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Background Pigeonpea has considerable extent of insect-aided natural out-crossing that impedes genetic purity of seeds. Pre-anthesis cleistogamy in pigeonpea promotes self-pollination which helps in maintaining genetic purity. The cleistogamous flowers are linked with shriveled seeds, an undesirable trait from variety adoption point of view, and breeding using genomics tools can help in overcoming this constraint. Therefore, in order to identify genomic regions governing these target traits, one recombinant inbred line (RIL) population was developed using contrasting parents (ICPL 99010 and ICP 5529) for flower shape and shriveled seeds. The RILs were phenotyped for two years and genotyped using the Axiom Cajanus SNP Array. Results Out of the 56,512 unique sequence variations on the array, the mapping population showed 8634 single nucleotide polymorphism (SNPs) segregating across the genome. These data facilitated generation of a high density genetic map covering 6818 SNPs in 974 cM with an average inter-marker distance of 0.1 cM, which is the lowest amongst all pigeonpea genetic maps reported. Quantitative trait loci (QTL) analysis using this genetic map and phenotyping data identified 5 QTLs associated with cleistogamous flower, 3 QTLs for shriveled seed and 1 QTL for seed size. The phenotypic variance explained by these QTLs ranged from 9.1 to 50.6%. A consistent QTL “qCl3.2” was identified for cleistogamous flower on CcLG03 covering a span of 42 kb in the pigeonpea genome. Epistatic QTLs were also identified for cleistogamous flower and shriveled seed traits. Conclusion Identified QTLs and genomic interactions for cleistogamous flower, shriveled seed and seed size will help in incorporating the required floral architecture in pigeonpea varieties/lines. Besides, it will also be useful in understanding the molecular mechanisms, and map-based gene cloning for the target traits.

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

Fusarium wilt (FW) is one of the most important biotic stresses causing yield losses in pigeonpea. Genetic improvement of pigeonpea through genomics-assisted breeding (GAB) is an economically feasible option for the development of high yielding FW resistant genotypes. In this context, two recombinant inbred lines (RILs) (ICPB 2049 × ICPL 99050 designated as PRIL_A and ICPL 20096 × ICPL 332 designated as PRIL_B) and one F2 (ICPL 85063 × ICPL 87119) populations were used for the development of high density genetic maps. Genotyping-by-sequencing (GBS) approach was used to identify and genotype SNPs in three mapping populations. As a result, three high density genetic maps with 964, 1101 and 557 SNPs with an average marker distance of 1.16, 0.84 and 2.60 cM were developed in PRIL_A, PRIL_B and F2, respectively. Based on the multi-location and multi-year phenotypic data of FW resistance a total of 14 quantitative trait loci (QTLs) including six major QTLs explaining >10% phenotypic variance explained (PVE) were identified. Comparative analysis across the populations has revealed three important QTLs (qFW11.1, qFW11.2 and qFW11.3) with upto 56.45% PVE for FW resistance. This is the first report of QTL mapping for FW resistance in pigeonpea and identified genomic region could be utilized in GAB.

As one of the major outputs of next-generation sequencing (NGS), a large number of genome-wide single-nucleotide polymorphisms (SNPs) have been developed in pigeonpea [Cajanus cajan (L.) Huth.]. However, SNPs require a genotyping platform or assay to be used in different evolutionary studies or in crop improvement programs. Therefore, we developed an Axiom Cajanus SNP array with 56K SNPs uniformly distributed across the genome and assessed its utility in a genetic diversity study. From the whole-genome resequencing (WGRS) data on 104 pigeonpea lines, ∼2 million sequence variations (SNPs and insertion–deletions [InDels]) were identified, from which a subset of 56,512 unique and informative sequence variations were selected to develop the array. The Axiom Cajanus SNP array developed was used for genotyping 103 pigeonpea lines encompassing 63 cultivars released between 1960 and 2014 and 40 breeding, germplasm, and founder lines. Genotyping data thus generated on 103 pigeonpea lines provided 51,201 polymorphic SNPs and InDels. Genetic diversity analysis provided in-depth insights into the genetic architecture and trends in temporal diversity in pigeonpea cultivars. Therefore, the continuous use of the high-density Axiom Cajanus SNP array developed will accelerate high-resolution trait mapping, marker-assisted breeding, and genomic selection efforts in pigeonpea.

