A large sequenced mutant library - valuable reverse genetic resource that covers 98% of sorghum genes.

Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the Ka /Ks value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

Molecular, Phenotypical, and Host-Range Characterization of Robbsia andropogonis Strains Isolated from Bougainvillea spp. in Mexico.

Characteristic leaf spot and blight symptoms caused by Robbsia andropogonis on bougainvillea plants were found in three locations in different provinces of Mexico from 2019 to 2020. Eleven bacterial isolates with morphology similar to R. andropogonis were obtained from the diseased bougainvillea leaves. The isolates were confirmed as R. andropogonis by phenotypic tests and 16S rRNA, rpoD, and gyrB gene sequencing. In addition to bougainvillea, the strains were pathogenic to 10 agriculturally significant crops, including maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), coffee (Coffea arabiga), carnation (Dianthus caryophilus), Mexican lime (Citrus × aurantifolia), common bean (Phaseolus vulgaris), broadbeans (Vicia faba), and pea (Pisum sativum), but not runner bean (Phaseolus coccineus). The haplotypes network reveals the genetic variability among Mexican strains and its phylogeographic relationship with Japan, the U.S.A., and China. The presence of this pathogen represents a challenge for plant protection strategies in Mexico.

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.

Genome-wide identification and characterization of InDels and SNPs in Glycine max and Glycine soja for contrasting seed permeability traits.

BACKGROUND: Water permeability governed by seed coat is a major facet of seed crops, especially soybean, whose seeds lack physiological dormancy and experience rapid deterioration in seed viability under prolonged storage. Moreover, the physiological and chemical characteristics of soybean seeds are known to vary with seed coat color. Thus, to underpin the genes controlling water permeability in soybean seeds, we carried out an in-depth characterization of the associated genomic variation. RESULTS: In the present study, we have analyzed genomic variation between cultivated soybean and its wild progenitor with implications on seed permeability, a trait related to seed storability. Whole genome resequencing of G.max and G. soja, identified SNPs and InDels which were further characterized on the basis of their genomic location and impact on gene expression. Chromosomal density distribution of the variation was assessed across the genome and genes carrying SNPs and InDels were characterized into different metabolic pathways. Seed hardiness is a complex trait that is affected by the allelic constitution of a genetic locus as well as by a tricky web of plant hormone interactions. Seven genes that hold a probable role in the determination of seed permeability were selected and their expression differences at different stages of water imbibition were analyzed. Variant interaction network derived 205 downstream interacting partners of 7 genes confirmed their role in seed related traits. Interestingly, genes encoding for Type I- Inositol polyphosphate 5 phosphatase1 and E3 Ubiquitin ligase could differentiate parental genotypes, revealed protein conformational deformations and were found to segregate among RILs in coherence with their permeability scores. The 2 identified genes, thus showed a preliminary association with the desirable permeability characteristics. CONCLUSION: In the light of above outcomes, 2 genes were identified that revealed preliminary, but a relevant association with soybean seed permeability trait and hence could serve as a primary material for understanding the molecular pathways controlling seed permeability traits in soybean.

Genome-wide discovery of tissue-specific miRNAs in clusterbean (Cyamopsis tetragonoloba) indicates their association with galactomannan biosynthesis.

Owing to the presence of 80% soluble dietary fibre, high protein content and high value gum, clusterbean (Cyamopsis tetragonoloba) has recently emerged as an economically important legume. The developing clusterbean seeds accumulate 90% galactomannans in the endosperm and, therefore, can be used as a model crop to understand galactomannan biosynthesis and its regulation. miRNAs are tiny master regulators of their corresponding target genes, resulting in variations in the amounts of their metabolic end products. To understand the role of these regulators in galactomannan biosynthesis regulation, small RNA libraries were prepared and sequenced from five tissues of clusterbean genotype RGC-936, and miRanalyzer and DSAP programs were used to identify conserved miRNAs and novel small RNAs. A total of 187 known and 171 novel miRNAs were found to be differentially expressed, of which 10 miRNAs were validated. A complicated network topology and 35% sharing of the target mRNAs between known and novel miRNAs suggest random evolution of novel miRNAs. The gene ontology (GO) annotation of potential target genes revealed the genes coding for signalling and carbohydrate metabolism (50.10%), kinases and other enzymes (20.75%), transcription factors (10.20%), transporters (8.35%) and other targets (10.6%). Two novel unigenes were annotated as ManS (mannosyltransferase/mannan synthase) and UGE (UDP- D-glucose 4-epimerase) and validated as targets for three novel miRNAs, that is Ct-miR3130, Ct-miR3135 and Ct-miR3157. Our findings reveal that these novel miRNAs could play an important role in the regulation of the galactomannan pathway in C. tetragonoloba and possibly other galactomannan-producing species.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

Global nucleosome positioning regulates salicylic acid mediated transcription in Arabidopsis thaliana.

