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.

Genome-wide identification of potassium channels in maize showed evolutionary patterns and variable functional responses to abiotic stresses.

Potassium (K) channels are essential components of plant biology, mediating not only K ion (K+) homeostasis but also regulating several physiological processes and stress tolerance. In the current investigation, we identified 27 K+ channels in maize and deciphered the evolution and divergence pattern with four monocots and five dicot species. Chromosomal localization and expansion of K+ channel genes showed uneven distribution and were independent of genome size. The dispersed duplication is the major force in expanding K+ channels in the target genomes. The mean Ka/Ks ratio of <0.5 in paralogs and orthologs indicates horizontal and vertical expansions of K+ channel genes under strong purifying selection. The one-to-one K+ channel orthologs were prominent among the closely related species, with higher synteny between maize and the rest of the monocots. Comprehensive K+ channels promoter analysis revealed various cis-regulatory elements mediating stress tolerance with the predominance of MYB and STRE binding sites. The regulatory network showed AP2-EREBP TFs, miR164 and miR399 are prominent regulatory elements of K+ channels. The qRT-PCR analysis of K+ channels and regulatory miRNAs showed significant expressions in response to drought and waterlogging stresses. The present study expanded the knowledge on K+ channels in maize and will serve as a basis for an in-depth functional analysis.

Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomics.

The entomopathogenic nematode, Heterorhabditis indica, is a popular biocontrol agent of high commercial significance. It possesses tremendous genetic architecture to survive desiccation stress by undergoing anhydrobiosis to increase its lifespan-an attribute exploited in the formulation technology. The comparative transcriptome of unstressed and anhydrobiotic H. indica revealed several previously concealed metabolic events crucial for adapting towards the moisture stress. During the induction of anhydrobiosis in the infective juveniles (IJ), 1584 transcripts were upregulated and 340 downregulated. As a strategy towards anhydrobiotic survival, the IJ showed activation of several genes critical to antioxidant defense, detoxification pathways, signal transduction, unfolded protein response and molecular chaperones and ubiquitin-proteasome system. Differential expression of several genes involved in gluconeogenesis - ß-oxidation of fatty acids, glyoxylate pathway; glyceroneogenesis; fatty acid biosynthesis; amino-acid metabolism - shikimate pathway, sachharopine pathway, kyneurine pathway, lysine biosynthesis; one-carbon metabolism-polyamine pathway, transsulfuration pathway, folate cycle, methionine cycle, nucleotide biosynthesis; mevalonate pathway; and glyceraldehyde-3-phosphate dehydrogenase were also observed. We report the role of shikimate pathway, sachharopine pathway and glyceroneogenesis in anhydrobiotes, and seven classes of repeat proteins, specifically in H. indica for the first time. These results provide insights into anhydrobiotic survival strategies which can be utilized to strengthen the development of novel formulations with enhanced and sustained shelf-life.

Whole-genome sequence analysis of Bipolaris sorokiniana infecting wheat in India and characterization of ToxA gene in different isolates as pathogenicity determinants.

Spot blotch disease of wheat caused by Bipolaris sorokiniana Boerma (Sacc.) is an emerging problem in South Asian countries. Whole genome of a highly virulent isolate of B. sorokiniana BS112 (BHU, Uttar Pradesh; Accession no. GCA_004329375.1) was sequenced using a hybrid assembly approach. Secreted proteins, virulence gene(s), pathogenicity-related gene(s) were identified and the role of ToxA gene present in this genome, in the development of disease was recognized. ToxA gene (535 bp) was analyzed and identified in the genome of B. sorokiniana (BS112) which revealed 100% homology with the ToxA gene of Pyrenophora tritici repentis (Accession no. MH017419). Furthermore, ToxA gene was amplified, sequenced and validated in 39 isolates of B. sorokiniana which confirmed the presence of ToxA gene in all the isolates taken for this study. All ToxA sequences were submitted in NCBI database (MN601358-MN601396). As ToxA gene interacts with Tsn1 gene of host, 13 wheat genotypes were evaluated out of which 5 genotypes (38.4%) were found to be Tsn1 positive with more severe necrotic lesions compared to Tsn1-negative wheat genotypes. In vitro expression analysis of ToxA gene in the pathogen B. sorokiniana using qPCR revealed maximum upregulation (14.67 fold) at 1st day after inoculation (DAI) in the medium. Furthermore, in planta expression analysis of ToxA gene in Tsn1-positive and Tsn1-negative genotypes, revealed maximum expression (7.89-fold) in Tsn1-positive genotype, Agra local at 5th DAI compared to Tsn1-negative genotype Chiriya 7 showing minimum expression (0.048-fold) at 5th DAI. In planta ToxA-Tsn1 interaction studies suggested that spot blotch disease is more severe in Tsn1-positive genotypes, which will be helpful in better understanding and management of spot blotch disease of wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03213-3.

