Novel insights into drought-induced regulation of ribosomal genes through DNA methylation in chickpea.

Modifications within the epigenome of an organism in response to external environmental conditions allow it to withstand the hostile stress factors. Drought in chickpea is a severely limiting abiotic stress factor which is known to cause huge yield loss. To analyse the methylome of chickpea in response to drought stress conditions and how it affects gene expression, we performed whole-genome bisulfite sequencing (WGBS) and RNA-seq of two chickpea genotypes which contrast for drought tolerance. It was observed that the mCHH was most variable under drought stress and the drought tolerant (DT) genotype exhibited substantial genome-wide hypomethylation as compared to the drought sensitive (DS) genotype. Specifically, there was substantial difference in gene expression and methylation for the ribosomal genes for the tolerant and sensitive genotypes. The differential expression of these genes was in complete agreement with earlier reported transcriptomes in chickpea. Many of these genes were hypomethylated (q < 0.01) and downregulated under drought stress (p < 0.01) in the sensitive genotype. The gene RPS6 (ribosomal protein small subunit) was found to be downregulated and hypomethylated in the drought sensitive genotype which could possibly lead to reduced ribosomal biosynthesis. This study provides novel insights into regulation of drought-responsive genes in chickpea.

Discovering the regulators of heat stress tolerance in Ziziphus nummularia (Burm.f) wight and walk.-arn.

Ziziphus nummularia an elite heat-stress tolerant shrub, grows in arid regions of desert. However, its molecular mechanism responsible for heat stress tolerance is unexplored. Therefore, we analysed whole transcriptome of Jaisalmer (heat tolerant) and Godhra (heat sensitive) genotypes of Z. nummularia to understand its molecular mechanism responsible for heat stress tolerance. De novo assembly of 16,22,25,052 clean reads yielded 276,029 transcripts. A total of 208,506 unigenes were identified which contains 4290 and 1043 differentially expressed genes (DEG) in TGO (treated Godhra at 42 °C) vs. CGO (control Godhra) and TJR (treated Jaisalmer at 42 °C) vs. CJR (control Jaisalmer), respectively. A total of 987 (67 highly enriched) and 754 (34 highly enriched) pathways were obsorved in CGO vs. TGO and CJR vs. TJR, respectively. Antioxidant pathways and TFs like Homeobox, HBP, ARR, PHD, GRAS, CPP, and E2FA were uniquely observed in Godhra genotype and SET domains were uniquely observed in Jaisalmer genotype. Further transposable elements were highly up-regulated in Godhra genotype but no activation in Jaisalmer genotype. A total of 43,093 and 39,278 simple sequence repeats were identified in the Godhra and Jaisalmer genotypes, respectively. A total of 10 DEGs linked to heat stress were validated in both genotypes for their expression under different heat stresses using quantitative real-time PCR. Comparing expression patterns of the selected DEGs identified ClpB1 as a potential candidate gene for heat tolerance in Z. nummularia. Here we present first characterized transcriptome of Z. nummularia in response to heat stress for the identification and characterization of heat stress-responsive genes. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01431-y.

Predictive Role of Cluster Bean (Cyamopsis tetragonoloba) Derived miRNAs in Human and Cattle Health.

