Celebrating Professor Rajeev K. Varshney's transformative research odyssey from genomics to the field on his induction as Fellow of the Royal Society.

Professor Rajeev K. Varshney's transformative impact on crop genomics, genetics, and agriculture is the result of his passion, dedication, and unyielding commitment to harnessing the potential of genomics to address the most pressing challenges faced by the global agricultural community. Starting from a small town in India and reaching the global stage, Professor Varshney's academic and professional trajectory has inspired many scientists active in research today. His ground-breaking work, especially his effort to list orphan tropical crops to genomic resource-rich entities, has been transformative. Beyond his scientific achievements, Professor Varshney is recognized by his colleagues as an exemplary mentor, fostering the growth of future researchers, building institutional capacity, and strengthening scientific capability. His focus on translational genomics and strengthening seed system in developing countries for the improvement of agriculture has made a tangible impact on farmers' lives. His skills have been best utilized in roles at leading research centres where he has applied his expertise to deliver a new vision for crop improvement. These efforts have now been recognized by the Royal Society with the award of the Fellowship (FRS). As we mark this significant milestone in his career, we not only celebrate Professor Varshney's accomplishments but also his wider contributions that continue to transform the agricultural landscape.

Chromosome-length genome assemblies of six legume species provide insights into genome organization, evolution, and agronomic traits for crop improvement.

INTRODUCTION: Legume crops are an important source of protein and oil for human health and in fixing atmospheric N2 for soil enrichment. With an objective to accelerate much-needed genetic analyses and breeding applications, draft genome assemblies were generated in several legume crops; many of them are not high quality because they are mainly based on short reads. However, the superior quality of genome assembly is crucial for a detailed understanding of genomic architecture, genome evolution, and crop improvement. OBJECTIVES: Present study was undertaken with an objective of developing improved chromosome-length genome assemblies in six different legumes followed by their systematic investigation to unravel different aspects of genome organization and legume evolution. METHODS: We employed in situ Hi-C data to improve the existing draft genomes and performed different evolutionary and comparative analyses using improved genome assemblies. RESULTS: We have developed chromosome-length genome assemblies in chickpea, pigeonpea, soybean, subterranean clover, and two wild progenitor species of cultivated groundnut (A. duranensis and A. ipaensis). A comprehensive comparative analysis of these genome assemblies offered improved insights into various evolutionary events that shaped the present-day legume species. We highlighted the expansion of gene families contributing to unique traits such as nodulation in legumes, gravitropism in groundnut, and oil biosynthesis in oilseed legume crops such as groundnut and soybean. As examples, we have demonstrated the utility of improved genome assemblies for enhancing the resolution of "QTL-hotspot" identification for drought tolerance in chickpea and marker-trait associations for agronomic traits in pigeonpea through genome-wide association study. Genomic resources developed in this study are publicly available through an online repository, 'Legumepedia'. CONCLUSION: This study reports chromosome-length genome assemblies of six legume species and demonstrates the utility of these assemblies in crop improvement. The genomic resources developed here will have significant role in accelerating genetic improvement applications of legume crops.

Improved pea reference genome and pan-genome highlight genomic features and evolutionary characteristics.

Complete and accurate reference genomes and annotations provide fundamental resources for functional genomics and crop breeding. Here we report a de novo assembly and annotation of a pea cultivar ZW6 with contig N50 of 8.98 Mb, which features a 243-fold increase in contig length and evident improvements in the continuity and quality of sequence in complex repeat regions compared with the existing one. Genome diversity of 118 cultivated and wild pea demonstrated that Pisum abyssinicum is a separate species different from P. fulvum and P. sativum within Pisum. Quantitative trait locus analyses uncovered two known Mendel's genes related to stem length (Le/le) and seed shape (R/r) as well as some candidate genes for pod form studied by Mendel. A pan-genome of 116 pea accessions was constructed, and pan-genes preferred in P. abyssinicum and P. fulvum showed distinct functional enrichment, indicating the potential value of them as pea breeding resources in the future.