BACKGROUND: The nucleosome positioning regulates the gene expression and many other DNA-related processes in eukaryotes. Genome-wide mapping of nucleosome positions and correlation of genome-wide nucleosomal remodeling with the changes in the gene expression can help us understanding gene regulation on genome level. RESULTS: In the present study, we correlate the gene expression and the genomic nucleosomal remodeling in response to salicylic acid (SA) treatment in A. thaliana. We have mapped genome-wide nucleosomes by performing tiling microarray using 146 bp mononucleosomal template DNA. The average nucleosomal coverage is approximately 346 bp per nucleosome both under the control and the SA-treated conditions. The nucleosomal coverage is more in the coding region than in the 5' regulatory regions. We observe approximately 50% nucleosomal remodeling on SA treatment where significant nucleosomal depletion and nucleosomal enrichment around the transcription start site (TSS) occur in SA induced genes and SA repressed genes respectively in response to SA treatment. Especially in the case of the SA-induced group, the nucleosomal remodeling over the minimal promoter in response to SA is especially significant in the Non-expresser of PR1 (NPR1)-dependent genes. A detailed investigation of npr1-1 mutant confirms a distinct role of NPR1 in the nucleosome remodeling over the core promoter. We have also identified several motifs for various hormonal responses; including ABRE elements in the remodeled nucleosomal regions around the promoter region in the SA regulated genes. We have further identified that the W-box and TGACG/C motif, reported to play an important role in SA-mediated induction, are enriched in nucleosome free regions (NFRs) of the promoter region of the SA induced genes. CONCLUSIONS: This is the first study reporting genome-wide effects of SA treatment on the chromatin architecture of A. thaliana. It also reports significant role of NPR1 in genome-wide nucleosomal remodeling in response to SA.

Transcriptome dynamics during fibre development in contrasting genotypes of Gossypium hirsutum L.

Understanding the contribution of genetic background in fibre quality traits is important for the development of future cotton varieties with superior fibre quality. We used Affymetrix microarray (Santa Clara, CA) and Roche 454 GSFLX (Branford, CT) for comparative transcriptome analysis between two superior and three inferior genotypes at six fibre developmental stages. Microarray-based analysis of variance (ANOVA) for 89 microarrays encompassing five contrasting genotypes and six developmental stages suggests that the stages of the fibre development have a more pronounced effect on the differentially expressed genes (DEGs) than the genetic background of genotypes. Superior genotypes showed enriched activity of cell wall enzymes, such as pectin methyl esterase, at early elongation stage, enriched metabolic activities such as lipid, amino acid and ribosomal protein subunits at peak elongation, and prolonged combinatorial regulation of brassinosteroid and auxin at later stages. Our efforts on transcriptome sequencing were focused on changes in gene expression at 25 DPA. Transcriptome sequencing resulted in the generation of 475 658 and 429 408 high-quality reads from superior and inferior genotypes, respectively. A total of 24 609 novel transcripts were identified manually for Gossypium hirsutum with no hits in NCBI 'nr' database. Gene ontology analyses showed that the genes for ribosome biogenesis, protein transport and fatty acid biosynthesis were over-represented in superior genotype, whereas salt stress, abscisic acid stimuli and water deprivation leading to the increased proteolytic activity were more pronounced in inferior genotype.

Comparative Genome Analysis of Old World and New World TYLCV Reveals a Biasness toward Highly Variable Amino Acids in Coat Protein.

