcane genome

RB975952 – Early maturing sugarcane cultivar

RB975952 is an early maturing sugarcane cultivar released for the South-Central region of Brazil. It should be harvested between April and May, and it is recommended for planting in environments with medium to high production potential. RB975952 has high resistance levels to the main diseases of the crop, it also has a good shoot development after mechanical harvesting, and high sucrose yields

RB975242 and RB975201 - Late maturation sugarcane varieties

The sugarcane varieties RB975201 and RB975242 were developed and released for harvest at the end of the season (late maturation) in the CentralSouth region of Brazil. In specific environments, these varieties were compared with commercial standards in sugar yield per area. They are resistant to major sugarcane diseases and present the Bru1 gene of resistance to brown rust

Differential expression in leaves of Saccharum genotypes contrasting in biomass production provides evidence of genes involved in carbon partitioning

The development of biomass crops aims to meet industrial yield demands, in order to optimize profitability and sustainability. Achieving these goals in an energy crop like sugarcane relies on breeding for sucrose accumulation, fiber content and stalk number. To expand the understanding of the biological pathways related to these traits, we evaluated gene expression of two groups of genotypes contrasting in biomass composition.First visible dewlap leaves were collected from 12 genotypes, six per group, to perform RNA-Seq. We found a high number of differentially expressed genes, showing how hybridization in a complex polyploid system caused extensive modifications in genome functioning. We found evidence that differences in transposition and defense related genes may arise due to the complex nature of the polyploid Saccharum genomes. Genotypes within both biomass groups showed substantial variability in genes involved in photosynthesis. However, most genes coding for photosystem components or those coding for phosphoenolpyruvate carboxylases (PEPCs) were upregulated in the high biomass group. Sucrose synthase (SuSy) coding genes were upregulated in the low biomass group, showing that this enzyme class can be involved with sucrose synthesis in leaves, similarly to sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP). Genes in pathways related to biosynthesis of cell wall components and expansins coding genes showed low average expression levels and were mostly upregulated in the high biomass group.Together, these results show differences in carbohydrate synthesis and carbon partitioning in the source tissue of distinct phenotypic groups. Our data from sugarcane leaves revealed how hybridization in a complex polyploid system resulted in noticeably different transcriptomic profiles between contrasting genotypes

CULTIVAR RELEASE - RB965902 and RB965917 – Early/medium maturing sugarcane varieties

The varieties RB965902 and RB965917 were developed for harvesting at the beginning to the middle of the sucroseextraction period (early/medium maturity) and released for the South-Central region of Brazil. In specific environments, the tons ofPol per area (sucrose yield) of these varieties is higher than of the commercial standard RB855453 and they are resistant to the maindiseases of the crop

<i>De Novo</i> Assembly and Transcriptome Analysis of Contrasting Sugarcane Varieties

<div><p>Sugarcane is an important crop and a major source of sugar and alcohol. In this study, we performed <i>de novo</i> assembly and transcriptome annotation for six sugarcane genotypes involved in bi-parental crosses. The <i>de novo</i> assembly of the sugarcane transcriptome was performed using short reads generated using the Illumina RNA-Seq platform. We produced more than 400 million reads, which were assembled into 72,269 unigenes. Based on a similarity search, the unigenes showed significant similarity to more than 28,788 sorghum proteins, including a set of 5,272 unigenes that are not present in the public sugarcane EST databases; many of these unigenes are likely putative undescribed sugarcane genes. From this collection of unigenes, a large number of molecular markers were identified, including 5,106 simple sequence repeats (SSRs) and 708,125 single-nucleotide polymorphisms (SNPs). This new dataset will be a useful resource for future genetic and genomic studies in this species.</p></div