Search

Novel mutant alleles related to isoflavone content are useful for breeding programs to improve the disease resistance and nutritional content of soybean. However, identification of mutant alleles from high-density mutant libraries is expensive and time-consuming because soybean has a large, complicated genome. Here, we identified the gene responsible for increased genistein-to-daidzein ratio in seed of the mutant line F333ES017D9. For this purpose, we used a time- and cost-effective approach based on selective genotyping of a small number of F2 plants showing the mutant phenotype with nearest-neighboring-nucleotide substitution-high-resolution melting analysis markers, followed by alignment of short reads obtained by next-generation sequencing analysis with the identified locus. In the mutant line, GmCHR5 harbored a single-base deletion that caused a change in the substrate flow in the isoflavone biosynthetic pathway towards genistein. Mutated GmCHR5 was expressed at a lower level during seed development than wild-type GmCHR5. Ectopic overexpression of GmCHR5 increased the production of daidzein derivatives in both the wild-type and mutant plants. The present strategy will be useful for accelerating identification of mutant alleles responsible for traits of interest in agronomically important crops.

RNA silencing is mediated through RNA interference (RNAi) pathway gene families, i.e., Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) and their cis-acting regulatory elements. The RNAi pathway is also directly connected with the post-transcriptional gene silencing (PTGS) mechanism, and the pathway controls eukaryotic gene regulation during growth, development, and stress response. Nevertheless, genome-wide identification of RNAi pathway gene families such as DCL, AGO, and RDR and their regulatory network analyses related to transcription factors have not been studied in many fruit crop species, including banana (Musa acuminata). In this study, we studied in silico genome-wide identification and characterization of DCL, AGO, and RDR genes in bananas thoroughly via integrated bioinformatics approaches. A genome-wide analysis identified 3 MaDCL, 13 MaAGO, and 5 MaRDR candidate genes based on multiple sequence alignment and phylogenetic tree related to the RNAi pathway in banana genomes. These genes correspond to the Arabidopsis thaliana RNAi silencing genes. The analysis of the conserved domain, motif, and gene structure (exon-intron numbers) for MaDCL, MaAGO, and MaRDR genes showed higher homogeneity within the same gene family. The Gene Ontology (GO) enrichment analysis exhibited that the identified RNAi genes could be involved in RNA silencing and associated metabolic pathways. A number of important transcription factors (TFs), e.g., ERF, Dof, C2H2, TCP, GATA and MIKC_MADS families, were identified by network and sub-network analyses between TFs and candidate RNAi gene families. Furthermore, the cis-acting regulatory elements related to light-responsive (LR), stress-responsive (SR), hormone-responsive (HR), and other activities (OT) functions were identified in candidate MaDCL, MaAGO, and MaRDR genes. These genome-wide analyses of these RNAi gene families provide valuable information related to RNA silencing, which would shed light on further characterization of RNAi genes, their regulatory elements, and functional roles, which might be helpful for banana improvement in the breeding program.