Soybean F-Box-Like Protein GmFBL144 Interacts With Small Heat Shock Protein and Negatively Regulates Plant Drought Stress Tolerance.

The F-box gene family is one of the largest gene families in plants. These genes regulate plant growth and development, as well as biotic and abiotic stress responses, and they have been extensively researched. Drought stress is one of the major factors limiting the yield and quality of soybean. In this study, bioinformatics analysis of the soybean F-box gene family was performed, and the role of soybean F-box-like gene GmFBL144 in drought stress adaptation was characterized. We identified 507 F-box genes in the soybean genome database, which were classified into 11 subfamilies. The expression profiles showed that GmFBL144 was highly expressed in plant roots. Overexpression of GmFBL144 increased the sensitivity of transgenic Arabidopsis to drought stress. Under drought stress, the hydrogen peroxide (H2O2) and malonaldehyde (MDA) contents of transgenic Arabidopsis were higher than those of the wild type (WT) and empty vector control, and the chlorophyll content was lower than that of the control. Y2H and bimolecular fluorescence complementation (BiFC) assays showed that GmFBL144 can interact with GmsHSP. Furthermore, our results showed that GmFBL144 can form SCF FBL144 (E3 ubiquitin ligase) with GmSkp1 and GmCullin1. Altogether, these results indicate that the soybean F-box-like protein GmFBL144 may negatively regulate plant drought stress tolerance by interacting with sHSP. These findings provide a basis for molecular genetics and breeding of soybean.

Tonoplast inositol transporters: Roles in plant abiotic stress response and crosstalk with other signals.

Inositol transporters (INT) are thought to be the pivotal transporters for vital metabolites, in particular lipids, minerals, and sugars. These transporters play an important role in transitional metabolism and various signaling pathways in plants through regulating the transduction of messages from hormones, neurotransmitters, and immunologic and growth factors. Extensive studies have been conducted on animal INT, with promising outcomes. However, only few recent studies have highlighted the importance and complexity of INT genes in the regulation of plant physiology stages, including growth and tolerance to stress conditions. The present review summarizes the most recent findings concerning the role of INT or inositol genes in plant metabolism and the response mechanisms triggered by external stressors. Moreover, we highlight the emerging role of vacuoles and vacuolar INT in plant molecular transition and their related roles in plant growth and development. INTs are the essential mediators of inositol uptake and its intracellular broadcasting for various metabolic pathways where they play crucial roles. Additionally, we report evidence on Na+/inositol transporters, which until now have only been characterized in animals, as well as H+/inositol symporters and their kinetic functions and physiological role and suggest their roles and operating mode in plants. A more comprehensive understanding of the INT functioning system, in particular the coordinated movement of inositol and the relation between inositol generation and other important plant signaling pathways, would greatly advance the study of plant stress adaptation.

QTL-seq for the identification of candidate genes for days to flowering and leaf shape in pigeonpea.

To identify genomic segments associated with days to flowering (DF) and leaf shape in pigeonpea, QTL-seq approach has been used in the present study. Genome-wide SNP profiling of extreme phenotypic bulks was conducted for both the traits from the segregating population (F2) derived from the cross combination- ICP 5529 × ICP 11605. A total of 126.63 million paired-end (PE) whole-genome resequencing data were generated for five samples, including one parent ICP 5529 (obcordate leaf and late-flowering plant), early and late flowering pools (EF and LF) and obcordate and lanceolate leaf shape pools (OLF and LLS). The QTL-seq identified two significant genomic regions, one on CcLG03 (1.58 Mb region spanned from 19.22 to 20.80 Mb interval) for days to flowering (LF and EF pools) and another on CcLG08 (2.19 Mb region spanned from 6.69 to 8.88 Mb interval) for OLF and LLF pools, respectively. Analysis of genomic regions associated SNPs with days to flowering and leaf shape revealed 5 genic SNPs present in the unique regions. The identified genomic regions for days to flowering were also validated with the genotyping-by-sequencing based classical QTL mapping method. A comparative analysis of the identified seven genes associated with days to flowering on 12 Fabaceae genomes, showed synteny with 9 genomes. A total of 153 genes were identified through the synteny analysis ranging from 13 to 36. This study demonstrates the usefulness of QTL-seq approach in precise identification of candidate gene(s) for days to flowering and leaf shape which can be deployed for pigeonpea improvement.