Begomoviruses, belonging to the family Geminiviridae and the genus Begomovirus, are DNA viruses that are transmitted by whitefly Bemisia tabaci (Gennadius) in a circulative persistent manner. They can easily adapt to new hosts and environments due to their wide host range and global distribution. However, the factors responsible for their adaptability and coevolutionary forces are yet to be explored. Among BGVs, TYLCV exhibits the broadest range of hosts. In this study, we have identified variable and coevolving amino acid sites in the proteins of Tomato yellow leaf curl virus (TYLCV) isolates from Old World (African, Indian, Japanese, and Oceania) and New World (Central and Southern America). We focused on mutations in the coat protein (CP), as it is highly variable and interacts with both vectors and host plants. Our observations indicate that some mutations were accumulating in Old World TYLCV isolates due to positive selection, with the S149N mutation being of particular interest. This mutation is associated with TYLCV isolates that have spread in Europe and Asia and is dominant in 78% of TYLCV isolates. On the other hand, the S149T mutation is restricted to isolates from Saudi Arabia. We further explored the implications of these amino acid changes through structural modeling. The results presented in this study suggest that certain hypervariable regions in the genome of TYLCV are conserved and may be important for adapting to different host environments. These regions could contribute to the mutational robustness of the virus, allowing it to persist in different host populations.

Genome wide expression profiling of two accession of G. herbaceum L. in response to drought.

BACKGROUND: Genome-wide gene expression profiling and detailed physiological investigation were used for understanding the molecular mechanism and physiological response of Gossypium herbaceum, which governs the adaptability of plants in drought conditions. Recently, microarray-based gene expression analysis is commonly used to decipher genes and genetic networks controlling the traits of interest. However, the results of such an analysis are often plagued due to a limited number of genes (probe sets) on microarrays. On the other hand, pyrosequencing of a transcriptome has the potential to detect rare as well as a large number of transcripts in the samples quantitatively. We used Affymetrix microarray as well as Roche's GS-FLX transcriptome sequencing for a comparative analysis of cotton transcriptome in leaf tissues under drought conditions. RESULTS: Fourteen accessions of Gossypium herbaceum were subjected to mannitol stress for preliminary screening; two accessions, namely Vagad and RAHS-14, were selected as being the most tolerant and most sensitive to osmotic stress, respectively. Affymetrix cotton arrays containing 24,045 probe sets and Roche's GS-FLX transcriptome sequencing of leaf tissue were used to analyze the gene expression profiling of Vagad and RAHS-14 under drought conditions. The analysis of physiological measurements and gene expression profiling showed that Vagad has the inherent ability to sense drought at a much earlier stage and to respond to it in a much more efficient manner than does RAHS-14. Gene Ontology (GO) studies showed that the phenyl propanoid pathway, pigment biosynthesis, polyketide biosynthesis, and other secondary metabolite pathways were enriched in Vagad under control and drought conditions as compared with RAHS-14. Similarly, GO analysis of transcriptome sequencing showed that the GO terms responses to various abiotic stresses were significantly higher in Vagad. Among the classes of transcription factors (TFs) uniquely expressed in both accessions, RAHS-14 showed the expression of ERF and WRKY families. The unique expression of ERFs in response to drought conditions reveals that RAHS-14 responds to drought by inducing senescence. This was further supported by transcriptome analysis which revealed that RAHS-14 responds to drought by inducing many transcripts related to senescence and cell death. CONCLUSION: The comparative genome-wide gene expression profiling study of two accessions of G.herbaceum under drought stress deciphers the differential patterns of gene expression, including TFs and physiologically relevant processes. Our results indicate that drought tolerance observed in Vagad is not because of a single molecular reason but is rather due to several unique mechanisms which Vagad has developed as an adaptation strategy.

Genomic Variation and Diversification in Begomovirus Genome in Implication to Host and Vector Adaptation.

Begomoviruses (family Geminiviridae, genus Begomovirus) are DNA viruses transmitted in a circulative, persistent manner by the whitefly Bemisia tabaci (Gennadius). As revealed by their wide host range (more than 420 plant species), worldwide distribution, and effective vector transmission, begomoviruses are highly adaptive. Still, the genetic factors that facilitate their adaptation to a diverse array of hosts and vectors remain poorly understood. Mutations in the virus genome may confer a selective advantage for essential functions, such as transmission, replication, evading host responses, and movement within the host. Therefore, genetic variation is vital to virus evolution and, in response to selection pressure, is demonstrated as the emergence of new strains and species adapted to diverse hosts or with unique pathogenicity. The combination of variation and selection forms a genetic imprint on the genome. This review focuses on factors that contribute to the evolution of Begomovirus and their global spread, for which an unforeseen diversity and dispersal has been recognized and continues to expand.

Variation Profile of the Orthotospovirus Genome.