MicroRNAs (miRNAs) are small non-coding conserved molecules with lengths varying between 18-25nt. Plants miRNAs are very stable, and probably they might have been transferred across kingdoms via food intake. Such miRNAs are also called exogenous miRNAs, which regulate the gene expression in host organisms. The miRNAs present in the cluster bean, a drought tolerant legume crop having high commercial value, might have also played a regulatory role for the genes involved in nutrients synthesis or disease pathways in animals including humans due to dietary intake of plant parts of cluster beans. However, the predictive role of miRNAs of cluster beans for gene-disease association across kingdoms such as cattle and humans are not yet fully explored. Thus, the aim of the present study is to (i) find out the cluster bean miRNAs (cb-miRs) functionally similar to miRNAs of cattle and humans and predict their target genes' involvement in the occurrence of complex diseases, and (ii) identify the role of cb-miRs that are functionally non-similar to the miRNAs of cattle and humans and predict their targeted genes' association with complex diseases in host systems. Here, we predicted a total of 33 and 15 functionally similar cb-miRs (fs-cb-miRs) to human and cattle miRNAs, respectively. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the participation of targeted genes of fs-cb-miRs in 24 and 12 different pathways in humans and cattle, respectively. Few targeted genes in humans like LCP2, GABRA6, and MYH14 were predicted to be associated with disease pathways of Yesinia infection (hsa05135), neuroactive ligand-receptor interaction (hsa04080), and pathogenic Escherichia coli infection (hsa05130), respectively. However, targeted genes of fs-cb-miRs in humans like KLHL20, TNS1, and PAPD4 are associated with Alzheimer's, malignant tumor of the breast, and hepatitis C virus infection disease, respectively. Similarly, in cattle, targeted genes like ATG2B and DHRS11 of fs-cb-miRs participate in the pathways of Huntington disease and steroid biosynthesis, respectively. Additionally, the targeted genes like SURF4 and EDME2 of fs-cb-miRs are associated with mastitis and bovine osteoporosis, respectively. We also found a few cb-miRs that do not have functional similarity with human and cattle miRNAs but are found to target the genes in the host organisms and as well being associated with human and cattle diseases. Interestingly, a few genes such as NRM, PTPRE and SUZ12 were observed to be associated with Rheumatoid Arthritis, Asthma and Endometrial Stromal Sarcoma diseases, respectively, in humans and genes like SCNN1B associated with renal disease in cattle.

Genomic insight into variations associated with flowering-time and early-maturity in pigeonpea mutant TAT-10 and its wild type parent T21.

Pigeonpea [Cajanus cajan (L.) Millspaugh] is an important grain legume crop with a broad range of 90 to 300 days for maturity. To identify the genomic variations associated with the early maturity, we conducted whole-genome resequencing of an early-maturing pigeonpea mutant TAT-10 and its wild type parent T21. A total of 135.67 and 146.34 million sequencing reads were generated for T21 and TAT-10, respectively. From this resequencing data, 1,397,178 and 1,419,904 SNPs, 276,741 and 292,347 InDels, and 87,583 and 92,903 SVs were identified in T21 and TAT-10, respectively. We identified 203 genes in the pigeonpea genome that are homologs of flowering-related genes in Arabidopsis and found 791 genomic variations unique to TAT-10 linked to 94 flowering-related genes. We identified three candidate genes for early maturity in TAT-10; Suppressor of FRI 4 (SUF4), Early Flowering In Short Days (EFS), and Probable Lysine-Specific Demethylase ELF6. The variations in ELF6 were predicted to be possibly damaging and the expression profiles of EFS and ELF6 also supported their probable role during early flowering in TAT-10. The present study has generated information on genomic variations associated with candidate genes for early maturity, which can be further studied and exploited for developing the early-maturing pigeonpea cultivars.

Comparative transcriptome analysis of two contrasting genotypes provides new insights into the drought response mechanism in pigeon pea (Cajanus cajan L. Millsp.).

