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

Abstract

Background: Pigeonpea [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.

Results: A 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.

Conclusion: In 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.

Figure 1

Annotation pipeline for analysis of BESs. This pipeline resulted in selection of non-redundant genomic BAC-ends which excluded organeller sequences, and further identification, annotation of non-redundant sequences together with SSR discovery, selection and primer designing.

Figure 2

Distribution of BAC end categories according to BES cluster depth. Cluster depth supported the repetitive nature of mobile genetic elements while genic regions were mostly associated with less repetitive sequences.

Figure 3

Distribution and frequency of SSRs in differing genome fractions. Maximum frequency and maximum amount of SSRs was exhibited by non annotated regions followed by the regions containing 'genes'.

Figure 4

Reference genetic map of pigeonpea derived from an inter-specific F₂ population (ICP 28 × ICPW 94). Initially, a skeleton map with normally segregating markers was constructed using MAPMAKER/EXP 3.0 while further integration of additional markers was performed with Joinmap 3.0 by keeping the mapmaker order as "fixed". Distances between the loci (in cM) are shown to the left of the linkage group and all the loci at the right side of the map.

Figure 5

Electropherogram display for the multiplex set MG 1 for purity assessment of hybrid ICPH 2671. This figure shows the analysis (GENEMAPPER output) of seven SSR markers of MG1 for ICPH 2671 in a single capillary. SSR markers labeled with the same fluorescence dye are analyzed in individual panels. A. Analysis of two VIC (green) labeled SSR markers, B. Two NED (black) labeled SSR markers, C. One PET (red) labeled SSR markers, and D. Analysis of two FAM (blue) labeled SSR markers.