Global gene expression analysis of pigeonpea with male sterility conditioned by A2 cytoplasm.

Cytoplasmic male sterility(CMS), a maternally inherited trait, provides a promising means to harness yield gains associated with hybrid vigor. In pigeonpea [Cajanus cajan (L.) Huth], nine types of sterility-inducing cytoplasm have been reported, of which A2 and A4 have been successfully deployed in hybrid breeding. Unfortunately, molecular mechanism of the CMS trait is poorly understood because of limited research invested. More recently, an association between a mitochondrial gene (nad7) and A4 -CMS has been demonstrated in pigeonpea; however, the mechanism underlying A2 -CMS still remains obscure. The current investigation aimed to analyze the differences in A2 -CMS line (ICPL 88039A) and its isogenic maintainer line (ICPL 88039B) at transcriptome level using next-generation sequencing. Gene expression profiling uncovered a set of 505 genes that showed altered expression in response to CMS, of which, 412 genes were upregulated while 93 were downregulated in the fertile maintainer line vs. the CMS line. Further, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) network analyses revealed association of CMS in pigeonpea with four major pathways: glucose and lipid metabolism, ATP production, pollen development and pollen tube growth, and reactive oxygen species (ROS) scavenging. Patterns of digital gene expression were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) of six candidate genes. This study elucidates candidate genes and metabolic pathways having potential associations with pollen development and male sterility in pigeonpea A2 -CMS. New insights on molecular mechanism of CMS trait in pigeonpea will be helpful to accelerate heterosis utilization for enhancing productivity gains in pigeonpea.

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.

Genome-wide comparative transcriptome analysis of the A4-CMS line ICPA 2043 and its maintainer ICPB 2043 during the floral bud development of pigeonpea.

Cytoplasmic male sterility (CMS) offers a unique system to understand cytoplasmic nuclear crosstalk, and is also employed for exploitation of hybrid vigor in various crops. Pigeonpea A4-CMS, a predominant source of male sterility, is being used for efficient hybrid seed production. The molecular mechanisms of CMS trait remain poorly studied in pigeonpea. We performed genome-wide transcriptome profiling of A4-CMS line ICPA 2043 and its isogenic maintainer ICPB 2043 at two different stages of floral bud development (stage S1 and stage S2). Consistent with the evidences from some other crops, we also observed significant difference in the expression levels of genes in the later stage, i.e., stage S2. Differential expression was observed for 143 and 55 genes within the two stages of ICPA 2043 and ICPB 2043, respectively. We obtained only 10 differentially expressed genes (DEGs) between the stage S1 of the two genotypes, whereas expression change was significant for 582 genes in the case of stage S2. The qRT-PCR assay of randomly selected six genes supported the differential expression of genes between ICPA 2043 and ICPB 2043. Further, GO and KEGG pathway mapping suggested a possible compromise in key bioprocesses during flower and pollen development. Besides providing novel insights into the functional genomics of CMS trait, our results were in strong agreement with the gene expression atlas of pigeonpea that implicated various candidate genes like sucrose-proton symporter 2 and an uncharacterized protein along with pectate lyase, pectinesterase inhibitors, L-ascorbate oxidase homolog, ATPase, ß-galactosidase, polygalacturonase, and aldose 1-epimerase for pollen development of pigeonpea. The dataset presented here provides a rich genomic resource to improve understanding of CMS trait and its deployment in heterosis breeding in pigeonpea.

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.

Multiomics approach unravels fertility transition in a pigeonpea line for a two-line hybrid system.

