Transcriptome analysis for molecular landscaping of genes controlling diterpene andrographolide biosynthesis in Andrographis paniculata (Burm . f.) Nees.

Kalmegh [Andrographis paniculata (Burm. f.) Nees.] is one of the essential medicinal plants due to an important terpenoid, i.e. andrographolide possesses immense therapeutic and pharmacological uses. The experiment was performed to elucidate the expression of candidate genes associated with andrographolide biosynthesis. Based on results obtained in chromatography for andrographolide content analysis of six genotypes, two contrast genotypes, i.e. IC-520361 (maximum andrographolide content-2.33%) and Anand Local (lowest andrographolide content-1.01%) were selected for the transcriptome analysis. A total of 1.04 Gb of raw data were produced using MiSeq Illumina platform, in which IC 520361 generated 645 million base pairs sequence along with 4,524,251 raw reads and Anand Local produced 419 million base pairs sequence along with 3,021,316 raw reads. The combined assembly of high quality reads generated for both the samples had 33,247,454 bp of total assembled bases and 38,292 of transcripts. The GC percent of assembled transcripts was 44.79%, an average read length was 800 bp and N50 value was 1186 bp. Species-specific distribution using BLAST X (Nr), showed the highest Blast hits with Sesamum indicum. Out of 23,346 transcripts, 87% of transcripts annotated in UniProt KB (Universal Protein Resource KnowledgeBase) database and only 0.21% of transcripts were annotated in TAIR (The Arabidopsis Information Resources). Biological processes gene ontology classified based on Blast2GO showed, out of 6853 transcripts, 1370 of transcripts were represented by terpenoid biosynthetic pathway, which involved in secondary metabolite andrographolide biosynthesis. The heat map showed 1016 transcripts were differentially expressed between two kalmegh genotypes, in which nine important differentially expressed transcripts related to MEP (2C methyl-d-erythritol 4-phosphate) and MVA (Mevalonic acid) andrographolide biosynthesis pathways such as, geranyl diphosphate synthase small subunit, Isopentenyl-diphosphate delta-isomerase i-like, 4, 13-hydroxy-3-methylglutaryl-coenzyme a reductase etc. were upregulated in IC 520361 as compared to Anand Local, which were validated through RT-qPCR. The highest expression of gene 13-hydroxy-3-methylglutaryl-coenzyme a reductase (HMGR) was reported, which is responsible for accumulation of andrographolide in leaf. This comparative transcriptome analysis confirmed the expression level of genes were higher in accession IC 520361 as compare to Anand Local related to andrographolide biosynthesis pathways i.e. MEP and MVA. These up-regulated genes could be over-expressed to enhance the andrographolide content using genetic engineering of these metabolic pathways. It will also give an idea to the breeder for development of molecular markers for direct screening of the genotypes.

Metagenomic characterisation of ruminal bacterial diversity in buffaloes from birth to adulthood using 16S rRNA gene amplicon sequencing.

Microbial colonisation in the forestomach of a ruminant is one of the most crucial factors in determining many of its physiological developments and digestive capabilities. The present study attempts to identify establishment pattern of microbes in relation to food, age and rumen development in the buffalo calves at every fortnight interval from birth to 6 months of age, followed by every month till animals became 1 year of age. Diversity study based on 16S rRNA gene sequencing identified rapidly changing bacterial population during initial 60 days of life, which got assemblage as rumen became physiologically mature with increasing age of animals. A lactate fermenting aerobic to facultative anaerobic genera found during initial 30 days of life were expeditiously replaced by strict anaerobic cellulolytic bacterial population with increasing age. The study confirms that initial colonisation mainly depends on the oral cavity and skin of the mother, followed by the surrounding environment and feed offered, which is reversed in order once animal gets older. Some of the well-described genera based on culture-dependent studies like Ruminococcus spp. were found to be in lesser proportion suggesting an additional role of other microbes or niche in cellulose degradation. We report the presence of Porphyromonas spp. and Mannheimia glucosidal for the first time in bovine infants.

Transcriptomic profiling of developing fiber in levant cotton (Gossypium herbaceum L.).

Cotton (Gossypium spp.) is an imperative economic crop of the globe due to its natural textile fiber. Molecular mechanisms of fiber development have been greatly revealed in allotetraploid cotton but remained unexplored in Gossypium herbaceum. G. herbaceum can withstand the rigors of nature like drought and pests but produce coarse lint. This undesirable characteristic strongly needs the knowledge of fiber development at molecular basis. The present study reported the transcriptome sequence of the developing fiber of G. herbaceum on pyrosequencing and its analysis. About 1.38 million raw and 1.12 million quality trimmed reads were obtained followed by de novo assembly-generated 20,125 unigenes containing 14,882 coding sequences (CDs). BLASTx-based test of homology indicated that A1-derived transcripts shared a high similarity with Gossypium arboreum (A2). Functional annotation of the CDs using the UniProt categorized them into biological processes, cellular components, and molecular function, COG classification showed that a large number of CDs have significant homology in COG database (6215 CDs), and mapping of CDs with Kyoto Encyclopedia of Genes and Genomes (KEGG) database generated 200 pathways ultimately showing predominant engagement in the fiber development process. Transcription factors were predicted by comparison with Plant Transcription Factor Database, and their differential expression between stages exposed their important regulatory role in fiber development. Differential expression analysis based on reads per kilobase of transcript per million mapped reads (RPKM) value revealed activities of specific gene related to carbohydrate and lipid synthesis, carbon metabolism, energy metabolism, signal transduction, etc., at four stages of fiber development, and was validated by qPCR. Overall, this study will help as a valuable foundation for diploid cotton fiber improvement.

Molecular diversity of Annona species and proximate fruit composition of selected genotypes.

Understanding the genetic variation in germplasm is of utmost importance for crop improvement. Therefore, efforts were made to analyse the molecular marker based genetic diversity of 20 Annona genotypes from five different species of family Annonaceae. During analysis, a set of 11 RAPD primers yielded a total of 152 bands with 80.01 % polymorphism and PIC for RAPD ranged from 0.86 to 0.92 with a mean of 0.89. With 93.05 % polymorphism, 12 SSR primers produced 39 amplicons. The PIC for SSRs ranged from 0.169 to 0.694 with of average of 0.339. The dendrogram produced from pooled molecular data of 11 RAPD and 12 SSR primers showed seven clusters at a cutoff value of 0.78. The dendrogram discriminated all the Annona genotypes suggesting that significant genetic diversity was present among the genotypes. Proximate fruit composition study of nine fruiting genotypes of Annona revealed that A. squamosa possessed significantly higher amount of most of studies biochemical which gives an opportunity to fruit breeders to improve the other Annona species. Likewise, A. muricata being rich in seed oil content can be exploited in oil industries.