栽培稻芽期耐低温全基因组关联分析

Genome-Wide Association Study of Cold Tolerance at the Germination Stage of Rice

【目的】水稻是重要的粮食作物,芽期是水稻生长发育过程中最脆弱的时期,直播稻遭遇冷害时发芽率大幅降低,减产严重。深入了解耐冷性的遗传机制,为培育芽期强耐受性水稻品种奠定基础。【方法】以世界范围内14个国家代表性的238份水稻种质资源为试验材料,于2021和2022年在沈阳开展表型鉴定试验,统计不同水稻品种在人工气候培养箱15℃低温条件下第1—10天的发芽率和相对发芽率,利用R语言绘制5—10 d的频率直方图,通过表型丰富度Hill值选择宜作关联分析的天数,将发芽率和相对发芽率表型数据与重测序数据相结合,进行基于混合线性模型MLM(QK)的全基因组关联分析,并对所获得的SNP位点进行耐冷候选基因的预测。【结果】发芽率频数分布直方图和表型丰富度计算结果显示第8天发芽率多态性最好,其Hill值为0.84,高于其他几天发芽率(0.48—0.83),可用于全基因组关联分析;主成分分析结果显示,这些水稻品种可以分为indica、aus、temperate japonica、tropical japonica和aromatic 5个亚群;2个指标进行的GWAS分析检测到3个相同的显著性SNP位点,均位于第4染色体,解释表型的11.9%—25.4%;在上下游各50 kb进行基因搜索,共发现24个相关候选基因,进一步开展LD和单倍型分析,发现LOC_Os04g24840和LOC_Os04g25140的不同单倍型耐冷性之间存在极显著差异。LOC_Os04g24840被编码区SNP分为5个单倍型,且Hap_3的耐冷性显著强于Hap_1;LOC_Os04g25140被编码区SNP分为18个单倍型,且77 bp处的氨基酸变异(S>L)存在籼粳差异。结果表明,编码糖基转移酶的基因LOC_Os04g24840和编码F-box蛋白基因LOC_Os04g25140可能与水稻芽期耐冷性密切相关。【结论】在238份水稻种质资源中共检测到3个与芽期耐冷性显著关联的SNP位点,筛选出2个与水稻芽期耐冷性相关的候选基因。

【Objective】Rice is an important food crop,and its growth and development are most vulnerable at the germination stage.Under cold stress,direct-seeded rice exhibited significantly reduced germination rates(GRs)and yield compared with normally grown plants.Thus,a better understanding of genetic mechanisms regulating cold tolerance will enable to develop rice varieties with improved tolerance during germination.【Method】238 representative rice germplasm resources from 14 countries worldwide were tested in phenotypic identification in Shenyang in 2021 and 2022;the low-temperature germination rate and relative low-temperature germination rate(LTGR and relative LTGR;1-10 days under 15℃)were evaluated in an artificial climate incubator,and a 5-10 day LTGR histogram was constructed using R.The day suitable for GWAS was determined by phenotypic variation(Hill)and a mixed linear model combining LTGR and relative LTGR phenotype data with resequencing data.【Result】LTGR histogram and phenotypic variation showed optimal GR on day 8(Hill=0.84),i.e.,it was higher than on other days(Hill=0.48-0.83),which could be used for GWAS.The principal component analysis results divided all germplasms into five groups-indica,aus,temperate japonica,tropical japonica,and aromatic.GWAS analysis of two indicators detected three identical significant single nucleotide polymorphisms(SNPs)related to cold tolerance in rice at the germination stage.These were located on chromosome 4,which could explain 11.9%-25.4%of the phenotype.In addition,24 candidate genes were screened in the 50-kb region upstream and downstream of these three SNPs.Further linkage disequilibrium analysis and haplotype analysis were carried out and highly significant differences were found between different haplotypes of the LOC_Os04g24840 and LOC_Os04g25140 genes for cold tolerance.LOC_Os04g24840 was divided into five haplotypes by the coding region SNP,and Hap_3 was significantly more cold tolerant than Hap_1;LOC_Os04g25140 was divided into 18 haplotypes by the coding region SNP and the amino acid variation(S>L)at 77 bp was different in japonica and indica rice.These results showed that the genes encoding glycosyltransferases(LOC_Os04g24840)and F-box protein(LOC_Os04g25140)might be closely related to cold tolerance in rice.【Conclusion】A total of three SNP loci were detected in 238 rice germplasm resources,and two candidate genes were screened for their association with cold tolerance during germination in rice.