Pigeonpea [Cajanus cajan (L.) Millsp.] is a pulse crop cultivated in the semi-arid regions of Asia and Africa. It is a rich source of protein and capable of alleviating malnutrition, improving soil health and the livelihoods of small-holder farmers. Hybrid breeding has provided remarkable improvements for pigeonpea productivity, but owing to a tedious and costly seed production system, an alternative two-line hybrid technology is being explored. In this regard, an environment-sensitive male sterile line has been characterized as a thermosensitive male sterile line in pigeonpea precisely responding to day temperature. The male sterile and fertile anthers from five developmental stages were studied by integrating transcriptomics, proteomics and metabolomics supported by precise phenotyping and scanning electron microscopic study. Spatio-temporal analysis of anther transcriptome and proteome revealed 17 repressed DEGs/DEPs in sterile anthers that play a critical role in normal cell wall morphogenesis and tapetal cell development. The male fertility to sterility transition was mainly due to a perturbation in auxin homeostasis, leading to impaired cell wall modification and sugar transport. Limited nutrient utilization thus leads to microspore starvation in response to moderately elevated day temperature which could be restored with auxin-treatment in the male sterile line. Our findings outline a molecular mechanism that underpins fertility transition responses thereby providing a process-oriented two-line hybrid breeding framework for pigeonpea.

Can pigeonpea hybrids negotiate stresses better than inbred cultivars?

Pigeonpea [Cajanus cajan (L.) Millsp.] is an important rainfed pulse crop of tropics and sub-tropics, and during its long growth cycle of 6-9 months it encounters a number of biotic and abiotic stresses. The recently developed CMS-based pigeonpea hybrids have demonstrated large gains in yield and stability over the traditional inbred cultivars. In this review, the authors argue that the heterosis expressed in traits like seed germination, radicle growth, root biomass production and moisture retention during water stress confers advantages to hybrid plants in negotiating a few abiotic and biotic stresses in much better way than pure line cultivars.

High resolution mapping of restoration of fertility (Rf) by combining large population and high density genetic map in pigeonpea [Cajanus cajan (L.) Millsp].

BACKGROUND: Restoration of fertility (Rf) is an important trait for pigeonpea hybrid breeding. Few coarse quantitative trait locus (QTL) studies conducted in the past identified QTLs with large confidence intervals on the genetic map and could not provide any information on possible genes responsible for Rf in pigeonpea. Therefore, a larger population comprising of 369 F2s derived from ICPA 2039 × ICPL 87119 was genotyped with high density Axiom Cajanus SNP Array with 56 K single nucleotide polymorphism (SNPs) for high resolution mapping of Rf. RESULTS: A genetic map with 4867 markers was developed and a total of four QTLs for Rf were identified. While one major effect QTL (qRf8.1) was co-localized with the QTL identified in two previous studies and its size was refined from 1.2 Mb to 0.41 Mb. Further analysis of qRf8.1 QTL with genome sequence provided 20 genes including two genes namely flowering locus protein T and 2-oxoglutarate/Fe (II)-dependent dioxygenases (2-ODDs) superfamily protein with known function in the restoration of fertility. CONCLUSION: The qRf8.1 QTL and the potential candidate genes present in this QTL will be valuable for genomics-assisted breeding and identification of causal genes/nucleotides for the restoration of fertility in the hybrid breeding program of pigeonpea.

Complete genome sequence of sixteen plant growth promoting Streptomyces strains.

The genome sequences of 16 Streptomyces strains, showing potential for plant growth-promotion (PGP) activities in rice, sorghum, chickpea and pigeonpea, isolated from herbal vermicompost, have been decoded. The genome assemblies of the 16 Streptomyces strains ranged from 6.8 Mb to 8.31 Mb, with a GC content of 72 to 73%. The extent of sequence similarity (in terms of shared ortholog) in 16 Streptomyces strains showed 70 to 85% common genes to the closest publicly available Streptomyces genomes. It was possible to identify ~1,850 molecular functions across these 16 strains, of which close to 50% were conserved across the genomes of Streptomyces strains, whereas, ~10% were strain specific and the rest were present in various combinations. Genome assemblies of the 16 Streptomyces strains have also provided genes involved in key pathways related to PGP and biocontrol traits such as siderophores, auxin, hydrocyanic acid, chitinase and cellulase. Further, the genome assemblies provided better understanding of genetic similarity among target strains and with the publically available Streptomyces strains.