Orthotospoviruses are plant-infecting members of the family Tospoviridae (order Bunyavirales), have a broad host range and are vectored by polyphagous thrips in a circulative-propagative manner. Because diverse hosts and vectors impose heterogeneous selection constraints on viral genomes, the evolutionary arms races between hosts and their pathogens might be manifested as selection for rapid changes in key genes. These observations suggest that orthotospoviruses contain key genetic components that rapidly mutate to mediate host adaptation and vector transmission. Using complete genome sequences, we profiled genomic variation in orthotospoviruses. Results show that the three genomic segments contain hypervariable areas at homologous locations across species. Remarkably, the highest nucleotide variation mapped to the intergenic region of RNA segments S and M, which fold into a hairpin. Secondary structure analyses showed that the hairpin is a dynamic structure with multiple functional shapes formed by stems and loops, contains sites under positive selection and covariable sites. Accumulation and tolerance of mutations in the intergenic region is a general feature of orthotospoviruses and might mediate adaptation to host plants and insect vectors.

Mutations in virus-derived small RNAs.

RNA viruses exist as populations of genome variants. Virus-infected plants accumulate 21-24 nucleotide small interfering RNAs (siRNAs) derived from viral RNA (virus-derived siRNAs) through gene silencing. This paper describes the profile of mutations in virus-derived siRNAs for three members of the family Potyviridae: Turnip mosaic virus (TuMV), Papaya ringspot virus (PRSV) and Wheat streak mosaic virus (WSMV). For TuMV in Arabidopsis thaliana, profiles were obtained for mechanically inoculated rosette leaves and systemically infected cauline leaves and inflorescence. Results are consistent with selection pressure on the viral genome imposed by local and systemic movement. By genetically removing gene silencing in the plant and silencing suppression in the virus, our results showed that antiviral gene silencing imposes selection in viral populations. Mutations in siRNAs derived from a PRSV coat protein transgene in the absence of virus replication showed the contribution of cellular RNA-dependent RNA polymerases to the generation of mutations in virus-derived siRNAs. Collectively, results are consistent with two sources of mutations in virus-derived siRNAs: viral RNA-dependent RNA polymerases responsible for virus replication and cellular RNA-dependent RNA polymerases responsible for gene silencing amplification.

Genome-Wide Variation in Potyviruses.

Potyviruses (family Potyviridae, genus Potyvirus) are the result of an initial radiation event that occurred 6,600 years ago. The genus currently consists of 167 species that infect monocots or dicots, including domesticated and wild plants. Potyviruses are transmitted in a non-persistent way by more than 200 species of aphids. As indicated by their wide host range, worldwide distribution, and diversity of their vectors, potyviruses have an outstanding capacity to adapt to new hosts and environments. However, factors that confer adaptability are poorly understood. Viral RNA-dependent RNA polymerases introduce nucleotide substitutions that generate genetic diversity. We hypothesized that selection imposed by hosts and vectors creates a footprint in areas of the genome involved in host adaptation. Here, we profiled genomic and polyprotein variation in all species in the genus Potyvirus. Results showed that the potyviral genome is under strong negative selection. Accordingly, the genome and polyprotein sequence are remarkably stable. However, nucleotide and amino acid substitutions across the potyviral genome are not randomly distributed and are not determined by codon usage. Instead, substitutions preferentially accumulate in hypervariable areas at homologous locations across potyviruses. At a frequency that is higher than that of the rest of the genome, hypervariable areas accumulate non-synonymous nucleotide substitutions and sites under positive selection. Our results show, for the first time, that there is correlation between host range and the frequency of sites under positive selection. Hypervariable areas map to the N terminal part of protein P1, N and C terminal parts of helper component proteinase (HC-Pro), the C terminal part of protein P3, VPg, the C terminal part of NIb (RNA-dependent RNA polymerase), and the N terminal part of the coat protein (CP). Additionally, a hypervariable area at the NIb-CP junction showed that there is variability in the sequence of the NIa protease cleavage sites. Structural alignment showed that the hypervariable area in the CP maps to the N terminal flexible loop and includes the motif required for aphid transmission. Collectively, results described here show that potyviruses contain fixed hypervariable areas in key parts of the genome which provide mutational robustness and are potentially involved in host adaptation.

Expressivity of the key genes associated with seed and pod development is highly regulated via lncRNAs and miRNAs in Pigeonpea.

