Genome-wide comparative transcriptome analysis of the A4-CMS line ICPA 2043 and its maintainer ICPB 2043 during the floral bud development of pigeonpea.

Cytoplasmic male sterility (CMS) offers a unique system to understand cytoplasmic nuclear crosstalk, and is also employed for exploitation of hybrid vigor in various crops. Pigeonpea A4-CMS, a predominant source of male sterility, is being used for efficient hybrid seed production. The molecular mechanisms of CMS trait remain poorly studied in pigeonpea. We performed genome-wide transcriptome profiling of A4-CMS line ICPA 2043 and its isogenic maintainer ICPB 2043 at two different stages of floral bud development (stage S1 and stage S2). Consistent with the evidences from some other crops, we also observed significant difference in the expression levels of genes in the later stage, i.e., stage S2. Differential expression was observed for 143 and 55 genes within the two stages of ICPA 2043 and ICPB 2043, respectively. We obtained only 10 differentially expressed genes (DEGs) between the stage S1 of the two genotypes, whereas expression change was significant for 582 genes in the case of stage S2. The qRT-PCR assay of randomly selected six genes supported the differential expression of genes between ICPA 2043 and ICPB 2043. Further, GO and KEGG pathway mapping suggested a possible compromise in key bioprocesses during flower and pollen development. Besides providing novel insights into the functional genomics of CMS trait, our results were in strong agreement with the gene expression atlas of pigeonpea that implicated various candidate genes like sucrose-proton symporter 2 and an uncharacterized protein along with pectate lyase, pectinesterase inhibitors, L-ascorbate oxidase homolog, ATPase, ß-galactosidase, polygalacturonase, and aldose 1-epimerase for pollen development of pigeonpea. The dataset presented here provides a rich genomic resource to improve understanding of CMS trait and its deployment in heterosis breeding in pigeonpea.

Genomics-assisted breeding in four major pulse crops of developing countries: present status and prospects.

KEY MESSAGE: Given recent advances in pulse molecular biology, genomics-driven breeding has emerged as a promising approach to address the issues of limited genetic gain and low productivity in various pulse crops. The global population is continuously increasing and is expected to reach nine billion by 2050. This huge population pressure will lead to severe shortage of food, natural resources and arable land. Such an alarming situation is most likely to arise in developing countries due to increase in the proportion of people suffering from protein and micronutrient malnutrition. Pulses being a primary and affordable source of proteins and minerals play a key role in alleviating the protein calorie malnutrition, micronutrient deficiencies and other undernourishment-related issues. Additionally, pulses are a vital source of livelihood generation for millions of resource-poor farmers practising agriculture in the semi-arid and sub-tropical regions. Limited success achieved through conventional breeding so far in most of the pulse crops will not be enough to feed the ever increasing population. In this context, genomics-assisted breeding (GAB) holds promise in enhancing the genetic gains. Though pulses have long been considered as orphan crops, recent advances in the area of pulse genomics are noteworthy, e.g. discovery of genome-wide genetic markers, high-throughput genotyping and sequencing platforms, high-density genetic linkage/QTL maps and, more importantly, the availability of whole-genome sequence. With genome sequence in hand, there is a great scope to apply genome-wide methods for trait mapping using association studies and to choose desirable genotypes via genomic selection. It is anticipated that GAB will speed up the progress of genetic improvement of pulses, leading to the rapid development of cultivars with higher yield, enhanced stress tolerance and wider adaptability.

Mapping QTL for important seed traits in an interspecific F2 population of pigeonpea.