BACKGROUND: Despite plant's ability to adapt and withstand challenging environments, drought poses a severe threat to their growth and development. Although pigeon pea is already quite resistant to drought, the prolonged dehydration induced by the aberrant climate poses a serious threat to their survival and productivity. OBJECTIVE: Comparative physiological and transcriptome analyses of drought-tolerant (CO5) and drought-sensitive (CO1) pigeon pea genotypes subjected to drought stress were carried out in order to understand the molecular basis of drought tolerance in pigeon pea. METHODS: The transcriptomic analysis allowed us to examine how drought affects the gene expression of C. cajan. Using bioinformatics tools, the unigenes were de novo assembled, annotated, and functionally evaluated. Additionally, a homology-based sequence search against the droughtDB database was performed to identify the orthologs of the DEGs. RESULTS: 1102 potential drought-responsive genes were found to be differentially expressed genes (DEGs) between drought-tolerant and drought-sensitive genotypes. These included Abscisic acid insensitive 5 (ABI5), Nuclear transcription factor Y subunit A-7 (NF-YA7), WD40 repeat-containing protein 55 (WDR55), Anthocyanidin reductase (ANR) and Zinc-finger homeodomain protein 6 (ZF-HD6) and were highly expressed in the tolerant genotype. Further, GO analysis revealed that the most enriched classes belonged to biosynthetic and metabolic processes in the biological process category, binding and catalytic activity in the molecular function category and nucleus and protein-containing complex in the cellular component category. Results of KEGG pathway analysis revealed that the DEGs were significantly abundant in signalling pathways such as plant hormone signal transduction and MAPK signalling pathways. Consequently, in our investigation, we have identified and validated by qPCR a group of genes involved in signal reception and propagation, stress-specific TFs, and basal regulatory genes associated with drought response. CONCLUSION: In conclusion, our comprehensive transcriptome dataset enabled the discovery of candidate genes connected to pathways involved in pigeon pea drought response. Our research uncovered a number of unidentified genes and transcription factors that could be used to understand and improve susceptibility to drought.

Nematode Genome Announcement: A Draft Genome of Seed Gall Nematode, Anguina tritici.

Anguina tritici is the first plant-parasitic nematode described in literature, dating back to the year 1743. It is responsible for causing earcockle (seed gall) and tundu diseases in wheat and rye. Notably, this nematode has been observed to survive in an anhydrobiotic state for up to 32 years within wheat seed galls. These exceptional characteristics have inspired the sequencing of the A. tritici genome. In this study, we present the initial draft genome of A. tritici, obtained using the Illumina MiSeq platform with coverage of 60-fold. The genome is estimated to have a size of 164 Mb and comprises 39,965 protein-coding genes, exhibiting a GC content of 39.1%. The availability of this genome data will serve as a foundation for future functional biological investigations, particularly for genes whose functions remain unknown to this day.

Whole chloroplast genome-specific non-synonymous SNPs reveal the presence of substantial diversity in the pigeonpea mini-core collection.

To unravel the plastid genome diversity among the cultivated groups of the pigeonpea germplasm, we characterized the SNP occurrence and distribution of 142 pigeonpea mini-core collections based on their reference-based assembly of the chloroplast genome. A total of 8921 SNPs were found, which were again filtered and finally 3871 non-synonymous SNPs were detected and used for diversity estimates. These 3871 SNPs were classified into 12 groups and were present in only 44 of the 125 genes, demonstrating the presence of a precise mechanism for maintaining the whole chloroplast genome throughout evolution. The Acetyl-CoA carboxylase D gene possesses the maximum number of SNPs (12.29%), but the Adenosine Tri-Phosphate synthatase cluster genes (atpA, atpB, atpE, atpF, atpH, and atpI) altogether bear 43.34% of the SNPs making them most diverse. Various diversity estimates, such as the number of effective alleles (1.013), Watterson's estimate (0.19), Tajima's D ( - 3.15), Shannon's information index (0.036), suggest the presence of less diversity in the cultivated gene pool of chloroplast genomes. The genetic relatedness estimates based on pairwise correlations were also in congruence with these diversity descriptors and indicate the prevalence of rare alleles in the accessions. Interestingly, no stratification was observed either through STRUCTURE, PCoA, or phylogenetic analysis, indicating the common origin of the chloroplast in all the accessions used, irrespective of their geographical distribution. Further 6194 Cleaved Amplified Polymorphic Sequences (CAPS) markers for 531 SNPs were developed and validated in a selected set of germplasm. Based on these results, we inferred that all of the cultivated gene pools of pigeonpea have a common origin for the chloroplast genome and they possess less diversity in protein-coding regions, indicating a stable and evolved plastid genome. At the same time, all diversity analysis indicates the occurrence of rare alleles, suggesting the suitability of the mini-core collection in future pigeonpea improvement programs. In addition, the development of chloroplast genome-based CAPS markers would have utility in pigeonpea breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03785-8.

