BackgroundPigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping.ResultsIn this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population.ConclusionWe developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.

BackgroundPigeonpea is an important grain legume of the semi-arid tropics and sub-tropical regions where it plays a crucial role in the food and nutritional security of the people. The average productivity of pigeonpea has remained very low and stagnant for over five decades due to lack of genomic information and intensive breeding efforts. Previous SSR-based linkage maps of pigeonpea used inter-specific crosses due to low inter-varietal polymorphism. Here our aim was to construct a high density intra-specific linkage map using genic-SNP markers for mapping of major quantitative trait loci (QTLs) for key agronomic traits, including plant height, number of primary and secondary branches, number of pods, days to flowering and days to maturity in pigeonpea.ResultsA population of 186 F2:3 lines derived from an intra-specific cross between inbred lines ‘Pusa Dwarf’ and ‘HDM04-1’ was used to construct a dense molecular linkage map of 296 genic SNP and SSR markers covering a total adjusted map length of 1520.22 cM for the 11 chromosomes of the pigeonpea genome. This is the first dense intra-specific linkage map of pigeonpea with the highest genome length coverage. Phenotypic data from the F2:3 families were used to identify thirteen QTLs for the six agronomic traits. The proportion of phenotypic variance explained by the individual QTLs ranged from 3.18% to 51.4%. Ten of these QTLs were clustered in just two genomic regions, indicating pleiotropic effects or close genetic linkage. In addition to the main effects, significant epistatic interaction effects were detected between the QTLs for number of pods per plant.ConclusionsA large amount of information on transcript sequences, SSR markers and draft genome sequence is now available for pigeonpea. However, there is need to develop high density linkage maps and identify genes/QTLs for important agronomic traits for practical breeding applications. This is the first report on identification of QTLs for plant type and maturity traits in pigeonpea. The QTLs identified in this study provide a strong foundation for further validation and fine mapping for utilization in the pigeonpea improvement.

Pigeonpea [Cajanus cajan (L.) Millspaugh] is an important food legume of the semi-arid tropics (SAT) sustaining livelihood of millions of people. Stagnant and unstable yield per hectare all over the world is the characteristic feature of this crop. This is primarily ascribed to its susceptibility/sensitivity to a number of biotic and abiotic factors. Among biotic factors, insects such as pod borer (Helicoverpa armigera), pod fly (Melanoagromyza obtusa) and spotted borer (Maruca vitrata) substantially damage the crop and result in significant economic losses. Management of these insects by genetic means has always been considered environment friendly approach. However, genetic improvement has always been impeded by limited genetic variability in the primary gene pool of pigeonpea. Wild species present in the secondary and tertiary gene pools have been reported to carry resistance for such insects. However, transfer of resistance through conventional backcrossing has not been much successful. It calls for gene introgression through marker assisted backcrossing (MABC) or advanced backcross breeding (AB breeding). In this review, we have attempted to assess the progress made through conventional and molecular breeding and suggested the ways to move further towards genetic enhancement for insects resistance in pigeonpea.

An understanding of the inheritance of aluminium (Al) tolerance is important to breed for Al-tolerant genotypes of chickpea (Cicer arietinum L.). Therefore, a study was undertaken to infer genes governing Al tolerance in chickpea. Tolerant lines ÔICC14880Õ and ÔIPC92-39Õ were crossed with sensitive lines ÔIPCK96-3Õ and ÔIPC99-4Õ. Parental, F 1 , F 2 , F 3 and backcross generations were evaluated in a nutrient solution containing 20 ppm Al for haematoxylin staining and root re-growth and classified for tolerance by staining of root tips and root re-growth. The F 1 hybrids responded similarly to the tolerant parents indicating dominance of Al tolerance over sensitivity. Segregation for tolerance vs. sensitivity in F 2 fitted well with the 3 : 1 ratio expected for a single gene. The backcross and F 3 data confirmed the presence of single dominant gene common in tolerant parents. Experimental findings showed that Al tolerance is a monogenic dominant trait that can be easily transferred to high yielding lines through a backcross breeding.

