中国农业科学2025，Vol.58Issue(11)：2062-2080,19.DOI:10.3864/j.issn.0578-1752.2025.11.002
一个新的水稻颖花开裂基因OsSG2的图位克隆与功能验证
Map-Based Cloning and Functional Verification of A Novel Split Glume Gene OsSG2 in Rice(Oryza sativa L.)
摘要
Abstract
[Objective]The sg2(split glume 2)is a naturally occurring rice mutant derived from an advanced-generation breeding population of the restorer line GK1327 originating from the Sanming Academy of Agricultural Sciences(SAAS).In this work,the OsSG2(Oryza sativa Split Glume 2)gene in the mutant was performed to conduct genetic analysis,map-based cloning and functional verification,which will provide a theoretical basis and an important germplasm resource for further study on the function of OsSG2 in the development of rice floral organs.[Method]The morphological characteristics of the sg2 mutant florets were anatomically observed to analyze the features of floral organ development.By investigating the phenotypic and agronomic characteristics of wild-type GK1327 and sg2 mutant,and observing the pollen staining in I2-KI,the effects of the mutant phenotype on yield-related traits and pollen fertility were analyzed.The F2 populations developed by crossing sg2 mutant with wild-type GK1327 and 9311,respectively,were used to perform genetic analysis and physical mapping of the OsSG2.Bulked segregant analysis(BSA)combined with next-generation sequencing(NGS)technology(BSA-seq)was used to screen the candidate genes of OsSG2,and a combination of RT-PCR(reverse-transcription PCR)and qRT-PCR(quantitative real-time PCR)was performed to analyze their expression patterns.The OsSG2 candidate genes were cloned and sequenced by PCR amplification and sequencing approaches,and the functional verification was conducted by knockout using CRISPR-cas9 and a complementation test.[Result]The sg2 mutant exhibited an abnormal spikelet phenotype at the reproductive stage,the palea and lemma of florets were weak,distorted,and dehiscent,and with the proportion of abnormal florets in the panicles were 100%.Compared with the wild-type GK1327,the seed setting rate,the 1000-grain weight,and the seed germination rate of sg2 mutant were markedly lower,whereas the pollen fertility was normal.Genetic analysis demonstrated that a recessive nuclear gene was responsible for the mutant phenotype of sg2.A map-based cloning strategy delimited OsSG2 to a 919.85 kb physical interval on the short arm of chromosome 3,flanked by two InDel(insertion/deletion)markers,InDel-12083 and InDel-17610,that included 75 putative ORFs(open read frames).Moreover,a BSA-seq technology identified a potential candidate gene LOC_Os03g11614 as the OsSG2 gene,which encodes an OsMADS1 transcription factor localized to the nucleus.Expression and sequence analyses revealed that the transcript levels of LOC_Os03g11614 were markedly lower in young panicles of the sg2 mutant compared with those in the wild-type GK1327 during the flowering stage,and a SNP(single nucleotide polymorphism)mutation(G-A)was identified at the third nucleotide position of exon 1 in the LOC_Os03g11614,resulting in the change of the start codon from ATG to ATA,which caused a transcriptional suppression of LOC_Os03g11614 and a functional loss of the OsMADS1 protein.These results further supported the tentative identification of LOC_Os03g11614 as the candidate OsSG2 gene,which was confirmed by knockout using CRISPR-cas9 and a functional complementation test.[Conclusion]The split glume gene OsSG2,which was successfully isolated from sg2 mutant in this work,was identified as a new allele of OsMADS1 in rice.关键词
水稻/花器官/OsSG2/图位克隆/基因编辑/OsMADS1Key words
rice(Oryza sativa L.)/floral organ/OsSG2/map-based cloning/gene editing/OsMADS1引用本文复制引用
曾跃辉,邹文广,赵福铭,肖长春,黄建鸿,马彬林,杨旺兴,韦新宇,许旭明..一个新的水稻颖花开裂基因OsSG2的图位克隆与功能验证[J].中国农业科学,2025,58(11):2062-2080,19.基金项目
财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-01)、福建省自然科学基金(2021J01535,2021J01536)、三明市科技计划(2022-N-7,2024-N-3) （CARS-01）