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.

Genome-wide identification and functional prediction of salt- stress related long non-coding RNAs (lncRNAs) in chickpea (Cicer arietinum L.).

LncRNAs (long noncoding RNAs) are 200 bp length crucial RNA molecules, lacking coding potential and having important roles in regulating gene expression, particularly in response to abiotic stresses. In this study, we identified salt stress-induced lncRNAs in chickpea roots and predicted their intricate regulatory roles. A total of 3452 novel lncRNAs were identified to be distributed across all 08 chickpea chromosomes. On comparing salt-tolerant (ICCV 10, JG 11) and salt-sensitive cultivars (DCP 92-3, Pusa 256), 4446 differentially expressed lncRNAs were detected under various salt  treatments. We predicted 3373 lncRNAs to be regulating their target genes in cis regulating manner and 80 unique lncRNAs were observed as interacting with 136 different miRNAs, as eTMs (endogenous target mimic) targets of miRNAs and implicated them in the regulatory network of salt stress response. Functional analysis of these lncRNA revealed their association in targeting salt stress response-related genes like potassium transporter, transporter family genes, serine/threonine-protein kinase, aquaporins like TIP1-2, PIP2-5 and transcription factors like, AP2, NAC, bZIP, ERF, MYB and WRKY. Furthermore, about 614 lncRNA-SSRs (simple sequence repeats) were identified as a new generation of molecular markers with higher efficiency and specificity in chickpea. Overall, these findings will pave the understanding of comprehensive functional role of potential lncRNAs, which can help in providing insight into the molecular mechanism of salt tolerance in chickpea. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01093-0.

Genome sequence analysis provides insights on genomic variation and late blight resistance genes in potato somatic hybrid (parents and progeny).

Wild Solanum species are the important resources for potato improvement. With the availability of potato genome and sequencing progress, knowledge about genomic resources is essential for novel genes discovery. Hence, the aim of this study was to decipher draft genome sequences of unique potato genotypes i.e. somatic hybrid P8 (J1), wild species S. pinnatisectum (J2), progeny MSH/14-112 (P8 × cv. Kufri Jyoti) (J3), and S. tuberosum dihaploid C-13 (J4). Draft genome sequencing using Illumina platform and reference-based assemblies with the potato genome yielded genome assembly size of 725.01 Mb (J1), 724.95 Mb (J2), 725.01 Mb (J3), and 809.59 Mb (J4). Further, 39,260 (J1), 25,711 (J2), 39,730 (J3) and 30,241 (J4) genes were identified and 17,411 genes were found common in the genotypes particularly late blight resistance genes (R3a, RGA2, RGA3, R1B-16, Rpi-blb2, Rpi and Rpi-vnt1). Gene ontology (GO) analysis showed that molecular function was predominant and signal transduction was major KEGG pathways. Further, gene enrichment analysis revealed dominance of metabolic process (GO: 0008152) in all the samples. Phylogeny analysis showed relatedness with potato and other plant species. Heterozygous single nucleotide polymorphism (SNP) was more than homozygous, and SNP in genic region was more than inter-genic region. Copy number variation (CNV) analysis indicated greater number of deletions than duplications. Sequence diversity and conserved motifs analysis revealed variation for late blight resistance genes. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed differential expression of late blight resistance genes. Our study provides insights on genome sequence, structural variation and late blight resistance genes in potato somatic hybrid (parents and progeny) for future research.

