G4008.47: Developing genomic resources for pigeonpea using next generation sequencing technologies

Abstract

Summary Pigeonpea (Cajanus cajan L.), an important legume crop in Indian subcontinent, ranks sixth in area and production. However, the productivity of pigeonpea crop in semi-arid regions is less than 750 kg/ha due to exposure of the crop with several diseases. Biotechnological tools especially molecular markers have been proven very useful for improving the breeding efficiency in several major crop species, however, few genomic resources and low level of genetic diversity in pigeonpea germplasm is another bottleneck to varietal improvement. To achieve the goal of generating large expressed sequence tag (EST) resources, Roche/FLX 454 sequencing was carried out on a normalized cDNA pool prepared from 31 tissues produced 494,353 short transcript reads (STRs). 150.8 million Illumina sequence tags were generated from 10 pigeonpea genotypes. For identification of SNPs, tags for two genotypes of a given mapping population were aligned with 127,754 TAs (the pigeonpea transcriptome assembly). The number of SNPs in an individual cross ranged from 704 to 6,263. In total, 12,141 SNPs were identified across these genotypes. In terms of developing the marker platforms, CAPS markers could not be validated at a good rated. As a result, a total of 1,834 SNPs were attempted for KASPar assays and successful assays were developed for 1,616 SNPs and tested on a set of 94 genotypes. In addition, four genetic maps were developed based on SSR markers and by using these maps, in addition to two earlier maps, a consensus genetic linkage map was developed that includes a total of 339 SSR marker loci. Activities wise progress made in this project have been given as follows: Activity 1: Develop pigeonpea EST resources using 454-FLX Based on its phenology and the utility in breeding programs Pusa Ageti (ICP 28) was chosen for developing these genomic/transcriptomic resources. Deep sequencing was undertaken on cDNAs pools of 31 different developmental stages. Roche/FLX454 sequencing of this normalized cDNA pool generated 494,353 short transcript reads (STRs). Publicly available ESTs and newly generated datasets were analyzed separately and in combination. In order to develop a transcriptome reference in pigeonpea, 505,170 Roche/454 STRs and Sanger ESTs were assembled in combination to yield a total of 127,754 pigeonpea transcript assemblies (CcTAs). Activity 2: SNP development through Solexa-based transcriptome sequencing from 10 pigeonpea genotypes For identification of SNPs, tags for two genotypes of a given mapping population were aligned with 127,754 TAs and variants were identified using the Alpheus program of NCGR. The number of SNPs in an individual cross ranged from 704 (BSMR 736 × TAT 10) to 6,263 (ICPL 87119 × ICPL 87091). In total, 12,141 SNPs were identified; however, only six SNPs were found in common across three populations (ICPL 20096 × ICPL 332, ICP 7035 × TTB7 and BSMR 736 × TAT 10) (Dubey et al. 2011). Activity 3: Genetically map within existing mapping populations SNPs Due to non availability of suitable restriction sites, CAPS assays could be designed for only 116 SNPs. A very low level i.e., only 10 SNPs out of 116 SNPs showed expected results. As a result, these efforts were abandoned. For developing the Illumina GoldenGate assays, ADT scores were calculated and submitted to Illumina pipleline. However because of inordinate delay in receiving reagents, it was decided not to undertake development of GoldenGate assays. As a result, a total of 1,834 SNPs were attempted for KASPar assays and successful assays were developed for 1,616 SNPs and tested on a set of 94 genotypes. In addition, four genetic maps were developed based on SSR markers and by using these maps, in addition to two earlier maps, a consensus genetic linkage map was developed that includes a total of 339 SSR marker loci. Activity 4: Develop a consensus map for pigeonpea Genotyping data generated for four intra-specific mapping populations (ICPB 2049 × ICPL 99050, ICPA 2039 × ICPR 2447, ICPA 2043 × ICPR 3467 and ICPA 2043 × ICPR 2671) together with earlier published two genetic linkage maps (ICP 8863 × ICPL 20097 and TTB 7 × ICP 7035) were used for developing the consensus genetic map in cultivated pigeonpea. Segregation data for 348 markers obtained on 6 different mapping populations was used for merging multiple genetic maps. All the common markers collectively led to the synthesis of a consensus map comprising 339 loci profiled on 11 LGs and covering a map distance of 1,058.98 cM (Bohra et al. communicated). References Dubey A, et al., Varshney RK (2011) Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.). DNA Research. doi: 10.1093/dnares/dsr007 Bohra A, et al., Varshney RK (2011) An intra-specific consensus genetic map of pigeonpea [Cajanus cajan (L.) Millspaugh] derived from six mapping populations. BMC Genomics, communicate