Structural and functional analysis of CCT family genes in pigeonpea.

BACKGROUND: Pigeonpea (Cajanus cajan L.) is a photoperiod-sensitive short-day plant. Understanding the flowering-related genes is critical to developing photoperiod insensitive cultivars. METHODS: The CCT family genes were identified using 'CCT DOMAIN PROTEIN' as a keyword and localized on the chromosomes using the BLAST search option available at the LIS database. The centromeric positions were identified through BLAST search using the centromeric repeat sequence of C. cajan as a query against the chromosome-wise FASTA files downloaded from the NCBI database. The CCT family genes were classified based on additional domains and/or CCT domains. The orthologous and phylogenetic relationships were inferred using the OrthoFinder and MEGA 10.1 software, respectively. The CCT family genes' expression level in photoperiod-sensitive and insensitive genotypes was compared using RNA-seq data and qRT-PCR analysis. RESULTS: We identified 33 CCT family genes in C. cajan distributed on ten chromosomes and nine genomic scaffolds. They were classified into CMF-type, COL-type, PRR-type, and GTCC- type. The CCT family genes of legumes exhibited an extensive orthologous relationship. Glycine max showed the maximum similarity of CCT family genes with C. cajan. The expression analysis of CCT family genes using photoperiod insensitive (ICP20338) and photoperiod sensitive (MAL3) genotypes of C. cajan demonstrated that CcCCT4 and CcCCT23 are the active CONSTANS in ICP20338. In contrast, only CcCCT23 is active in MAL3. CONCLUSION: The CCT family genes in C. cajan vary considerably in structure and domain types. They are maximally similar to soybean's CCT family genes. The differential photoperiod response of pigeonpea genotypes, ICP20338 and MAL3, is possibly due to the difference in the number and types of active CONSTANS in them.

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.

Comparative RNA editing profile of mitochondrial transcripts in cytoplasmic male sterile and fertile pigeonpea reveal significant changes at the protein level.

RNA editing is a process which leads to post-transcriptional alteration of the nucleotide sequence of the corresponding mRNA molecule which may or may not lead to changes at the protein level. Apart from its role in providing variability at the transcript and protein levels, sometimes, such changes may lead to abnormal expression of the mitochondrial gene leading to a cytoplasmic male sterile phenotype. Here we report the editing status of 20 major mitochondrial transcripts in both male sterile (AKCMS11) and male fertile (AKPR303) pigeonpea genotypes. The validation of the predicted editing sites was done by mapping RNA-seq reads onto the amplified mitochondrial genes, and 165 and 159 editing sites were observed in bud tissues of the male sterile and fertile plant respectively. Among the resulting amino acid alterations, the most frequent one was the conversion of hydrophilic amino acids to hydrophobic. The alterations thus detected in our study indicates differential editing, but no major change in terms of the abnormal protein structure was detected. However, the above investigation provides an insight into the behaviour of pigeonpea mitochondrial genome in native and alloplasmic state and could hold clues in identification of editing factors and their role in adaptive evolution in pigeonpea.

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.

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 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.

The chloroplast genome of black pepper (Piper nigrum L.) and its comparative analysis with related Piper species.

Piper nigrum, also known as black pepper, is an economically and ecologically important crop of the genus Piper. It has been titled as the king of spices due to its wide consumption throughout the world. In the present investigation, the chloroplast genome of P. nigrum has been assembled from a whole genome sequence by integrating the short and long reads generated through Illumina and PacBio platforms, respectively. The chloroplast genome was observed to be 161,522 bp in size, having a quadripartite structure with a large single copy (LSC) region of 89,153 bp and a small single copy (SSC) region of 18,255 bp separated by a copy of inverted repeats (IRs), each 27,057 bp in length. Taking into consideration all the duplicated genes, a total of 131 genes were observed, which included 81 protein-coding genes, 37 tRNAs, 4 rRNAs, and 1 pseudogene. Individually, the LSC region consisted of 83 genes, the SSC region had 13 genes, and 18 genes were present in each IR region. Additionally, 216 SSRs were detected and 11 of these were validated through amplification in 12 species of Piper. The features of the chloroplast genome have been compared with those of the genus Piper. Our results provide useful insights into evolutionary and molecular studies of black pepper which will contribute to its further genetic improvement and breeding.

Methods and Tools for Plant Organelle Genome Sequencing, Assembly, and Downstream Analysis.

Organelles play an important role in a eukaryotic cell. Among them, the two organelles, chloroplast and mitochondria, are responsible for the critical function of photosynthesis and aerobic respiration. Organellar genomes are also very important for plant systematic studies. Here we have described the methods for isolation of the mitochondrial and plastid DNA and its subsequent sequencing with the help of NGS technology. We have also discussed in detail the various tools available for assembly, annotation, and visualization of the organelle genome sequence.

Elucidation of Galactomannan Biosynthesis Pathway Genes through Transcriptome Sequencing of Seeds Collected at Different Developmental Stages of Commercially Important Indian Varieties of Cluster Bean (Cyamopsis tetragonoloba L.).