Physiological and genome-wide RNA-sequencing analyses identify candidate genes in a nitrogen-use efficient potato cv. Kufri Gaurav.

Nitrogen (N) is an important nutrient for plant growth. However, its excess application leads to environmental damage. Hence, improving nitrogen use efficiency (NUE) of plant is one of the plausible options to solve the problems. Aim of this study was to identify candidate genes involved in enhancing NUE in potato cv. Kufri Gaurav (N efficient). Plants were grown in aeroponic with two contrasting N regimes (low N: 0.75 mM, and high N: 7.5 mM). Higher NUE in Kufri Gaurav was observed in low N based on the parameters like NUE, NUpE (N uptake efficiency), NUtE (N utilization efficiency) and AgNUE (agronomic NUE). Further, global gene expression profiles in root, leaf and stolon tissues were analyzed by RNA-sequencing using Ion Proton™ System. Quality data (≥Q20) of 2.04-2.73 Gb per sample were mapped with the potato genome. Statistically significant (P ≤ 0.05) differentially expressed genes (DEGs) were identified such as 176 (up-regulated) and 30 (down-regulated) in leaves, 39 (up-regulated) and 105 (down-regulated) in roots, and 81 (up-regulated) and 694 (down-regulated) in stolons. The gene ontology (GO) terms like metabolic process, cellular process and catalytic activity were predominant. Our RT-qPCR analysis confirmed the gene expression profiles of RNA-seq. Overall, we identified candidate genes associated with improving NUE such as superoxide dismutase, GDSL esterase lipase, probable phosphatase 2C, high affinity nitrate transporters, sugar transporter, proline rich proteins, transcription factors (VQ motif, SPX domain, bHLH) etc. Our findings suggest that these candidate genes probably play crucial roles in enhancing NUE in potato.

Transcriptome profiling of differentially expressed genes in cytoplasmic male-sterile line and its fertility restorer line in pigeon pea (Cajanus cajan L.).

BACKGROUND: Pigeon pea (Cajanus cajan L.) is the sixth major legume crop widely cultivated in the Indian sub-continent, Africa, and South-east Asia. Cytoplasmic male-sterility (CMS) is the incompetence of flowering plants to produce viable pollens during anther development. CMS has been extensively utilized for commercial hybrid seeds production in pigeon pea. However, the molecular basis governing CMS in pigeon pea remains unclear and undetermined. In this study transcriptome analysis for exploring differentially expressed genes (DEGs) between cytoplasmic male-sterile line (AKCMS11) and its fertility restorer line (AKPR303) was performed using Illumina paired-end sequencing. RESULTS: A total of 3167 DEGs were identified, of which 1432 were up-regulated and 1390 were down-regulated in AKCMS11 in comparison to AKPR303. By querying, all the 3167 DEGs against TAIR database, 34 pigeon pea homologous genes were identified, few involved in pollen development (EMS1, MS1, ARF17) and encoding MYB and bHLH transcription factors with lower expression in the sterile buds, implying their possible role in pollen sterility. Many of these DEGs implicated in carbon metabolism, tricarboxylic acid cycle (TCA), oxidative phosphorylation and elimination of reactive oxygen species (ROS) showed reduced expression in the AKCMS11 (sterile) buds. CONCLUSION: The comparative transcriptome findings suggest the potential role of these DEGs in pollen development or abortion, pointing towards their involvement in cytoplasmic male-sterility in pigeon pea. The candidate DEGs identified in this investigation will be highly significant for further research, as they could lend a comprehensive basis in unravelling the molecular mechanism governing CMS in pigeon pea.