Non-coding RNA's like miRNA, lncRNA, have gained immense importance as a significant regulatory factor in different physiological and developmental processes in plants. In an effort to understand the molecular role of these regulatory agents, in the present study, 3019 lncRNAs and 227 miRNAs were identified from different seed and pod developmental stages in Pigeonpea, a major grain legume of Southeast Asia and Africa. Target analysis revealed that 3768 mRNAs, including 83 TFs were targeted by lncRNAs; whereas 3060 mRNA, including 154 TFs, were targeted by miRNAs. The targeted transcription factors majorly belong to WRKY, MYB, bHLH, etc. families; whereas the targeted genes were associated with the embryo, seed, and flower development. Total 302 lncRNAs interact with miRNAs and formed endogenous target mimics (eTMs) which leads to sequestering of the miRNAs present in the cell. Expression analysis showed that notably, Cc_lncRNA-2830 expression is up-regulated and sequestrates miR160h in pod leading to higher expression of the miR160h target gene, Auxin responsive factor-18. A similar pattern was observed for SPIKE, Auxin signaling F-box-2, Bidirectional sugar transporter, and Starch synthetase-2 eTMs. All the identified target mRNAs code for transcription factor and genes are involved in the processes like cell division, plant growth and development, starch synthesis, sugar transportation and accumulation of storage proteins which are essential for seed and pod development. On a combinatorial basis, our study provides a lncRNA and miRNA based regulatory insight into the genes governing seed and pod development in Pigeonpea.

Whitefly and aphid inducible promoters of Arabidopsis thaliana L.

Lack of regulated expression and tissue specificity are the major drawbacks of plant and virus-derived constitutive promoters. A precise tissue or site-specific expression, facilitate regulated expression of proteins at the targeted time and site. Publically available microarray data on whitefly and aphid infested Arabidopsis thaliana L. was used to identify whitefly and aphid-inducible genes. The qRT-PCR further validated the inducible behaviour of these genes under artificial infestation. Promoter sequences of genes were retrieved from the Arabidopsis Information Resources database with their corresponding 5'UTR and cloned from the A. thaliana genome. Promoter reporter transcriptional fusions were developed with the beta-glucuronidase (GUS) gusA gene in a binary expression vector to validate the inducible behaviour of these promoters in eight independent transgenic Nicotiana tabaccum lines. Histochemical analysis of the reporter gene in T2 transgenic tobacco lines confirmed promoter driven expression at the sites of aphid and whitefly infestation. The qRT-PCR and GUS expression analysis of transgenic lines revealed that abscisic acid largely influenced the expression of both aphid and whitefly inducible promoters. Further, whitefly-specific promoter respond to salicylic acid and jasmonic acid (JA), whereas aphid-specific promoters to JA and 1-aminocyclopropane carboxylic acid. The response of promoters to phytohormones correlated to the presence of corresponding conserved cis-regulatory elements.

De novo Assembly and Characterization of Cajanus scarabaeoides (L.) Thouars Transcriptome by Paired-End Sequencing.

Pigeonpea [Cajanus cajan (L.) Millsp.] is a heat and drought resilient legume crop grown mostly in Asia and Africa. Pigeonpea is affected by various biotic (diseases and insect pests) and abiotic stresses (salinity and water logging) which limit the yield potential of this crop. However, resistance to all these constraints is not readily available in the cultivated genotypes and some of the wild relatives have been found to withstand these resistances. Thus, the utilization of crop wild relatives (CWR) in pigeonpea breeding has been effective in conferring resistance, quality and breeding efficiency traits to this crop. Bud and leaf tissue of Cajanus scarabaeoides, a wild relative of pigeon pea were used for transcriptome profiling. Approximately 30 million clean reads filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (3.02 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were pooled and assembled de novo into 1,17,007 transcripts using the Trinity. Finally, a total of 98,664 unigenes were derived with mean length of 396 bp and N50 values of 1393. The assembly produced significant mapping results (73.68%) in BLASTN searches of the Glycine max CDS sequence database (Ensembl). Further, uniprot database of Viridiplantae was used for unigene annotation; 81,799 of 98,664 (82.90%) unigenes were finally annotated with gene descriptions or conserved protein domains. Further, a total of 23,475 SSRs were identified in 27,321 unigenes. This data will provide useful information for mining of functionally important genes and SSR markers for pigeonpea improvement.

Regulation of Isoflavone Biosynthesis by miRNAs in Two Contrasting Soybean Genotypes at Different Seed Developmental Stages.

