Genome-wide characterization of small RNA, gene expression and DNA methylation changes in response to salt stress in Musa acuminata / Gudimella Ranganath

Gudimella , Ranganath (2018) Genome-wide characterization of small RNA, gene expression and DNA methylation changes in response to salt stress in Musa acuminata / Gudimella Ranganath. PhD thesis, Universiti Malaya.

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Abstract

Banana, a commercially important crop which serves as a staple food in several countries worldwide, faces threats from abiotic stress especially related to soil and water salinity due to climate change. Most banana cultivars are salt sensitive, which results in low productivity and fruit of low quality. Physiological responses to salt stress are regulated by underlying gene expression which is influenced by microRNA, small interfering RNA and methylations of genic regions. This study integrated data from transcriptomes, small RNA transcriptomes, degradomes and methylomes using high-throughput sequencing of RNA and DNA extracted from the roots of salt-stressed and non-salt-stressed banana plantlets. Various bioinformatics approaches were adopted for analysis of multi-omics data, for miRNA prediction using small RNA transcriptome a customized pipeline was designed using miRDeep2, miRNA target validation using degradomes was performed by cleaveland4 tool, methylomes were analysed using Bismark and MethPipe tools. Data integration for small RNA and degradome data was performed using network mapping by cytoscape tool. Similarly, data integration for small RNA, transcriptome and methylomes was performed by using statistical approach by custom scripts and visualized data using genome browser. Genome-wide microRNAs were annotated using small RNA transcriptome data and the most recent banana genome sequence. A total of 180 mature miRNAs belonging to 20 orthologous miRNA families and 39 Musa-specific miRNA families were identified. Candidate microRNA targets genes were predicted using bioinformatics tools and validated using degradome data. Profiling of transcription factor binding sites (TFBS) motifs across miRNA promoter regions showed that transcription factors belonging to TCP, AP2; ERF, GATA, NF-YB, DOF, B3, bZIP, trihelix, ZF-HD, bHLH and Dehydrin are likely abundant in the Musa acuminata genome. A putative miRNA-mediated regulatory network is proposed for miR156, miR164, miR166, miR171, miR319 miR396, miR528, mac-miR6, mac-miR-new14 and mac-miR-new20 and their respective transcription factor targets. Genome-wide association between DNA methylation, expression of genes and of 21nt and 24nt small RNAs in response to salt stress was determined using methylome, transcriptome and small RNA transcriptome libraries. DNA methylation in genic regions showed transcriptional repression in several stress-responsive gene candidates such as DRE2, DHN1, AP2, ion-transport related genes, i.e. calcium permeable stress-gated cation channel 1-like and cation/H+ antiporter 20-like, and peroxidases (PER1, PER67 and PNC1), which are ROS-related antioxidants during salt stress. Salt-stressed root samples displayed symmetric CG methylation and CHH demethylation adjacent to differentially expressed genes, while 21 and 24nt siRNA clusters on genomic loci showed increased methylation levels in CG, CHG and CHH contexts. This research contributes Musa- specific miRNA”ome” and small RNA-targeted differentially methylated genic regions which serve as molecular and epigenetic markers to support improvement of banana to address cultivation in salinized soil. Musa-specific genomic markers will serve as an important knowledge base for crop improvement and plant breeding programs.

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