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

<p>Abstract</p> <p>Background</p> <p>Pigeonpea [<it>Cajanus cajan </it>(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.</p> <p>Results</p> <p>A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using <it>Hin</it>dIII (34,560 clones) and <it>Bam</it>HI (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 <it>first </it>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.</p> <p>Conclusion</p> <p>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.</p

Mitochondrial SSRs and their utility in distinguishing wild species, CMS lines and maintainer lines in pigeonpea (Cajanus cajan L.)

Analysis of the pigeonpea mitochondrial genome sequence identified 25 SSRs. Mononucleotide SSR motifs were the most abundant repeats followed by dinucleotide and trinucleotide repeats. Primer pairs could be designed for 24 SSRs, 23 of which were polymorphic amongst the 22 genotypes consisting of cytoplasmic male sterile (CMS or A) line, maintainer or B line and wild Cajanus species representing six different CMS systemsviz.,A1,A2,A 4,A5,A6 andA8. These markers amplified a total of 107 alleles ranging from 2 to 10 with an average of 4.65 alleles per locus. The polymorphic information content for these markers ranged from 0.09 to 0.84 with an average of 0.52 per marker. Hence, the present study adds a novel set of 24 mitochondrial SSR markers to the markers repository in pigeonpea, which would be useful to distinguish the genotypes based on mitochondrial genome types in evolutionary and phylogenetic studies

Characterization of heterosis and genomic prediction-based establishment of heterotic patterns for developing better hybrids in pigeonpea

Whole-genome resequencing (WGRS) of 396 lines, consisting of 104 hybrid parental lines and 292 germplasm lines, were used to study the molecular basis of mid-parent heterosis (MPH) and to identify complementary heterotic patterns in pigeonpea [Cajanus cajan (L.) Millsp.] hybrids. The lines and hybrids were assessed for yield and yield-related traits in multiple environments. Our analysis showed positive MPH values in 78.6% of hybrids, confirming the potential of hybrid breeding in pigeonpea. By using genome-wide prediction and association mapping approaches, we identified 129 single nucleotide polymorphisms and 52 copy number variations with significant heterotic effects and also established a high-yielding heterotic pattern in pigeonpea. In summary, our study highlights the role of WGRS data in the study and use of heterosis in crops where hybrid breeding is expected to boost selection gain in order to ensure global food security

SNP marker polymorphism status across cultivated and wild <i>Cajanus</i> accessions.

<p>SNP marker polymorphism status across cultivated and wild <i>Cajanus</i> accessions.</p

Geographical distribution of the collection sites for cultivated and wild <i>Cajanus</i> accessions.

<p>Geographical distribution of the collection sites for cultivated and wild <i>Cajanus</i> accessions.</p

Polymorphism information content (PIC) value range of 1,616 PKAM screened over 184 <i>Cajanus</i> accessions.

<p>Polymorphism information content (PIC) value range of 1,616 PKAM screened over 184 <i>Cajanus</i> accessions.</p

Population analysis of <i>Cajanus</i> accessions present in Indian regions and provinces

<p><i>a)</i> Principal coordinates analysis of domesticated pigeonpea and wild relatives in 11 defined zones <i>b)</i> Analysis of molecular variance (AMOVA) in 11 defined zones <i>c)</i> Structure results across gene pools at the province scale</p

Diversity in three different gene pools (GP) of pigeonpea germplasm.

<p>Na  =  No. of Different Alleles, Ne  =  No. of Effective Alleles  =  1 / (Sum pî2), I  =  Shannon's Information Index  =  −1* Sum (pi * Ln (pi)), Ho  =  Observed Heterozygosity  =  No. of Hets / N, He  =  Expected Heterozygosity  =  1 - Sum pî2, UHe  =  Unbiased Expected Heterozygosity  =  (2N / (2N-1)) * He, F  =  Fixation Index  =  (He − Ho) / He  =  1 − (Ho / He) (Where pi is the frequency of the ith allele for the population & Sum pî2 is the sum of the squared population allele frequencies), %P =  percent of loci polymorphic.</p