Owing to the presence of nutritionally important, health-promoting bioactive compounds, especially isoflavones, soybean has acquired the status of a functional food. miRNAs are tiny riboregulator of gene expression by either decreasing and/or increasing the expression of their corresponding target genes. Despite several works on identification and functional characterization of plant miRNAs, the role of miRNAs in the regulation of isoflavones metabolism is still a virgin field. In the present study, we identified a total of 31 new miRNAs along with their 245 putative target genes from soybean seed-specific ESTs using computational approach. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates metabolism and genetic information processing. Out of that, a total of 5 miRNAs (Gma-miRNA12, Gma-miRNA24, Gma-miRNA26, Gma-miRNA28, and Gma-miRNA29) were predicted and validated for their probable role during isoflavone biosynthesis. We also validated their five target genes using RA-PCR, which is as good as 5'RLM-RACE. Temporal regulation [35 days after flowering, 45, 55, and 65 DAF] of miRNAs and their targets showed differential expression schema. Differential expression of Gma-miR26 and Gma-miRNA28 along with their corresponding target genes (Glyma.10G197900 and Glyma.09G127200) showed a direct relationship with the total isoflavone content. Therefore, understanding the miRNA-based genetic regulation of isoflavone pathway would assist in selection and manipulation to get high-performing soybean genotypes with better isoflavone yield.

High Quality Unigenes and Microsatellite Markers from Tissue Specific Transcriptome and Development of a Database in Clusterbean (Cyamopsis tetragonoloba, L. Taub).

Clusterbean (Cyamopsis tetragonoloba L. Taub), is an important industrial, vegetable and forage crop. This crop owes its commercial importance to the presence of guar gum (galactomannans) in its endosperm which is used as a lubricant in a range of industries. Despite its relevance to agriculture and industry, genomic resources available in this crop are limited. Therefore, the present study was undertaken to generate RNA-Seq based transcriptome from leaf, shoot, and flower tissues. A total of 145 million high quality Illumina reads were assembled using Trinity into 127,706 transcripts and 48,007 non-redundant high quality (HQ) unigenes. We annotated 79% unigenes against Plant Genes from the National Center for Biotechnology Information (NCBI), Swiss-Prot, Pfam, gene ontology (GO) and KEGG databases. Among the annotated unigenes, 30,020 were assigned with 116,964 GO terms, 9984 with EC and 6111 with 137 KEGG pathways. At different fragments per kilobase of transcript per millions fragments sequenced (FPKM) levels, genes were found expressed higher in flower tissue followed by shoot and leaf. Additionally, we identified 8687 potential simple sequence repeats (SSRs) with an average frequency of one SSR per 8.75 kb. A total of 28 amplified SSRs in 21 clusterbean genotypes resulted in polymorphism in 13 markers with average polymorphic information content (PIC) of 0.21. We also constructed a database named 'ClustergeneDB' for easy retrieval of unigenes and the microsatellite markers. The tissue specific genes identified and the molecular marker resources developed in this study is expected to aid in genetic improvement of clusterbean for its end use.

Expression of GhNAC2 from G. herbaceum, improves root growth and imparts tolerance to drought in transgenic cotton and Arabidopsis.

NAC proteins are plant-specific transcription factors that play essential roles in regulating development and responses to abiotic and biotic stresses. We show that over-expression of the cotton GhNAC2 under the CaMV35S promoter increases root growth in both Arabidopsis and cotton under unstressed conditions. Transgenic Arabidopsis plants also show improved root growth in presence of mannitol and NaCl while transgenic cotton expressing GhNAC2 show reduced leaf abscission and wilting upon water stress compared to control plants. Transgenic Arabidopsis plants also have larger leaves, higher seed number and size under well watered conditions, reduced transpiration and higher relative leaf water content. Micro-array analysis of transgenic plants over-expressing GhNAC2 reveals activation of the ABA/JA pathways and a suppression of the ethylene pathway at several levels to reduce expression of ERF6/ERF1/WRKY33/ MPK3/MKK9/ACS6 and their targets. This probably suppresses the ethylene-mediated inhibition of organ expansion, leading to larger leaves, better root growth and higher yields under unstressed conditions. Suppression of the ethylene pathway and activation of the ABA/JA pathways also primes the plant for improved stress tolerance by reduction in transpiration, greater stomatal control and suppression of growth retarding factors.