Development and validation of whole genome-wide and genic microsatellite markers in oil palm (Elaeis guineensis Jacq.): First microsatellite database (OpSatdb).

The availability of large expressed sequence tag (EST) and whole genome databases of oil palm enabled the development of a data base of microsatellite markers. For this purpose, an EST database consisting of 40,979 EST sequences spanning 27 Mb and a chromosome-wise whole genome databases were downloaded. A total of 3,950 primer pairs were identified and developed from EST sequences. The tri and tetra nucleotide repeat motifs were most prevalent (each 24.75%) followed by di-nucleotide repeat motifs. Whole genome-wide analysis found a total of 245,654 SSR repeats across the 16 chromosomes of oil palm, of which 38,717 were compound microsatellite repeats. A web application, OpSatdb, the first microsatellite database of oil palm, was developed using the PHP and MySQL database ( https://ssr.icar.gov.in/index.php ). It is a simple and systematic web-based search engine for searching SSRs based on repeat motif type, repeat type, and primer details. High synteny was observed between oil palm and rice genomes. The mapping of ESTs having SSRs by Blast2GO resulted in the identification of 19.2% sequences with gene ontology (GO) annotations. Randomly, a set of ten genic SSRs and five genomic SSRs were used for validation and genetic diversity on 100 genotypes belonging to the world oil palm genetic resources. The grouping pattern was observed to be broadly in accordance with the geographical origin of the genotypes. The identified genic and genome-wide SSRs can be effectively useful for various genomic applications of oil palm, such as genetic diversity, linkage map construction, mapping of QTLs, marker-assisted selection, and comparative population studies.

Transcriptome analysis reveals interplay between hormones, ROS metabolism and cell wall biosynthesis for drought-induced root growth in wheat.

The ability of roots to grow under drought stress is an adaptive trait for crop plants especially under rain fed and restricted irrigation regime. To unravel the molecular mechanism of drought induced-root growth, root transcriptomes of two wheat genotypes viz. Raj3765 and HD2329, with contrasting root growth under drought stress were analyzed. Drought stress significantly enhanced total root length in Raj3765 as compared to that of HD2329. RNA-seq analysis led to the identification of 2783 and 2638 differentially expressed genes (DEGs) in Raj3765 and HD2329, respectively, under drought stress as compared with non-stress conditions. Functional annotation, gene ontology and MapMan analysis of the DEGs revealed differential regulation of genes for pathways associated with root growth and stress tolerance. Drought stress significantly upregulated auxin receptor (AFB2) and ABA responsive transcription factors (MYB78, WRKY18 and GBF3) in roots of Raj3765. Although certain genes for ethylene pathway were downregulated in both the genotypes, ACC oxidase and 2OG-Fe(II) oxygenase were upregulated only in Raj3765 which might contribute to maintenance of a basal ethylene level to maintain root growth. Several genes related to cell wall biosynthesis and ROS metabolism were significantly upregulated in Raj3765. Genes related to gibberellic acid, jasmonic acid and phenylpropanoid pathways were downregulated in roots of both the genotypes under drought stress. Our analysis suggests that a coordinated yet complex interplay between hormones, cellular tolerance, cell wall synthesis and ROS metabolism are required for drought induced root growth in wheat.

GinMicrosatDb: a genome-wide microsatellite markers database for sesame (Sesamum indicum L.).

Molecular breeding in sesame is still at infancy due to limited number of microsatellite markers available and the low level of polymorphism exhibited by them. Therefore, whole genome sequencing was used for development of microsatellite markers so as to ensure availability of substantial number of polymorphic markers for use in marker assisted breeding programs. Whole genome sequencing of sesame variety 'Swetha' was done using Illumina paired-end sequencing and Roche 454 shotgun sequencing technologies (GCA_000975565.1 in GenBank). 'GinMicrosatDb', a genome-wide microsatellite marker database has been developed using the whole genome sequence data of sesame variety 'Swetha'. The database consists of microsatellites localized on both linkage groups and scaffolds with their genomic co-ordinates. It provides five sets of forward and reverse primers for each of the microsatellite loci along with the flanking sequences, primer GC content, product size and melting temperature etc. The distribution of microsatellites can be viewed and selected through a genome browser as well as through a physical map. The newly identified microsatellite markers are expected to help sesame breeders in developing marker tags for traits of economic importance thereby bringing about greater efficiency in marker-assisted selection programs.

