Utilizing Wild Cajanus platycarpus, a Tertiary Genepool Species for Enriching Variability in the Primary Genepool for Pigeonpea Improvement.

The use of crop wild relatives in the breeding program has been well recognized to diversify the genetic base along with introgression of useful traits. Cajanus platycarpus (Benth.) Maesen, an annual wild relative belonging to the tertiary genepool of pigeonpea, possesses many useful traits such as early maturity, high protein content, photoperiod insensitivity, and pod borer tolerance for the genetic improvement of cultivated pigeonpea. Using this cross incompatible wild Cajanus species, an advanced backcross population was developed following the embryo rescue technique. In the present study, a pre-breeding population consisting of 136 introgression lines (ILs) along with five popular varieties (used as checks) was evaluated for important agronomic traits during 2016 and 2017 rainy seasons and for grain nutrient content during 2016, 2017, and 2018 rainy seasons. Large genetic variation was observed for agronomic traits such as days to 50% flowering, number of pods per plant, pod weight per plant, grain yield per plant, and grain nutrients [protein content, grain iron (Fe), zinc (Zn), calcium (Ca), and magnesium (Mg)] in the pre-breeding population. Significant genotype × environment interaction was also observed for agronomic traits as well as grain nutrients indicating the sensitivity of these traits to the environments. No significant correlations were observed between grain yield and grain nutrients except grain Zn content which was negatively correlated with grain yield. Overall, 28 promising high-yielding ILs with high grain nutrient content were identified. These ILs, in particular, ICPP # 171012, 171004, 171102, 171087, 171006, and 171050 flowered significantly earlier than the popular mega variety, ICPL 87119 (Asha) and thus hold potential in developing new short-duration cultivars. The comprehensive multi-site assessment of these high-yielding, nutrient-rich accessions would be useful in identifying region-specific promising lines for direct release as cultivars. Moreover, these ILs are expected to replace the popular existing cultivars or for use as new and diverse sources of variations in hybridization programs for pigeonpea improvement.

Genome-wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea.

Hybrids are extensively used in agriculture to deliver an increase in yield, yet the molecular basis of heterosis is not well understood. Global DNA methylation analysis, transcriptome analysis and small RNA profiling were aimed to understand the epigenetic effect of the changes in gene expression level in the two hybrids and their parental lines. Increased DNA methylation was observed in both the hybrids as compared to their parents. This increased DNA methylation in hybrids showed that majority of the 24-nt siRNA clusters had higher expression in hybrids than the parents. Transcriptome analysis revealed that various phytohormones (auxin and salicylic acid) responsive hybrid-MPV DEGs were significantly altered in both the hybrids in comparison to MPV. DEGs associated with plant immunity and growth were overexpressed whereas DEGs associated with basal defence level were repressed. This antagonistic patterns of gene expression might contribute to the greater growth of the hybrids. It was also noticed that some common as well as unique changes in the regulatory pathways were associated with heterotic growth in both the hybrids. Approximately 70% and 67% of down-regulated hybrid-MPV DEGs were found to be differentially methylated in ICPH 2671 and ICPH 2740 hybrid, respectively. This reflected the association of epigenetic regulation in altered gene expressions. Our findings also revealed that miRNAs might play important roles in hybrid vigour in both the hybrids by regulating their target genes, especially in controlling plant growth and development, defence and stress response pathways. The above finding provides an insight into the molecular mechanism of pigeonpea heterosis.

Molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea (Cajanus cajan (L.) Millsp.).