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 prebreeding 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.

With the exception of Cajanus cajan (L.) Millspaugh (pigeonpea), the remaining species of genus Cajanus have not been domesticated. For millennia these taxa have persisted in natural habitats through self-sown seeds. These wild species are an asset for sustaining future pigeonpea breeding programmes since they contain certain traits (genes) that are necessary for encountering various breeding challenges related to crop improvement and adaptation. In this review we identify the key traits from wild Cajanus species, and discuss various physical and genetic constraints encountered in their utilization in introgression breeding. Some noteworthy achievements recorded from inter-specific breeding programmes in pigeonpea are also discussed. These include the development of (1) high protein (> 28%) genotypes (2) cytoplasmic nuclear male sterility systems (3) highly (> 95%) self-pollinating genotypes, and (4) resistance sources to sterility mosaic disease, nematodes, salinity, photo-insensitivity, pod borers, podfly, bruchids, and Phytophthora blight. To help pigeonpea breeders engaged in inter-specific breeding programmes, we suggest the division of the secondary gene pool germplasm into two sub-group/tiers on the basis of ease in hybridization.

Pigeonpea (Cajanus cajan) is one of the most important legume crops grown in arid and semi-arid regions of the world. It is characterized with few unique features compared with other legume species, such as Lotus, Medicago, and Glycine. One of them is growth habit, an important agronomic trait. In the present study, identification of mutations affecting growth habit accompanied by a precise analysis of phenotype has been done which will shed more light upon developmental regulation in pigeonpea. A genetic study was conducted to examine the inheritance of growth habit and a genotyping by sequencing (GBS)-based genetic map constructed using F2 mapping population derived from crossing parents ICP 5529 and ICP 11605. Inheritance studies clearly demonstrated the dominance of indeterminate (IDT) growth habit over determinate (DT) growth habit in F2 and F2:3 progenies. A total of 787 SNP markers were mapped in the genetic map of 1454 cM map length. Growth habit locus (Dt1) was mapped on the CcLG03 contributing more than 61% of total phenotypic variations. Subsequently, QTL analysis highlighted one gene, CcTFL1, as a candidate for determinacy in pigeonpea, since an Indel marker derived from this gene co-segregated with the Dt1 locus. Ability of this Indel-derived marker to differentiate DT/IDT lines was also validated on 262 pigeonpea lines. This study clearly demonstrated that CcTFL1 is a candidate gene for growth habit in pigeonpea and a user-friendly marker was developed in the present study which will allow low-cost genotyping without need of automation.

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.

Key messageWe 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.

The genetic architecture of seed protein content (SPC) and its relationships to agronomic traits in pigeonpea is poorly understood. Accordingly, five F2 populations segregating for SPC and four agronomic traits (seed weight (SW), seed yield (SY), growth habit (GH) and days to first flowering (DFF)) were phenotyped and genotyped using genotyping-by-sequencing approach. Five high-density population-specific genetic maps were constructed with an average inter-marker distance of 1.6 to 3.5 cM, and subsequently, integrated into a consensus map with average marker spacing of 1.6 cM. Based on analysis of phenotyping data and genotyping data, 192 main effect QTLs (M-QTLs) with phenotypic variation explained (PVE) of 0.7 to 91.3% were detected for the five traits across the five populations. Major effect (PVE ≥ 10%) M-QTLs included 14 M-QTLs for SPC, 16 M-QTLs for SW, 17 M-QTLs for SY, 19 M-QTLs for GH and 24 M-QTLs for DFF. Also, 573 epistatic QTLs (E-QTLs) were detected with PVE ranging from 6.3 to 99.4% across traits and populations. Colocalization of M-QTLs and E-QTLs explained the genetic basis of the significant (P < 0.05) correlations of SPC with SW, SY, DFF and GH. The nature of genetic architecture of SPC and its relationship with agronomic traits suggest that genomics-assisted breeding targeting genome-wide variations would be effective for the simultaneous improvement of SPC and other important traits.