Cyamopsis tetragonoloba (L) endosperm predominantly contains guar gum a polysaccharide, which has tremendous industrial applications in food, textile, paper, oil drilling and water treatment. In order to understand the genes controlling galactomannan biosynthesis, mRNA was isolated from seeds collected at different developmental stages; young pods, mature pods and young leaf from two guar varieties, HG365 and HG870 and subjected to Illumina sequencing. De novo assembly of fourteen individual read files from two varieties of guar representing seven developmental stages gave a total of 1,13,607 contigs with an N50 of 1,244 bases. Annotation of assemblies with GO mapping revealed three levels of distribution, namely, Biological Processes, Molecular Functions and Cellular Components. GO studies identified major genes involved in galactomannan biosynthesis: Cellulose synthase D1 (CS D1) and GAUT-like gene families. Among the polysaccharide biosynthetic process (GO:0000271) genes the transcript abundance for CS was found to be predominantly more in leaf samples, whereas, the transcript abundance for GAUT-like steadily increased from 65% to 90% and above from stage1 to stage5 indicating accumulation of galactomannan in developing seeds; and validated by qRT-PCR analysis. Galactomannan quantification by HPLC showed HG365 (12.98-20.66%) and HG870 (7.035-41.2%) gradually increasing from stage1 to stage 5 (10-50 DAA) and highest accumulation occurred in mature and dry seeds with 3.8 to 7.1 fold increase, respectively. This is the first report of transcriptome sequencing and complete profiling of guar seeds at different developmental stages, young pods, mature pods and young leaf material from two commercially important Indian varieties and elucidation of galactomannan biosynthesis pathway. It is envisaged that the data presented herein will be very useful for improvement of guar through biotechnological interventions in future.

Chloroplast Genome Sequence of Clusterbean (Cyamopsis tetragonoloba L.): Genome Structure and Comparative Analysis.

Clusterbean (Cyamopsis tetragonoloba L.), also known as guar, belongs to the family Leguminosae, and is an annual herbaceous legume. Guar is the main source of galactomannan for gas mining industries. In the present study, the draft chloroplast genome of clusterbean was generated and compared to some of the previously reported legume chloroplast genomes. The chloroplast genome of clusterbean is 152,530 bp in length, with a quadripartite structure consisting of large single copy (LSC) and small single copy (SSC) of 83,025 bp and 17,879 bp in size, respectively, and a pair of inverted repeats (IRs) of 25,790 bp in size. The chloroplast genome contains 114 unique genes, which includes 78 protein coding genes, 30 tRNAs, 4 rRNAs genes, and 2 pseudogenes. It also harbors a 50 kb inversion, typical of the Leguminosae family. The IR region of the clusterbean chloroplast genome has undergone an expansion, and hence, the whole rps19 gene is included in the IR, as compared to other legume plastid genomes. A total of 220 simple sequence repeats (SSRs) were detected in the clusterbean plastid genome. The analysis of the clusterbean plastid genome will provide useful insights for evolutionary, molecular and genetic engineering studies.

Chloroplast Genome Sequence of Pigeonpea (Cajanus cajan (L.) Millspaugh) and Cajanus scarabaeoides (L.) Thouars: Genome Organization and Comparison with Other Legumes.

Pigeonpea (Cajanus cajan (L.) Millspaugh), a diploid (2n = 22) legume crop with a genome size of 852 Mbp, serves as an important source of human dietary protein especially in South East Asian and African regions. In this study, the draft chloroplast genomes of Cajanus cajan and Cajanus scarabaeoides (L.) Thouars were generated. Cajanus scarabaeoides is an important species of the Cajanus gene pool and has also been used for developing promising CMS system by different groups. A male sterile genotype harboring the C. scarabaeoides cytoplasm was used for sequencing the plastid genome. The cp genome of C. cajan is 152,242bp long, having a quadripartite structure with LSC of 83,455 bp and SSC of 17,871 bp separated by IRs of 25,398 bp. Similarly, the cp genome of C. scarabaeoides is 152,201bp long, having a quadripartite structure in which IRs of 25,402 bp length separates 83,423 bp of LSC and 17,854 bp of SSC. The pigeonpea cp genome contains 116 unique genes, including 30 tRNA, 4 rRNA, 78 predicted protein coding genes and 5 pseudogenes. A 50 kb inversion was observed in the LSC region of pigeonpea cp genome, consistent with other legumes. Comparison of cp genome with other legumes revealed the contraction of IR boundaries due to the absence of rps19 gene in the IR region. Chloroplast SSRs were mined and a total of 280 and 292 cpSSRs were identified in C. scarabaeoides and C. cajan respectively. RNA editing was observed at 37 sites in both C. scarabaeoides and C. cajan, with maximum occurrence in the ndh genes. The pigeonpea cp genome sequence would be beneficial in providing informative molecular markers which can be utilized for genetic diversity analysis and aid in understanding the plant systematics studies among major grain legumes.