Genome-wide identification and characterization of GRAS gene family in pigeonpea (Cajanus cajan (L.) Millspaugh).

The GRAS proteins are plant-specific transcription factors (TFs) that play a crucial role in various plant physiological processes, including tissue development and stress responses. To date, GRAS family has been comprehensively characterized in Arabidopsis, soybean, rice, chickpea and other plant species. To understand the structural and functional aspects of pigeonpea (C. cajan), we identified 60 putative GRAS (CcGRAS) genes from pigeonpea genome and further analysed their physicochemical properties, subcellular locations, evolutionary classification, exon-intron structures, conserved domains, gene duplication events and cis-promoter regions. Based on the sequence similarity, CcGRAS family was clustered into 9 subfamilies and the genes with a similar structure and motif distribution were clustered in the same group. The gene duplication studies revealed that these genes were derived from tandem and dispersed duplication events. The cis-promoter regulatory analysis of CcGRAS genes indicated the presence of three types of cis-acting elements including light-responsive, hormone-responsive and plant growth and development related. The expression profiling of CcGRAS genes revealed their tissue-specific functions and differential nature. Collectively, this study highlights relevant functional and regulatory elements of GRAS family in pigeonpea creating a significant resource for future functional studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03782-x.

Unravelling the due importance of pseudogenes and their resurrection in plants.

The complexities of a genome are underpinned to the vast expanses of the intergenic region, which constitutes ∼97-98% of the genome. This region is essentially composed of what is colloquially referred to as the "junk DNA" and is composed of various elements like transposons, repeats, pseudogenes, etc. The latter have long been considered as dead elements merely contributing to transcriptional noise in the genome. Many studies now describe the previously unknown regulatory functions of these genes. Recent advances in the Next-generation sequencing (NGS) technologies have allowed unprecedented access to these regions. With the availability of whole genome sequences of more than 788 different plant species in past 20 years, genome annotation has become feasible like never before. Different bioinformatic pipelines are available for the identification of pseudogenes. However, still little is known about their biological functions. The functional validation of these genes remains challenging and research in this area is still in infancy, particularly in plants. CRISPR/Cas-based genome editing could provide solutions to understand the biological roles of these genes by allowing creation of precise edits within these genes. The possibility of pseudogene reactivation or resurrection as has been demonstrated in a few studies might open new avenues of genetic manipulation to yield a desirable phenotype. This review aims at comprehensively summarizing the progress made with regards to the identification of pseudogenes and understanding their biological functions in plants.

Genome-wide survey, molecular evolution and expression analysis of Auxin Response Factor (ARF) gene family indicating their key role in seed number per pod in pigeonpea (C. cajan L. Millsp.).

Auxin Response Factors (ARF) are a family of transcription factors that mediate auxin signalling and regulate multiple biological processes. Their crucial role in increasing plant biomass/yield influenced this study, where a systematic analysis of ARF gene family was carried out to identify the key proteins controlling embryo/seed developmental pathways in pigeonpea. A genome-wide scan revealed the presence of 12 ARF genes in pigeonpea, distributed across the chromosomes 1, 3, 4, 8 and 11. Domain analysis of ARF proteins showed the presence of B3 DNA binding, AUX response, and IAA domains. Majority of them are of nuclear origin, and do not exhibit the level of genomic expansion as observed in Glycine max (51 members). The duplication events seem to range from 31.6 to 42.3 million years ago (mya). Promoter analysis revealed the presence of multiple cis-acting elements related to stress responses, hormone signalling and other development processes. The expression atlas data highlighted the expression of CcARF8 in hypocotyl, bud and flower whereas, CcARF7 expression was significantly high in pod. The real-time expression of CcARF2, CcARF3 and CcARF18 was highest in genotypes with high seed number indicating their key role in regulating embryo development and determining seed set in pigeonpea.