KEY MESSAGE: We report molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea. We have also developed PCR-based markers amenable to low-cost genotyping to identify fertility restorer lines. Commercial hybrids in pigeonpea are based on A4 cytoplasmic male sterility (CMS) system, and their fertility restoration is one of the key prerequisites for breeding. In this context, an effort has been made to understand the genetics and identify quantitative trait loci (QTL) associated with restoration of fertility (Rf). One F2 population was developed by crossing CMS line (ICPA 2039) with fertility restorer line (ICPL 87119). Genetic analysis has shown involvement of two dominant genes in regulation of restoration of fertility. In parallel, the genotyping-by-sequencing (GBS) approach has generated ~ 33 Gb data on the F2 population. GBS data have provided 2457 single nucleotide polymorphism (SNPs) segregating across the mapping population. Based on these genotyping data, a genetic map has been developed with 306 SNPs covering a total length 981.9 cM. Further QTL analysis has provided the region flanked by S8_7664779 and S8_6474381 on CcLG08 harboured major QTL explained up to 28.5% phenotypic variation. Subsequently, sequence information within the major QTLs was compared between the maintainer and the restorer lines. From this sequence information, we have developed two PCR-based markers for identification of restorer lines from non-restorer lines and validated them on parental lines of hybrids as well as on another F2 mapping population. The results obtained in this study are expected to enhance the efficiency of selection for the identification of restorer lines in hybrid breeding and may reduce traditional time-consuming phenotyping activities.

Plant growth-promotion and biofortification of chickpea and pigeonpea through inoculation of biocontrol potential bacteria, isolated from organic soils.

Seven strains of bacteria [Pseudomonas plecoglossicida SRI-156, Brevibacterium antiquum SRI-158, Bacillus altitudinis SRI-178, Enterobacter ludwigii SRI-211, E. ludwigii SRI-229, Acinetobacter tandoii SRI-305 and Pseudomonas monteilii SRI-360; demonstrated previously for control of charcoal rot disease in sorghum and plant growth-promotion (PGP) in rice] were evaluated for their PGP and biofortification traits in chickpea and pigeonpea under field conditions. When treated on seed, the seven selected bacteria significantly enhanced the shoot height and root length of both chickpea and pigeonpea over the un-inoculated control. Under field conditions, in both chickpea and pigeonpea, the plots inoculated with test bacteria enhanced the nodule number, nodule weight, root and shoot weights, pod number, pod weight, leaf weight, leaf area and grain yield over the un-inoculated control plots. Among the seven bacteria, SRI-229 was found to significantly and consistently enhance all the studied PGP and yield traits including nodule number (24 and 36%), nodule weight (11 and 44%), shoot weight (22 and 20%), root weight (23 and 16%) and grain yield (19 and 26%) for both chickpea and pigeonpea, respectively. When the harvested grains were evaluated for their mineral contents, iron (up to 18 and 12%), zinc (up to 23 and 5%), copper (up to 19 and 8%), manganese (up to 2 and 39%) and calcium (up to 22 and 11%) contents in chickpea and pigeonpea, respectively, were found enhanced in test bacteria inoculated plots over the un-inoculated control plots. This study further confirms that the selected bacterial isolates not only have the potential for PGP in cereals and legumes but also have the potential for biofortification of mineral nutrients.

Association of nad7a Gene with Cytoplasmic Male Sterility in Pigeonpea.

Cytoplasmic male sterility (CMS) has been exploited in the commercial pigeonpea [Cajanus cajan (L.) Millsp.] hybrid breeding system; however, the molecular mechanism behind this system is unknown. To understand the underlying molecular mechanism involved in A4 CMS system derived from C. cajanifolius (Haines) Maesen, 34 mitochondrial genes were analyzed for expression profiling and structural variation analysis between CMS line (ICRISAT Pigeonpea A line, ICPA 2039) and its cognate maintainer (ICPB 2039). Expression profiling of 34 mitochondrial genes revealed nine genes with significant fold differential gene expression at P ≤ 0.01, including one gene, nad4L, with 1366-fold higher expression in CMS line as compared with the maintainer. Structural variation analysis of these mitochondrial genes identified length variation between ICPA 2039 and ICPB 2039 for nad7a (subunit of nad7 gene). Sanger sequencing of nad4L and nad7a genes in the CMS and the maintainer lines identified two single nucleotide polymorphisms (SNPs) in upstream region of nad4L and a deletion of 10 bp in nad7a in the CMS line. Protein structure evaluation showed conformational changes in predicted protein structures for nad7a between ICPA 2039 and ICPB 2039 lines. All above analyses indicate association of nad7a gene with the CMS for A4 cytoplasm in pigeonpea. Additionally, one polymerase chain reaction (PCR) based Indel marker (nad7a_del) has been developed and validated for testing genetic purity of A4 derived CMS lines to strengthen the commercial hybrid breeding program in pigeonpea.