Transcriptome and metabolite analyses in Azadirachta indica: identification of genes involved in biosynthesis of bioactive triterpenoids

Bhambhani S, Lakhwani D, Gupta P, et al. Transcriptome and metabolite analyses in Azadirachta indica: identification of genes involved in biosynthesis of bioactive triterpenoids. SCIENTIFIC REPORTS. 2017;7(1): 5043.Azadirachta indica A. Juss, commonly known as Neem, is the reservoir of triterpenoids of economic importance. Metabolite analysis of different developmental stages of leaf and fruit suggests tissue-specific accumulation of the major triterpenoids in this important tree. Though biosynthesis of these complex molecules requires substrate flux from the isoprenoid pathway, enzymes involved in late biosynthetic steps remain uncharacterized. We established and analyzed transcriptome datasets from leaf and fruit and identified members of gene families involved in intermediate steps of terpenoid backbone biosynthesis and those related to secondary transformation leading to the tissue-specific triterpenoid biosynthesis. Expression analysis suggests differential expression of number of genes between leaf and fruit and probable participation in the biosynthesis of fruit-specific triterpenoids. Genome-wide analysis also identified members of gene families putatively involved in secondary modifications in late biosynthetic steps leading to the synthesis of highly oxygenated triterpenoids. Expression and molecular docking analyses suggest involvement of specific members of CYP450 family in secondary modifications for the biosynthesis of bioactive triterpenoids. This study generated rich genomic resource and identified genes involved in biosynthesis of important molecules, which will aid in the advancement of tools for functional genomics and elucidation of the biosynthesis of triterpenoid from this important tree

Association mapping reveals novel genes and genomic regions controlling grain size architecture in mini core accessions of Indian National Genebank wheat germplasm collection

Wheat (Triticum aestivum L.) is a staple food crop for the global human population, and thus wheat breeders are consistently working to enhance its yield worldwide. In this study, we utilized a sub-set of Indian wheat mini core germplasm to underpin the genetic architecture for seed shape-associated traits. The wheat mini core subset (125 accessions) was genotyped using 35K SNP array and evaluated for grain shape traits such as grain length (GL), grain width (GW), grain length, width ratio (GLWR), and thousand grain weight (TGW) across the seven different environments (E1, E2, E3, E4, E5, E5, E6, and E7). Marker-trait associations were determined using a multi-locus random-SNP-effect Mixed Linear Model (mrMLM) program. A total of 160 non-redundant quantitative trait nucleotides (QTNs) were identified for four grain shape traits using two or more GWAS models. Among these 160 QTNs, 27, 36, 38, and 35 QTNs were associated for GL, GW, GLWR, and TGW respectively while 24 QTNs were associated with more than one trait. Of these 160 QTNs, 73 were detected in two or more environments and were considered reliable QTLs for the respective traits. A total of 135 associated QTNs were annotated and located within the genes, including ABC transporter, Cytochrome450, Thioredoxin_M-type, and hypothetical proteins. Furthermore, the expression pattern of annotated QTNs demonstrated that only 122 were differentially expressed, suggesting these could potentially be related to seed development. The genomic regions/candidate genes for grain size traits identified in the present study represent valuable genomic resources that can potentially be utilized in the markers-assisted breeding programs to develop high-yielding varieties

Analysis of the Rdr1 gene family in different Rosaceae genomes reveals an origin of an R-gene cluster after the split of Rubeae within the Rosoideae subfamily

The Rdr1 gene confers resistance to black spot in roses and belongs to a large TNL gene family, which is organized in two major clusters at the distal end of chromosome 1. We used the recently available chromosome scale assemblies for the R. chinensis ‘Old Blush’ genome, re-sequencing data for nine rose species and genome data for Fragaria, Rubus, Malus and Prunus to identify Rdr1 homologs from different taxa within Rosaceae. Members of the Rdr1 gene family are organized into two major clusters in R. chinensis and at a syntenic location in the Fragaria genome. Phylogenetic analysis indicates that the two clusters existed prior to the split of Rosa and Fragaria and that one cluster has a more recent origin than the other. Genes belonging to cluster 2, such as the functional Rdr1 gene muRdr1A, were subject to a faster evolution than genes from cluster 1. As no Rdr1 homologs were found in syntenic positions for Prunus persica, Malus x domestica and Rubus occidentalis, a translocation of the Rdr1 clusters to the current positions probably happened after the Rubeae split from other groups within the Rosoideae approximately 70–80 million years ago during the Cretaceous period

Comparative transcriptomic analysis of roots of contrasting <it>Gossypium herbaceum</it> genotypes revealing adaptation to drought