Integration of miRNA dynamics and drought tolerant QTLs in rice reveals the role of miR2919 in drought stress response.

To combat drought stress in rice, a major threat to global food security, three major quantitative trait loci for 'yield under drought stress' (qDTYs) were successfully exploited in the last decade. However, their molecular basis still remains unknown. To understand the role of secondary regulation by miRNA in drought stress response and their relation, if any, with the three qDTYs, the miRNA dynamics under drought stress was studied at booting stage in two drought tolerant (Sahbaghi Dhan and Vandana) and one drought sensitive (IR 20) cultivars. In total, 53 known and 40 novel differentially expressed (DE) miRNAs were identified. The primary drought responsive miRNAs were Osa-MIR2919, Osa-MIR3979, Osa-MIR159f, Osa-MIR156k, Osa-MIR528, Osa-MIR530, Osa-MIR2091, Osa-MIR531a, Osa-MIR531b as well as three novel ones. Sixty-one target genes that corresponded to 11 known and 4 novel DE miRNAs were found to be co-localized with the three qDTYs, out of the 1746 target genes identified. We could validate miRNA-mRNA expression under drought for nine known and three novel miRNAs in eight different rice genotypes showing varying degree of tolerance. From our study, Osa-MIR2919, Osa-MIR3979, Osa-MIR528, Osa-MIR2091-5p and Chr01_11911S14Astr and their target genes LOC_Os01g72000, LOC_Os01g66890, LOC_Os01g57990, LOC_Os01g56780, LOC_Os01g72834, LOC_Os01g61880 and LOC_Os01g72780 were identified as the most promising candidates for drought tolerance at booting stage. Of these, Osa-MIR2919 with 19 target genes in the qDTYs is being reported for the first time. It acts as a negative regulator of drought stress tolerance by modulating the cytokinin and brassinosteroid signalling pathway.

Genome wide identification and characterization of the amino acid transporter (AAT) genes regulating seed protein content in chickpea (Cicer arietinum L.).

Amino acid transporters (AATs), besides, being a crucial component for nutrient partitioning system are also vital for growth and development of the plants and stress resilience. In order to understand the role of AAT genes in seed quality proteins, a comprehensive analysis of AAT gene family was carried out in chickpea leading to identification of 109 AAT genes, representing 10 subfamilies with random distribution across the chickpea genome. Several important stress responsive cis-regulatory elements like Myb, ABRE, ERE were detected in the promoter region of these CaAAT genes. Most of the genes belonging to the same sub-families shared the intron-exon distribution pattern owing to their conserved nature. Random distribution of these CaAAT genes was observed on plasma membrane, vacuolar membrane, Endoplasmic reticulum and Golgi membranes, which may be associated to distinct biochemical pathways. In total 92 out 109 CaAAT genes arise as result of duplication, among which segmental duplication was more prominent over tandem duplication. As expected, the phylogenetic tree was divided into 2 major clades, and further sub-divided into different sub-families. Among the 109 CaAAT genes, 25 were found to be interacting with 25 miRNAs, many miRNAs like miR156, miR159 and miR164 were interacting only with single AAT genes. Tissues specific expression pattern of many CaAAT genes was observed like CaAAP7 and CaAVT18 in nodules, CaAAP17, CaAVT5 and CaCAT9 in vegetative tissues while CaCAT10 and CaAAP23 in seed related tissues as per the expression analysis. Mature seed transcriptome data revealed that genotypes having high protein content (ICC 8397, ICC 13461) showed low CaAATs expression as compared to the genotypes having low protein content (FG 212, BG 3054). Amino acid profiling of these genotypes revealed a significant difference in amount of essential and non-essential amino acids, probably due to differential expression of CaAATs. Thus, the present study provides insights into the biological role of AAT genes in chickpea, which will facilitate their functional characterization and role in various developmental stages, stress responses and involvement in nutritional quality enhancement.

The chromosome-scale genome assembly of cluster bean provides molecular insight into edible gum (galactomannan) biosynthesis family genes.

