Microsatellite Development and Application in Pigeonpea (<i>Cajanus cajan </i>(L.) Millsp.)

Abstract

Pigeonpea is a major legume crop grown in the semi-arid tropics but has been relatively neglected in terms of genomic research. This study aimed at developing molecular markers as a basic requirement towards initiating marker assisted breeding techniques in pigeonpea. Simple Sequence Repeat (SSR) loci of pigeonpea were isolated by screening non-enriched (library A) and enriched (library B) partial genomic libraries with SSR probes. Positive clones were sequenced and primers designed for 152 microsatellite loci, 39 from library A and 113 from library B. Optimisation of reaction conditions was achieved for 51% and 65% of primers designed from library A and B, respectively. For the purpose of exploiting the transferability of SSRs across genera within the legume species, 220 soybean primers were tested in pigeonpea, 39 of which amplified interpretable bands.<br /> Nineteen out of 20 amplified primers from library A were polymorphic among 15 cultivated and 9 wild species. The diversity analysis revealed contrasted levels of variability within cultivated and wild accessions. A total of 98 alleles were detected at the 19 polymorphic loci with an average of 4.9 alleles per locus while the observed heterozygosity ranged from 0.17 – 0.80 with a mean of 0.42 per locus. Substantially less allelic variation (31 alleles) was observed within the cultivated species than across the wild species (92 alleles). Primers from library B were not tested for amplification in wild species but 35 out of the amplified 73 revealed polymorphism among 24 pigeonpea genotypes. The number of alleles detected ranged from 2 to 6 with a total of 110 alleles and an average of 3.1 alleles per locus. Only one of the 39 amplified soybean primers revealed polymorphism among 24 cultivated pigeonpea accessions. No significant relationship was detected between the class of repeats and heterozygosity values.<br /> AT and TG class of repeats were the most abundant di-nucleotide repeats in library A and B respectively while TAA and GAA were the most abundant trinucleotide repeats in both libraries. Protein database searches provided putative functions for 21 SSR-containing pigeonpea sequences that would be useful in functional marker development. UPGMA and MDS cluster analysis revealed genetic relationships among recently bred varieties, old varieties and wild accessions. Nine of the markers developed were polymorphic to the parental lines of a F6 Fusarium wilt RIL mapping population that had been developed by ICRISAT breeders. Analysis of allele segregation in the RIL population revealed that all the 9 SSRs segregated in the expected 1:1 ratio and were further tested for any possible linkage with a QTL for resistance to Fusarium wilt. All the polymorphic markers derived from this study are now being used for characterisation and evaluation of pigeonpea germplasm collection at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) headquarters, India. SSRs provide a powerful tool for genomic studies and are recommended for systematic fingerprinting of pigeonpea germplasm.