<p>Abstract</p> <p>Background</p> <p>Root length and its architecture govern the adaptability of plants to various stress conditions, including drought stress. Genetic variations in root growth, length, and architecture are genotypes dependent. In this study, we compared the drought-induced transcriptome of four genotypes of <it>Gossypium herbaceum</it> that differed in their drought tolerance adaptability. Three different methodologies, namely, microarray, pyrosequencing, and qRT–PCR, were used for transcriptome analysis and validation.</p> <p>Results</p> <p>The variations in root length and growth were found among four genotypes of <it>G.herbaceum</it> when exposed to mannitol-induced osmotic stress. Under osmotic stress, the drought tolerant genotypes Vagad and GujCot-21 showed a longer root length than did by drought sensitive RAHS-14 and RAHS-IPS-187. Further, the gene expression patterns in the root tissue of all genotypes were analyzed. We obtained a total of 794 differentially expressed genes by microarray and 104928 high-quality reads representing 53195 unigenes from the root transcriptome. The Vagad and GujCot-21 respond to water stress by inducing various genes and pathways such as response to stresses, response to water deprivation, and flavonoid pathways. Some key regulatory genes involved in abiotic stress such as AP2 EREBP, MYB, WRKY, ERF, ERD9, and LEA were highly expressed in Vagad and GujCot-21. The genes RHD3, NAP1, LBD, and transcription factor WRKY75, known for root development under various stress conditions, were expressed specifically in Vagad and GujCot-21. The genes related to peroxidases, transporters, cell wall-modifying enzymes, and compatible solutes (amino acids, amino sugars, betaine, sugars, or sugar alcohols) were also highly expressed in Vagad and Gujcot-21.</p> <p>Conclusion</p> <p>Our analysis highlights changes in the expression pattern of genes and depicts a small but highly specific set of drought responsive genes induced in response to drought stress. Some of these genes were very likely to be involved in drought stress signaling and adaptation, such as transmembrane nitrate transporter, alcohol dehydrogenase, pyruvate decarboxylase, sucrose synthase, and LEA. These results might serve as the basis for an in-depth genomics study of <it>Gossypium herbaceum</it>, including a comparative transcriptome analysis and the selection of genes for root traits and drought tolerance.</p

Histogram of gene ontology classification.

<p>The results are summarized in three main categories: Biological Process, Cellular Component and Molecular Function. Bars represent the assign percent of unigenes in <i>pap1</i> (white bars) and BR086 (black bars) with BLAST matches in the TAIR9 database to each GO term.</p

Comparative Transcriptome Analysis Using High Papaverine Mutant of <i>Papaver somniferum</i> Reveals Pathway and Uncharacterized Steps of Papaverine Biosynthesis

<div><p>The benzylisoquinoline alkaloid papaverine, synthesized in low amount in most of the opium poppy varieties of <i>Papaver somniferum</i>, is used as a vasodilator muscle relaxant and antispasmodic. Papaverine biosynthesis remains controversial as two different routes utilizing either (S)-coclaurine or (S)-reticuline have been proposed with uncharacterized intermediate steps. In an attempt to elucidate papaverine biosynthesis and identify putative genes involved in uncharacterized steps, we carried out comparative transcriptome analysis of high papaverine mutant (<i>pap1</i>) and normal cultivar (BR086) of <i>P. somniferum</i>. This natural mutant synthesizes more than 12-fold papaverine in comparison to BR086. We established more than 238 Mb transcriptome data separately for <i>pap1</i> and BR086. Assembly of reads generated 127,342 and 106,128 unigenes in <i>pap1</i> and BR086, respectively. Digital gene expression analysis of transcriptomes revealed 3,336 differentially expressing unigenes. Enhanced expression of (S)-norcoclaurine-6-O-methyltransferase (6OMT), (S)-3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT), norreticuline 7-O-methyltransferase (N7OMT) and down-regulation of reticuline 7-O-methyltransferase (7OMT) in <i>pap1</i> in comparison to BR086 suggest (S)-coclaurine as the route for papaverine biosynthesis. We also identified several methyltransferases and dehydrogenases with enhanced expression in <i>pap1</i> in comparison to BR086. Our analysis using natural mutant, <i>pap1</i>, concludes that (S)-coclaurine is the branch-point intermediate and preferred route for papaverine biosynthesis. Differentially expressing methyltransferases and dehydrogenases identified in this study will help in elucidating complete biosynthetic pathway of papaverine. The information generated will be helpful in developing strategies for enhanced biosynthesis of papaverine through biotechnological approaches.</p></div

Differential expression of methyltransferases (a) and dehydrogenases (b) with their differential expression in <b><i>pap1</i></b> and BR086.

<p>Two columns in each represent <i>pap1</i> and BR086, while each row represents contigs encoding members of these families (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065622#pone.0065622.s014" target="_blank">Table S10</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065622#pone.0065622.s015" target="_blank">S11</a>). Clustering was carried out with log tpm value of each contig in <i>pap1</i> and BR086 transcriptome to visualize differential expression.</p

Phenotype of fresh latex and specific alkaloid content in <b><i>pap1</i></b><b> and BR086.</b>

<p>Fresh latex of BR086 is brown or red whereas <i>pap1</i> exuded white latex (a). Content of specific alkaloids in latex was measured in <i>pap1</i> and BR086 (b) using High Pressure Liquid Chromatography. *** indicate values that differ significantly from BR086.</p

Sequencing reads, assembly and annotation.

<p>Size distribution of 454 high-quality reads (a) and assembled contigs (b). Black and white bars represents reads and contigs from <i>pap1</i> and BR086 transcriptome data respectively. Annotation of unigenes of <i>pap1</i> (c) and BR086 (d) was carried out against TAIR, NR and CDD databases. Out of all annotated unigenes, 25,343 and 19,225 unigenes from <i>pap1</i> and BR086, respectively, had common BLAST hits to annotated proteins by different databases.</p

Combined assembly and annotation.

<p>Size distribution of combined assembled contig length and frequency (a) using high–quality reads from <i>pap1</i> and BR086. Annotation statistics (b) of unigenes of combined assembly queried against TAIR, NR and CDD databases.</p