Cluster bean (Cyamopsis tetragonoloba (L.) Taub 2n = 14, is commonly known as Guar. Apart from being a vegetable crop, it is an abundant source of a natural hetero-polysaccharide called guar gum or galactomannan. Here, we are reporting a chromosome-scale reference genome assembly of a popular cluster bean cultivar RGC-936, by combining sequencing data from Illumina, 10X Genomics, Oxford Nanopore technologies. An initial assembly of 1580 scaffolds with an N50 value of 7.12 Mb was generated and these scaffolds were anchored to a high density SNP linkage map. Finally, a genome assembly of 550.31 Mb (94% of the estimated genome size of ~ 580 Mb (through flow cytometry) with 58 scaffolds was obtained, including 7 super scaffolds with a very high N50 value of 78.27 Mb. Phylogenetic analysis using single copy orthologs among 12 angiosperms showed that cluster bean shared a common ancestor with other legumes 80.6 MYA. No evidence of recent whole genome duplication event in cluster bean was found in our analysis. Further comparative transcriptomics analyses revealed pod-specific up-regulation of genes encoding enzymes involved in galactomannan biosynthesis. The high-quality chromosome-scale cluster bean genome assembly will facilitate understanding of the molecular basis of galactomannan biosynthesis and aid in genomics-assisted improvement of cluster bean.

Genomic survey of high-throughput RNA-Seq data implicates involvement of long intergenic non-coding RNAs (lincRNAs) in cytoplasmic male-sterility and fertility restoration in pigeon pea.

BACKGROUND: Long-intergenic non-coding RNAs (lincRNAs) originate from intergenic regions and have no coding potential. LincRNAs have emerged as key players in the regulation of various biological processes in plant development. Cytoplasmic male-sterility (CMS) in association with restorer-of-fertility (Rf) systems makes it a highly reliable tool for exploring heterosis for producing commercial hybrid seeds. To date, there have been no reports of lincRNAs during pollen development in CMS and fertility restorer lines in pigeon pea. OBJECTIVE: Identification of lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines. METHODS: We employed a computational approach to identify lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines using RNA-Seq data. RESULTS: We predicted a total of 2145 potential lincRNAs of which 966 were observed to be differentially expressed between the sterile and fertile pollen. We identified, 927 cis-regulated and 383 trans-regulated target genes of the lincRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the target genes revealed that these genes were specifically enriched in pathways like pollen and pollen tube development, oxidative phosphorylation, etc. We detected 23 lincRNAs that were co-expressed with 17 pollen-related genes with known functions. Fifty-nine lincRNAs were predicted to be endogenous target mimics (eTMs) for 25 miRNAs, and found to be associated with pollen development. The, lincRNA regulatory networks revealed that different lincRNA-miRNA-mRNA networks might be associated with CMS and fertility restoration. CONCLUSION: Thus, this study provides valuable information by highlighting the functions of lincRNAs as regulators during pollen development in pigeon pea and utilization in hybrid seed production.

The SPL transcription factor genes are potential targets for epigenetic regulation in response to drought stress in chickpea (C. arietinum L.).

BACKGROUND: Crop improvement for tolerance to various biotic and abiotic stress factors necessitates understanding the key gene regulatory mechanisms. One such mechanism of gene regulation involves changes in cytosine methylation at the gene body and flanking regulatory sequences. The present study was undertaken to identify genes which might be potential targets of drought-induced DNA methylation in chickpea. METHODS AND RESULTS: Two chickpea genotypes, which contrast for drought tolerance, were subjected to drought stress conditions and their differential response was studied by analysing different morpho-physiological traits. Utilizing the in-house, high throughput sequencing data, the SQUAMOSA promoter-binding (SBP) protein-like (SPL) transcription factor genes were identified to be differentially methylated and expressed amongst the two genotypes, in response to drought stress. The methylation status of one of these genes was examined and validated through bisulfite PCR (BS-PCR). The identified genes could be possible homologs to known epialleles and can therefore serve as potential epialleles which can be utilized for crop improvement in chickpea. CONCLUSION: The SPL TF genes are potential targets of epigenetic regulation in response to drought stress in chickpea. Since these are TFs, they might play important roles in controlling the expression of other genes, thus contributing to differential drought response of the two genotypes.

