Draft genome sequence in pigeonpea offers unprecedented opportunities for genomics assisted crop improvement via enabling access to genome-wide genetic markers. In the present study, 421 hypervariable simple sequence repeat (SSR) markers from the pigeonpea genome were screened on a panel of eight pigeonpea genotypes yielding marker validation and polymorphism percentages of 95.24 and 54.11%, respectively. The SSR marker assay uncovered a total of 570 alleles with three as an average number of alleles per marker. Similarly, the mean values for gene diversity and PIC were 0.44 and 0.37, respectively. The number of polymorphic markers ranged from 39 to 89 for different parental combinations. Further, 60 of these SSRs were assayed on 94 genotypes, and model based clustering using STRUCTURE resulted in the identification of the two subpopulations (K = 2). This remained in close agreement with the clustering patterns inferred from genetic distance (GD)-based approaches i.e., dendrogram, factorial and principal coordinate analysis (PCoA). The AMOVA accounted majority of the genetic variation within groups (89%) in comparison to the variation existing between the groups (11%). A subset of these markers was implicated for hybrid purity testing. We also demonstrated utility of these SSR markers in trait mapping through association and bi-parental linkage analyses. The general linear (GLM) and mixed linear (MLM) models both detected a single SSR marker (CcGM03681) with R2 = 16.4 as associated with the resistance to Fusarium wilt variant 2. Similarly, by using SSR data in a segregating backcross population, the corresponding restorer-of-fertility (Rf) locus was putatively mapped at 39 cM with the marker CcGM08896. However, The marker-trait associations (MTAs) detected here represent a very preliminary type and hence demand deeper investigations for conclusive evidence. Given their ability to reveal polymorphism in simple agarose gels, the hypervariable SSRs are valuable genomic resource for pigeonpea research community, particularly in South Asia and East Africa where pigeonpea is primarily grown.

ABSTRACT. Current DNA extraction protocols, which require liquid nitrogen, lyophilization and considerable infrastructure in terms of instrumentation, often impede the application of biotechnological tools in less researched crops in laboratories in developing countries. We modified and optimized the existing CTAB method for plant genomic DNA extraction by avoiding liquid nitrogen usage and lyophilization. DNA was extracted directly from freshly harvested leaves ground in pre-heated CTAB buffer. Chloroform:isoamyl alcohol (24:1) and RNase treatments followed by single-purification step decontaminated the samples thereby paving way for selective extraction of DNA. High molecular weight DNA yield in the range of 328 to 4776 ng/µL with an average of 1459 ng/µL was obtained from 45 samples of cultivated and wild Cajanus species. With an absorbance ratio at 260 to 280 nm, a range of 1.66 to 2.20, and a mean of 1.85, very low levels of protein and polysaccharide contamination were recorded. Forty samples can be extracted daily at a cost between 1.8 and US$2.0 per plant sample. This modified method is suitable for most plants especially members of the Leguminosae. Apart from Cajanus, it has been extensively applied in DNA extraction from Cicer and Vigna species.

The simple sequence repeat (SSR) survey of ‘Tunisia’ genome (296.85 Mb) identified a total of 365,279 perfect SSRs spanning eight chromosomes, with a mean marker density of 1,230.6 SSRs/Mb. We found a positive trend in chromosome length and the SSR abundance as marker density enhanced with a shorter chromosome length. The highest number of SSRs (60,708) was mined from chromosome 1 (55.56 Mb), whereas the highest marker density (1,294.62 SSRs/Mb) was recorded for the shortest chromosome 8 (27.99 Mb). Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. Across the eight chromosomes, the class III had maximum number of SSR motifs (301,684, 82.59%), followed by the class II (31,056, 8.50%) and the class I (5,003, 1.37%). Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides, and these results are consistent with ‘Tunisia’ genome features as a whole. Concerning major repeat types, AT/AG was the most frequent (14.16%), followed by AAAAAT/AAAAAG (7.89%), A/C (7.54%), AAT/AAG (5.23%), AAAT/AAAG (4.37%), and AAAAT/AAAAG (1.2%) types. We designed and validated a total of 3,839 class I SSRs in the ‘Tunisia’ genome through electronic polymerase chain reaction (ePCR) and found 1,165 (30.34%) SSRs producing a single amplicon. Then, we selected 906 highly variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across multiple draft genomes of pomegranate, which provided us a subset of 265 highly polymorphic SSRs. Of these, 235 primers were validated on six pomegranate genotypes through wet-lab experiment. We found 221 (94%) polymorphic SSRs on six genotypes, and 187 of these SSRs had ≥ 0.5 PIC values. The utility of the developed SSRs was demonstrated by analyzing genetic diversity of 30 pomegranate genotypes using 16 HvSSRs spanning eight pomegranate chromosomes. In summary, we developed a comprehensive set of highly polymorphic genome-wide SSRs. These chromosome-specific SSRs will serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate.

No abstract

Pigeonpea production is severely constrained by wilt disease caused by Fusarium udum. In the current study, we discover the putative genomic regions that control resistance response to variant 2 of fusarium wilt using association mapping approach. The association panel comprised of 89 diverse pigeonpea genotypes including seven varieties, three landraces and 79 germplasm lines. The panel was screened rigorously for 3 consecutive years (2013-14, 2014-15 and 2015-2016) against variant 2 in a wilt-sick field. A total of 65 pigeonpea specific hypervariable SSR markers (HASSRs) were screened representing seven linkage groups and 29 scaffolds of the pigeonpea genome. A total of 181 alleles were detected, with average values of gene diversity and polymorphism information content (PIC) of 0.55 and 0.47, respectively. Further analysis using model based (STRUCTURE) and distance based (clustering) approaches separated the entire pigeonpea collection into two distinct subgroups (K = 2). The marker trait associations (MTAs) were established based on three-year wilt incidence data and SSR dataset using a unified mixed linear model. Consequently, six SSR markers were identified, which were significantly associated with wilt resistance and explained up to 6% phenotypic variance (PV) across the years. Among these SSRs, HASSR18 was found to be the most stable and significant, accounting for 5-6% PV across the years. To the best of our knowledge, this is the first report of identification of favourable alleles for resistance to variant 2 of Fusarium udum in pigeonpea using association mapping. The SSR markers identified here will greatly facilitate marker assisted resistance breeding against fusarium wilt in pigeonpea.