Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea (Cajanus spp.)

Abstract

BackgroundPigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea.ResultsA set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme.ConclusionIn summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea.

Molecular diversity and phenotypic characterization of A, B and R lines in Pigeonpea [Cajanus cajan (L.) Millsp.]

“…Major allele frequency ranged from 0.34 (CcM0057) to 0.79 (CcM0402) with an average of 0.47 per marker (Table 2). Similar kinds of reports were reported by Singh et al (2008), Odeny et al (2009), Bohra et al (2011) and Indira et al (2014.…”

Legume Crops Phylogeny and Genetic Diversity for Science and Breeding

“…Other marker systems used to estimate the polymorphism in Cajanus were RAPD (Ratnaparkhe et al, 1995), AFLP (Punguluri et al, 2006), DArT (Yang et al, 2006), SSR (Saxena et al, 2010a, b;Bohra et al, 2011a), and, recently, single-nucleotide polymorphism (Saxena et al, 2012;Roorkiwal et al, 2013). All marker-based studies have revealed that a very low level of diversity is present in cultivated pigeonpea, whereas the wild relatives of pigeonpea showed enormous diversity (Ratnaparkhe et al, 1995;Punguluri et al, 2006;Saxena et al, 2010a, b;Bohra et al, 2011a). These studies also revealed that two of the wild relatives, C. cajanifolius and C. scarabaeoides, are closely related to pigeonpea.…”

Isolation and characterization of microsatellite markers in Garcinia gummi-gutta by next-generation sequencing and cross-species amplification

“…However, development of microsatellite markers have helped in fingerprinting unique trees, assessing degree of diversity in the populations, and identifying marker tightly linked to the important agronomic traits like active ingredients, and resistance to biotic and abiotic stresses. Therefore, the development of markers has become a prerequisite for genetic studies (Bohra et al 2011;Dutta et al 2011). Keeping this in view, here, we report the development and standardization of microsatellite markers of G. gummi-gutta using next-generation sequencing (NGS) and their cross species transferability.…”