Role of cytokinins in seed development in pulses and oilseed crops: Current status and future perspective.

Cytokinins constitutes a vital group of plant hormones regulating several developmental processes, including growth and cell division, and have a strong influence on grain yield. Chemically, they are the derivatives of adenine and are the most complex and diverse group of hormones affecting plant physiology. In this review, we have provided a molecular understanding of the role of cytokinins in developing seeds, with special emphasis on pulses and oilseed crops. The importance of cytokinin-responsive genes including cytokinin oxidases and dehydrogenases (CKX), isopentenyl transferase (IPT), and cytokinin-mediated genetic regulation of seed size are described in detail. In addition, cytokinin expression in germinating seeds, its biosynthesis, source-sink dynamics, cytokinin signaling, and spatial expression of cytokinin family genes in oilseeds and pulses have been discussed in context to its impact on increasing economy yields. Recently, it has been shown that manipulation of the cytokinin-responsive genes by mutation, RNA interference, or genome editing has a significant effect on seed number and/or weight in several crops. Nevertheless, the usage of cytokinins in improving crop quality and yield remains significantly underutilized. This is primarily due to the multigene control of cytokinin expression. The information summarized in this review will help the researchers in innovating newer and more efficient ways of manipulating cytokinin expression including CKX genes with the aim to improve crop production, specifically of pulses and oilseed crops.

Transcriptome profiling reveals the genes and pathways involved in thermo-tolerance in wheat (Triticum aestivum L.) genotype Raj 3765.

Wheat, one of the most widely consumed staple food crops globally, is relatively vulnerable to high temperature-induced heat stress. It is therefore essential to gain more insight into the comprehensive mechanism of thermotolerance of wheat in order to safeguard its production. In view of this, we analysed heat stress responsive transcriptome data of wheat to determine its gene expression level under heat stress. A total of 7990 DEGs, including 4483 up-regulated and 3507 down regulated genes were identified. Gene Ontology (GO) analysis categorized 3910 DEGs into different ontology families. 146 pathways involving 814 DEGs were enriched during KEGG analysis. Metabolic pathways and biosynthesis of secondary metabolites were the major pathways enriched. MYB (myeloblastosis) transcription factors (TFs) and many other TFs as bHLH, WRKY, NAC, ERF, were determined to be quite abundant in the DEGs. Since various reports indicate that these TFs play important role in plants abiotic stress, it is an indication that our DEGs are functional in heat stress tolerance. Verification of few selected DEGs using RT-qPCR produced expression levels similar to the transcriptome data. This indicates that the transcriptome data is reliable. These results could be helpful in enhancing our understanding of the mechanism underlying thermotolerance in wheat.

Evolutionary fates of gene-body methylation and its divergent association with gene expression in pigeonpea.

Pigeonpea (Cajanus cajan L. Huth) is an agronomically important legume cultivated worldwide. In this study, we extensively analyzed gene-body methylation (GbM) patterns in pigeonpea. We found a bimodal distribution of CG and CHG methylation patterns. GbM features- slow evolution rate and increased length remained conserved. Genes with moderate CG body methylation showed highest expression where as highly-methylated genes showed lowest expression. Transposable element (TE)-related genes were methylated in multiple contexts and hence classified as C-methylated genes. A low expression among C-methylated genes was associated with transposons insertion in gene-body and upstream regulatory regions. The CG methylation patterns were found to be conserved in orthologs compared with non-CG methylation. By comparing methylation patterns between differentially methylated regions (DMRs) of the three genotypes, we found that variably methylated marks are less likely to target evolutionary conserved sequences. Finally, our analysis showed enrichment of nitrogen-related genes in GbM orthologs of legumes, which could be promising candidates for generating epialleles for crop improvement.