Seed traits present important breeding targets for enhancing grain yield and quality in various grain legume crops including pigeonpea. The present study reports significant genetic variation for six seed traits including seed length (SL), seed width (SW), seed thickness (ST), seed weight (SWT), electrical conductivity (EC) and water uptake (WU) among Cajanus cajan (L.) Millspaugh acc. ICPL 20340 and Cajanus scarabaeoides (L.) Thouars acc. ICP 15739 and an F2 population derived from this interspecific cross. Maximum phenotypic values recorded for the F2 population were higher than observed in the parent ICPL 20340 [F2 max vs ICPL 20340: SW (7.05 vs 5.38), ST (4.63 vs 4.51), EC (65.17 vs 9.72), WU (213.17 vs 109.5)], which suggested contribution of positive alleles from the wild parent, ICP 15739. Concurrently, to identify the QTL controlling these seed traits, we assayed two parents and 94 F2 individuals with 113 polymorphic simple sequence repeat (SSR) markers. In the F2 population, 98 of the 113 SSRs showed Mendelian segregation ratio 1:2:1, whereas significant deviations were observed for 15 SSRs with their χ 2 values ranging between 6.26 and 20.62. A partial genetic linkage map comprising 83 SSR loci was constructed. QTL analysis identified 15 marker-trait associations (MTAs) for seed traits on four linkage groups i.e. LG01, LG02, LG04 and LG05. Phenotypic variations (PVs) explained by these QTL ranged from 4.4 (WU) to 19.91% (EC). These genomic regions contributing significantly towards observed variability of seed traits would serve as potential candidates for future research that aims to improve seed traits in pigeonpea.

Development and Application of High-Density Axiom Cajanus SNP Array with 56K SNPs to Understand the Genome Architecture of Released Cultivars and Founder Genotypes.

As one of the major outputs of next-generation sequencing (NGS), a large number of genome-wide single-nucleotide polymorphisms (SNPs) have been developed in pigeonpea [ (L.) Huth.]. However, SNPs require a genotyping platform or assay to be used in different evolutionary studies or in crop improvement programs. Therefore, we developed an Axiom SNP array with 56K SNPs uniformly distributed across the genome and assessed its utility in a genetic diversity study. From the whole-genome resequencing (WGRS) data on 104 pigeonpea lines, ∼2 million sequence variations (SNPs and insertion-deletions [InDels]) were identified, from which a subset of 56,512 unique and informative sequence variations were selected to develop the array. The Axiom SNP array developed was used for genotyping 103 pigeonpea lines encompassing 63 cultivars released between 1960 and 2014 and 40 breeding, germplasm, and founder lines. Genotyping data thus generated on 103 pigeonpea lines provided 51,201 polymorphic SNPs and InDels. Genetic diversity analysis provided in-depth insights into the genetic architecture and trends in temporal diversity in pigeonpea cultivars. Therefore, the continuous use of the high-density Axiom SNP array developed will accelerate high-resolution trait mapping, marker-assisted breeding, and genomic selection efforts in pigeonpea.

New Hypervariable SSR Markers for Diversity Analysis, Hybrid Purity Testing and Trait Mapping in Pigeonpea [Cajanus cajan (L.) Millspaugh].

Draft genome sequence in pigeonpea offers unprecedented opportunities for genomics assisted crop improvement via enabling access to genome-wide genetic markers. In the present study, 421 hypervariable simple sequence repeat (SSR) markers from the pigeonpea genome were screened on a panel of eight pigeonpea genotypes yielding marker validation and polymorphism percentages of 95.24 and 54.11%, respectively. The SSR marker assay uncovered a total of 570 alleles with three as an average number of alleles per marker. Similarly, the mean values for gene diversity and PIC were 0.44 and 0.37, respectively. The number of polymorphic markers ranged from 39 to 89 for different parental combinations. Further, 60 of these SSRs were assayed on 94 genotypes, and model based clustering using STRUCTURE resulted in the identification of the two subpopulations (K = 2). This remained in close agreement with the clustering patterns inferred from genetic distance (GD)-based approaches i.e., dendrogram, factorial and principal coordinate analysis (PCoA). The AMOVA accounted majority of the genetic variation within groups (89%) in comparison to the variation existing between the groups (11%). A subset of these markers was implicated for hybrid purity testing. We also demonstrated utility of these SSR markers in trait mapping through association and bi-parental linkage analyses. The general linear (GLM) and mixed linear (MLM) models both detected a single SSR marker (CcGM03681) with R2 = 16.4 as associated with the resistance to Fusarium wilt variant 2. Similarly, by using SSR data in a segregating backcross population, the corresponding restorer-of-fertility (Rf) locus was putatively mapped at 39 cM with the marker CcGM08896. However, The marker-trait associations (MTAs) detected here represent a very preliminary type and hence demand deeper investigations for conclusive evidence. Given their ability to reveal polymorphism in simple agarose gels, the hypervariable SSRs are valuable genomic resource for pigeonpea research community, particularly in South Asia and East Africa where pigeonpea is primarily grown.