Identification of microRNAs and their gene targets in cytoplasmic male sterile and fertile maintainer lines of pigeonpea.

MAIN CONCLUSION: Comparative analysis of genome-wide miRNAs and their gene targets between cytoplasmic male sterile (CMS) and fertile lines of pigeonpea suggests a possible role of miRNA-regulated pathways in reproductive development. Exploitation of hybrid vigor using CMS technology has delivered nearly 50% yield gain in pigeonpea. Among various sterility-inducing cytoplasms (A1-A9) reported so far in pigeonpea, A2 and A4 are the two major sources that facilitate hybrid seed production. Recent evidence suggests involvement of micro RNA in vast array of biological processes including plant reproductive development. In pigeonpea, information about the miRNAs is insufficient. In view of this, we sequenced six small RNA libraries of CMS line UPAS 120A and isogenic fertile line UPAS 120B using Illumina technology. Results revealed 316 miRNAs including 248 known and 68 novel types. A total of 637 gene targets were predicted for known miRNAs, while 324 genes were associated with novel miRNAs. Degradome analysis revealed 77 gene targets of predicted miRNAs, which included a variety of transcription factors playing key roles in plant reproduction such as F-box family proteins, apetala 2, auxin response factors, ethylene-responsive factors, homeodomain-leucine zipper proteins etc. Differential expression of both known and novel miRNAs implied roles for both conserved as well as species-specific players. We also obtained several miRNA families such as miR156, miR159, miR167 that are known to influence crucial aspects of plant fertility. Gene ontology and pathway level analyses of the target genes showed their possible implications for crucial events during male reproductive development such as tapetal degeneration, pollen wall formation, retrograde signaling etc. To the best of our knowledge, present study is first to combine deep sequencing of small RNA and degradome for elucidating the role of miRNAs in flower and male reproductive development in pigeonpea.

A diagnostic marker kit for Fusarium wilt and sterility mosaic diseases resistance in pigeonpea.

Fusarium wilt (FW) and sterility mosaic diseases (SMD) are key biotic constraints to pigeonpea production. Occurrence of these two diseases in congenial conditions is reported to cause complete yield loss in susceptible pigeonpea cultivars. Various studies to elucidate genomic architecture of the two traits have revealed significant marker-trait associations for use in breeding programs. However, these DNA markers could not be used effectively in genomics-assisted breeding for developing FW and SMD resistant varieties primarily due to pathogen variability, location or background specificity, lesser phenotypic variance explained by the reported QTL and cost-inefficiency of the genotyping assays. Therefore, in the present study, a novel approach has been used to develop a diagnostic kit for identification of suitable FW and SMD resistant lines. This kit was developed with 10 markers each for FW and SMD resistance. Investigation of the diversity of these loci has shown the role of different alleles in different resistant genotypes. Two genes (C.cajan_03691 and C.cajan_18888) for FW resistance and four genes (C.cajan_07858, C.cajan_20995, C.cajan_21801 and C.cajan_17341) for SMD resistance have been identified. More importantly, we developed a customized and cost-effective Kompetitive allele-specific PCR genotyping assay for the identified genes in order to encourage their downstream applications in pigeonpea breeding programs. The diagnostic marker kit developed here will offer great strength to pigeonpea varietal development program, since the resistance against these two diseases is essentially required for nominating an improved line in varietal release pipeline.

Mapping QTL for important seed traits in an interspecific F2 population of pigeonpea.

Seed traits present important breeding targets for enhancing grain yield and quality in various grain legume crops including pigeonpea. The present study reports significant genetic variation for six seed traits including seed length (SL), seed width (SW), seed thickness (ST), seed weight (SWT), electrical conductivity (EC) and water uptake (WU) among Cajanus cajan (L.) Millspaugh acc. ICPL 20340 and Cajanus scarabaeoides (L.) Thouars acc. ICP 15739 and an F2 population derived from this interspecific cross. Maximum phenotypic values recorded for the F2 population were higher than observed in the parent ICPL 20340 [F2 max vs ICPL 20340: SW (7.05 vs 5.38), ST (4.63 vs 4.51), EC (65.17 vs 9.72), WU (213.17 vs 109.5)], which suggested contribution of positive alleles from the wild parent, ICP 15739. Concurrently, to identify the QTL controlling these seed traits, we assayed two parents and 94 F2 individuals with 113 polymorphic simple sequence repeat (SSR) markers. In the F2 population, 98 of the 113 SSRs showed Mendelian segregation ratio 1:2:1, whereas significant deviations were observed for 15 SSRs with their χ 2 values ranging between 6.26 and 20.62. A partial genetic linkage map comprising 83 SSR loci was constructed. QTL analysis identified 15 marker-trait associations (MTAs) for seed traits on four linkage groups i.e. LG01, LG02, LG04 and LG05. Phenotypic variations (PVs) explained by these QTL ranged from 4.4 (WU) to 19.91% (EC). These genomic regions contributing significantly towards observed variability of seed traits would serve as potential candidates for future research that aims to improve seed traits in pigeonpea.