Transcriptome Analysis of Pennisetum glaucum (L.) R. Br. Provides Insight Into Heat Stress Responses.

Pennisetum glaucum (L.) R. Br., being widely grown in dry and hot weather, frequently encounters heat stress at various stages of growth. The crop, due to its inherent capacity, efficiently overcomes such stress during vegetative stages. However, the same is not always the case with the terminal (flowering through grain filling) stages of growth, where recovery from stress is more challenging. However, certain pearl millet genotypes such as 841-B are known to overcome heat stress even at the terminal growth stages. Therefore, we performed RNA sequencing of two contrasting genotypes of pearl millet (841-B and PPMI-69) subjected to heat stress (42°C for 6 h) at flowering stages. Over 274 million high quality reads with an average length of 150 nt were generated, which were assembled into 47,310 unigenes having an average length of 1,254 nucleotides, N50 length of 1853 nucleotides, and GC content of 53.11%. Blastx resulted in the annotation of 35,628 unigenes, and functional classification showed 15,950 unigenes designated to 51 Gene Ontology terms. A total of 13,786 unigenes were allocated to 23 Clusters of Orthologous Groups, and 4,255 unigenes were distributed to 132 functional Kyoto Encyclopedia of Genes and Genomes database pathways. A total of 12,976 simple sequence repeats and 305,759 SNPs were identified in the transcriptome data. Out of 2,301 differentially expressed genes, 10 potential candidate genes were selected based on log2 fold change and adjusted p value parameters for their differential gene expression by qRT-PCR. We were able to identify differentially expressed genes unique to either of the two genotypes, and also, some DEGs common to both the genotypes were enriched. The differential expression patterns suggested that 841-B 6 h has better ability to maintain homeostasis during heat stress as compared to PPMI-69 6 h. The sequencing data generated in this study, like the SSRs and SNPs, shall serve as an important resource for the development of genetic markers, and the differentially expressed heat responsive genes shall be used for the development of transgenic crops.

Single trait versus principal component based association analysis for flowering related traits in pigeonpea.

Pigeonpea, a tropical photosensitive crop, harbors significant diversity for days to flowering, but little is known about the genes that govern these differences. Our goal in the current study was to use genome wide association strategy to discover the loci that regulate days to flowering in pigeonpea. A single trait as well as a principal component based association study was conducted on a diverse collection of 142 pigeonpea lines for days to first and fifty percent of flowering over 3 years, besides plant height and number of seeds per pod. The analysis used seven association mapping models (GLM, MLM, MLMM, CMLM, EMLM, FarmCPU and SUPER) and further comparison revealed that FarmCPU is more robust in controlling both false positives and negatives as it incorporates multiple markers as covariates to eliminate confounding between testing marker and kinship. Cumulatively, a set of 22 SNPs were found to be associated with either days to first flowering (DOF), days to fifty percent flowering (DFF) or both, of which 15 were unique to trait based, 4 to PC based GWAS while 3 were shared by both. Because PC1 represents DOF, DFF and plant height (PH), four SNPs found associated to PC1 can be inferred as pleiotropic. A window of ± 2 kb of associated SNPs was aligned with available transcriptome data generated for transition from vegetative to reproductive phase in pigeonpea. Annotation analysis of these regions revealed presence of genes which might be involved in floral induction like Cytochrome p450 like Tata box binding protein, Auxin response factors, Pin like genes, F box protein, U box domain protein, chromatin remodelling complex protein, RNA methyltransferase. In summary, it appears that auxin responsive genes could be involved in regulating DOF and DFF as majority of the associated loci contained genes which are component of auxin signaling pathways in their vicinity. Overall, our findings indicates that the use of principal component analysis in GWAS is statistically more robust in terms of identifying genes and FarmCPU is a better choice compared to the other aforementioned models in dealing with both false positive and negative associations and thus can be used for traits with complex inheritance.