Pigeonpea is the second most important pulse legume crop for food and nutritional security of South Asia that requires accelerated breeding using high throughput genomic tools. Single nucleotide polymorphisms (SNPs) are highly suitable markers for this purpose because of their bi-allelic nature, reproducibility and high abundance in the genome. Here we report on development and use of a pigeonpea 62 K SNP chip array 'CcSNPnks' for Affymetrix GeneTitan ® platform. The array was designed after filtering 645,662 genic-SNPs identified by re-sequencing of 45 diverse genotypes and has 62,053 SNPs from 9629 genes belonging to five different categories, including 4314 single-copy genes unique to pigeonpea, 4328 single-copy genes conserved between soybean and pigeonpea, 156 homologs of agronomically important cloned genes, 746 disease resistance and defense response genes and 85 multi-copy genes of pigeonpea. This fully genic chip has 28.94% exonic, 33.04% intronic, 27.56% 5′UTR and 10.46% 3′UTR SNPs and incorporates multiple SNPs per gene allowing gene haplotype network analysis. It was used successfully for the analysis of genetic diversity and population structure of 95 pigeonpea varieties and high resolution mapping of 11 yield related QTLs for number of branches, pod bearing length and number of seeds per pod in a biparental RIL population. As an accurate highdensity genotyping tool, 'CcSNPnks' chip array will be useful for high resolution fingerprinting, QTL mapping and genome wide as well as gene-based association studies in pigeonpea.

Chickpea is predominantly an indeterminate (IDT) plant due to which high fertility and irrigation have adverse effect on seed yield. The semi-determinate (SDT) types are relatively early, lodging resistant and found to be similar in their yield potential to that of IDT genotypes. However, the inheritance of SDT stem type is not well understood in chickpea. The present investigation was aimed at studying the genetics of SDT stem type and identifying molecular marker linked to Dt1 locus through Bulked Segregant Analysis (BSA). The genetics of semi-determinacy was studied in BGD 72 (IDT) x BG 3078-1(SDT) cross in which the genetic constitution of BGD 72 was already known as Dt1Dt1Dt2Dt2 based on the previous study. The F1 was IDT indicating the dominance of indeterminacy over semideterminacy. The segregation in F2 and F3 revealed that SDT stem growth in the new-found genotype BG3078-1 was governed by a single dominant gene Dt2 and its genotype designated as dt1dt1Dt2Dt2. The study of polymorphic survey between BGD-72(IDT) and BG 3078- 1(SDT) using 581 SSR markers found 50 markers polymorphic. The BSA using 50 polymorphic markers has identified TA42 and TR29 as polymorphic between IDT and SDT parents as well as IDT and SDT bulks and hence considered putatively linked to Dt1 locus governing IDT stem growth in chickpea. The two linked markers were validated in 15 IDT and 15 SDT F2 plants individually. These two markers were also validated in 3 IDT, 8SDT and 3 DT genotypes. This is the first report on the identification of a molecular marker associated with stem growth habit in chickpea.

Pigeonpea is an important tropical grain legume widely cultivated in South and South‐East Asia for versatile end usage as food, feed, fodder and fuel. Recent publication of draft genome sequence of pigeonpea (‘Arhar’) has allowed mining of large numbers of genomic simple sequence repeat (SSR) markers most of which are either not validated or show very little polymorphism in simple agarose gel–based assays. Here, we describe a special category of 370 validated highly variable ‘Arhar’ genomic SSR (HASSR) markers, which provide much higher level of polymorphism than a random set of SSR markers. These markers were validated for consistent amplification in a set of eight pigeonpea varieties and 152 of these (41%) showed allelic polymorphism on agarose gel electrophoresis. Twenty‐four highest polymorphic HASSR markers were used on a larger set of forty pigeonpea varieties and eight wild Cajanus species for the analysis of genetic diversity and phylogenetic relationship. Genomic HASSR markers described here are highly suitable for genetic diversity and phylogenic studies in pigeonpea.

Pigeonpea (Cajanus cajan (L.) Millsp.) is a major food legume cultivated in semi-arid tropical regions including the Indian subcontinent, Africa, and Southeast Asia. It is an important source of protein, minerals, and vitamins for nearly 20% of the world population. Due to high carbon sequestration and drought tolerance, pigeonpea is an important crop for the development of climate resilient agriculture and nutritional security. However, pigeonpea productivity has remained low for decades because of limited genetic and genomic resources, and sparse utilization of landraces and wild pigeonpea germplasm. Here, we present a dense intraspecific linkage map of pigeonpea comprising 932 markers that span a total adjusted map length of 1,411.83 cM. The consensus map is based on three different linkage maps that incorporate a large number of single nucleotide polymorphism (SNP) markers derived from next generation sequencing data, using Illumina GoldenGate bead arrays, and genotyping with restriction site associated DNA (RAD) sequencing. The genotyping-by-sequencing enhanced the marker density but was met with limited success due to lack of common markers across the genotypes of mapping population. The integrated map has 547 bead-array SNP, 319 RAD-SNP, and 65 simple sequence repeat (SSR) marker loci. We also show here correspondence between our linkage map and published genome pseudomolecules of pigeonpea. The availability of a high-density linkage map will help improve the anchoring of the pigeonpea genome to its chromosomes and the mapping of genes and quantitative trait loci associated with useful agronomic traits.