Superior haplotypes for haplotype-based breeding for drought tolerance in pigeonpea (Cajanus cajan L.).

Haplotype-based breeding, a recent promising breeding approach to develop tailor-made crop varieties, deals with identification of superior haplotypes and their deployment in breeding programmes. In this context, whole genome re-sequencing data of 292 genotypes from pigeonpea reference set were mined to identify the superior haplotypes for 10 drought-responsive candidate genes. A total of 83, 132 and 60 haplotypes were identified in breeding lines, landraces and wild species, respectively. Candidate gene-based association analysis of these 10 genes on a subset of 137 accessions of the pigeonpea reference set revealed 23 strong marker-trait associations (MTAs) in five genes influencing seven drought-responsive component traits. Haplo-pheno analysis for the strongly associated genes resulted in the identification of most promising haplotypes for three genes regulating five component drought traits. The haplotype C. cajan_23080-H2 for plant weight (PW), fresh weight (FW) and turgid weight (TW), the haplotype C. cajan_30211-H6 for PW, FW, TW and dry weight (DW), the haplotype C. cajan_26230-H11 for FW and DW and the haplotype C. cajan_26230-H5 for relative water content (RWC) were identified as superior haplotypes under drought stress condition. Furthermore, 17 accessions containing superior haplotypes for three drought-responsive genes were identified. The identified superior haplotypes and the accessions carrying these superior haplotypes will be very useful for deploying haplotype-based breeding to develop next-generation tailor-made better drought-responsive pigeonpea cultivars.

Genomics-assisted breeding for pigeonpea improvement.

KEY MESSAGE: The review outlines advances in pigeonpea genomics, breeding and seed delivery systems to achieve yield gains at farmers' field. Pigeonpea is a nutritious and stress-tolerant grain legume crop of tropical and subtropical regions. Decades of breeding efforts in pigeonpea have resulted in development of a number of high-yielding cultivars. Of late, the development of CMS-based hybrid technology has allowed the exploitation of heterosis for yield enhancement in this crop. Despite these positive developments, the actual on-farm yield of pigeonpea is still well below its potential productivity. Growing needs for high and sustainable pigeonpea yields motivate scientists to improve the breeding efficiency to deliver a steady stream of cultivars that will provide yield benefits under both ideal and stressed environments. To achieve this objective in the shortest possible time, it is imperative that various crop breeding activities are integrated with appropriate new genomics technologies. In this context, the last decade has seen a remarkable rise in the generation of important genomic resources such as genome-wide markers, high-throughput genotyping assays, saturated genome maps, marker/gene-trait associations, whole-genome sequence and germplasm resequencing data. In some cases, marker/gene-trait associations are being employed in pigeonpea breeding programs to improve the valuable yield and market-preferred traits. Embracing new breeding tools like genomic selection and speed breeding is likely to improve genetic gains. Breeding high-yielding pigeonpea cultivars with key adaptation traits also calls for a renewed focus on systematic selection and utilization of targeted genetic resources. Of equal importance is to overcome the difficulties being faced by seed industry to take the new cultivars to the doorstep of farmers.

Trait associations in the pangenome of pigeon pea (Cajanus cajan).

Pigeon pea (Cajanus cajan) is an important orphan crop mainly grown by smallholder farmers in India and Africa. Here, we present the first pigeon pea pangenome based on 89 accessions mainly from India and the Philippines, showing that there is significant genetic diversity in Philippine individuals that is not present in Indian individuals. Annotation of variable genes suggests that they are associated with self-fertilization and response to disease. We identified 225 SNPs associated with nine agronomically important traits over three locations and two different time points, with SNPs associated with genes for transcription factors and kinases. These results will lead the way to an improved pigeon pea breeding programme.

Seed protein content and its relationships with agronomic traits in pigeonpea is controlled by both main and epistatic effects QTLs.

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.

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.