Genome-wide identification and characterization of lncRNAs and miRNAs in cluster bean (Cyamopsis tetragonoloba).

Long non coding RNAs (lncRNAs) are a class of non-protein coding RNAs that play a crucial role in most of the biological activities like nodule metabolism, flowering time and male sterility. Quite often, the function of lncRNAs is species-specific in nature. Thus an attempt has been made in cluster bean (Cyamopsis tetragonoloba) for the first time to computationally identify lncRNAs based on a proposed index and study their targeted genes. Further, these targeted genes of lncRNAs were identified and characterized for their role in various biological processes like stress mechanisms, DNA damage repair, cell wall synthesis. Besides, lncRNAs and miRNAs bearing Simple Sequence Repeats (SSRs) were identified that contribute towards biogenesis of small non-coding RNAs. Moreover, five novel endogenous Target Mimic lncRNAs (eTMs) were identified that may disrupt the miRNA-mRNA regulations. For easy understanding and usability, a database CbLncRNAdb has been developed and made available at http://cabgrid.res.in/cblncrnadb.

Genome-wide identification and functional annotation of miRNAs in anti-inflammatory plant and their cross-kingdom regulation in Homo sapiens.

MicroRNAs (miRNAs) are newly discovered non-coding small (~17-24 nucleotide) RNAs that regulate gene expression of its target mRNA at the post-transcriptional levels. In this study, total 12,593 ESTs of Curcuma longa were taken from database of expressed sequence tags (dbEST) and clustered into 2821 contigs using EGassembler web server. Precursor miRNAs (pre-miRNAs) were predicted from these contigs that folded into stem-loop structure using MFold server. Thirty-four mature C. longa miRNAs (clo-miRNAs) were identified from pre-miRNAs having targets involved in various important functions of plant such as self-defence, growth and development, alkaloid metabolic pathway and ethylene signalling process. Sequence analysis of identified clo-miRNAs indicated that 56% miRNAs belong to ORF and 44% belong to non-ORF region. clo-mir-5 and clo-mir-6 were established as the conserved miRNAs, whereas clo-mir-20 was predicted to be the most stable miRNA. Phylogenetic analysis carried out by molecular evolutionary genetics analysis (MEGA) software indicated close evolutionary relationship of clo-mir-5075 with osa-MIR5075. Further, identified clo-miRNAs were checked for their cross-kingdom regulatory potential. clo-mir-14 was found to regulate various gene transcripts in humans that has been further investigated for its biostability in foetal bovine serum (FBS). The results indicated higher degree of stability of clo-mir-14 (48 h) in FBS. Thus, contribution of this miRNA to the cellular immune response during the inflamed condition of rheumatoid arthritis and adequate stability may make it a good choice for the therapeutic agent in near future.

Computational identification of miRNAs in medicinal plant Senecio vulgaris (Groundsel).

RNAs Interference plays a very important role in gene silencing. In vitro identification of miRNAs is a slow process as it is difficult to isolate them. Nucleotide sequences of miRNAs are highly conserved among the plants and, this form the key feature behind the identification of miRNAs in plant species by homology alignment. In silico identification of miRNAs from EST database is emerging as a novel, faster and reliable approach. Here EST sequences of Senecio vulgaris (Groundsel) were searched against known miRNA sequences by using BLASTN tool. A total of 10 miRNAs were identified from 1956 EST sequences and 115 GSS sequences. The most stable miRNA identified is svu-mir-1. This approach will accelerate advance research in regulation of gene expression in Groundsel by interfering RNAs.