Mapping and Identifying a Candidate Gene Plr4, a Recessive Gene Regulating Purple Leaf in Rice, by Using Bulked Segregant and Transcriptome Analysis with Next-Generation Sequencing.

Ju Gao,Hao Wu,Guofu Deng,Haifu Liang,Dongjin Qing,Weiwei Chen,Danting Li,Gaoxing Dai,Juan Huang,Lijun Gao,Weiyong Zhou,Xinghai Yang

doi:10.3390/ijms20184335

Abstract

The anthocyanin biosynthesis of rice is a major concern due to the potential nutritional value. Purple appears in various organs and tissues of rice such as pericarp, flower organs, leaves, leaf sheaths, internodes, ligules, apex, and stigma. At present, there are many studies on the color of rice pericarp, but the gene and mechanism of other organs such as leaves are still unclear, and the gene regulatory network of specific organ coloring has not been systematically understood. In this study, genetic analysis demonstrated that the purple leaf traits of rice were regulated by a recessive gene. The green leaf cultivar Y58S and purple leaf cultivar XianHongB were used to construct the mapping population. A set of near isogenicline (NIL) (BC3F1) was bred via crossing and back-crossing. The generations of BC3F2 appeared to separate four phenotypes, pl1, pl2, pl3, and pl4, due to the occurrence of a purple color in different organs. We constructed three bulked segregant analysis (BSA) pools (pl1–pl2, pl1–pl3, and pl1–pl4) by using the separated generations of BC3F5 and mapped the purple leaf gene plr4 to the vicinity of 27.9–31.1 Mb on chromosome 4. Subsequently, transcriptome sequencing (RNA-Seq) for pl3 and pl2 was used to analyze the differentially expressed genes in the localization interval, where 12 unigenes exhibited differential expression in which two genes (Os04g0577800, Os04g0616400) were downregulated. The two downregulated genes (Os04g0577800 and Os04g0616400) are possible candidate genes because of the recessive genetic characteristics of the purple leaf genes. These results will facilitate the cloning of plr4 and illustrate the molecular mechanisms of the anthocyanin synthesis pathway.

Highlights

Anthocyanin is a strong antioxidant, which brings various colors to plant organs and exerts obvious effects on human health
Anthocyanin biosynthesis is jointly controlled by two types of genes: structural genes including CHS, CHI, F3H, F3 H, F3 5 H, dihydroflavonol-4-reductase(DFR), ANS, and 3GT [1,2,3,4,5,6,7], which encode various enzymes required for anthocyanin biosynthesis pathways [8]; and regulatory genes, which encode transcription factors that regulate the spatio-temporal expression of structural genes including the R2R3-MYB protein, MYC family of basic helix–loop–helix proteins, and WD40 protein gene family
The plr4 gene was mapped via genetic analysis, bulked segregant analysis (BSA)-Seq, RNA-Seq, and qRT-PCR

Summary

Introduction

Anthocyanin is a strong antioxidant, which brings various colors to plant organs and exerts obvious effects on human health. Ra is the homologous gene of the maize R/B gene that is located on chromosome 4 of rice, encodes a transcription factor of the bHLH domain, is composed of two genes Ra1 and Ra2, and plays an important role in the anthocyanin biosynthesis pathway. It is an important gene for black pericarpin rice [20]. Through bulked segregant analysis (BSA) with next-generation sequencing (BSA-Seq) and transcriptome sequencing (RNA-Seq) strategies, we identified plr, which is a new gene that regulates purple leaf in rice

Statistical Analysis of Phenotypes and Genetics of Rice’s Purple Leaf Trait

Verification of DEGs via qRT-PCR

RNA Extraction and Illumina Sequencing

Quantification of Gene Expression Level and Differential Expression Analysis

GO and KEGG Enrichment Analysis of DEGs

Conclusions