粳稻分蘖数全基因组关联分析及候选基因的挖掘

doi:10.3864/j.issn.0578-1752.2020.16.001

摘要/Abstract

摘要：

【目的】利用全基因组关联分析（genome-wide association study,GWAS）检测与粳稻分蘖数显著相关的SNP位点,筛选影响分蘖数的候选基因,为分子辅助育种提高产量提供理论基础。【方法】利用295份来自世界各地的粳稻品种,于2018和2019年分蘖盛期调查水稻分蘖数,结合高通量重测序获得的788 396个高质量多态性SNP,利用TASSEL 5.0软件的MLM模型进行全基因组关联分析,对GEC软件计算的有效独立SNP数目进行阈值确定,判定SNP标记与目标性状关联的显著性。根据2年检测到的峰值SNP和水稻每条染色体LD衰减距离,确定2年共定位分蘖数主效QTL,并进一步提取QTL区间内所有基因外显子区非同义突变SNP和启动子区SNP进行单倍型分析,结合基因注释筛选影响粳稻分蘖数候选基因。【结果】295份粳稻品种的分蘖数在2018和2019年总趋势基本一致,并且均具有较大的表型分布。通过全基因组关联分析,在P<5.46×10^-6阈值条件下,从第8、9和10染色体上共鉴定3个与粳稻分蘖数相关的QTL（qTiller8、qTiller9和qTiller10）,其中,在2年中均检测到qTiller9,在2018和2019年的表型贡献率分别为11.86%和10.61%,而qTiller8和qTiller10仅在2018年被检测到,表型贡献率分别为9.36%和9.10%。对qTiller9区间内的全部15个基因进行单倍型分析,结果表明,在qTiller9区间内共有6个基因（LOC_Os09g25090、LOC_Os09g25100、LOC_Os09g25150、LOC_Os09g25190、LOC_Os09g25200和LOC_Os09g25220）的不同单倍型分蘖数之间存在极显著差异,LOC_Os09g25090被启动子区SNP分为2种单倍型,hap2（TAA）分蘖数极显著大于hap1（AGG）;LOC_Os09g25100被非同义突变SNP分为2种单倍型,hap2（GAGA）分蘖数极显著大于hap1（AGCC）;LOC_Os09g25150被非同义突变SNP分为2种单倍型,hap2（ATG）分蘖数极显著大于hap1（GCC）;LOC_Os09g25190被启动子区SNP分为2种单倍型,hap2（GCATCGCATCGACGCCGA）分蘖数极显著大于hap1（ATGCTGATGAAGTCATCC）;LOC_Os09g25200被非同义突变SNP分为2种单倍型,hap2（TAG）分蘖数极显著大于hap1（AGA）;LOC_Os09g25220被非同义突变SNP分为2种单倍型,hap1（GG）分蘖数极其显著大于hap2（AA）。结合基因注释发现,LOC_Os09g25090和LOC_Os09g25100均预测编码钙调蛋白依赖性蛋白激酶,是脱落酸（ABA）表达所必需Ca²⁺的传感器,推测LOC_Os09g25090和LOC_Os09g25100为影响粳稻分蘖数的候选基因。【结论】筛选出LOC_Os09g25090和LOC_Os09g25100为影响粳稻分蘖数的候选基因。

关键词: 粳稻, 分蘖数, 全基因组关联分析, 单倍型分析, 候选基因

Abstract:

【Objective】Genome-wide association study (GWAS) was used to detect SNP loci that were significantly related to tillering number in japonica rice, and to screen candidate genes affecting tillering number 【Method】This study used 295 japonica rice varieties from around the world, in 2018 and 2019 survey from the tillering stage of rice tillering number, the combination of high throughput sequencing weight gain high quality 788396 polymorphism, SNP, using TASSEL 5.0 software genome-wide association analysis of MLM model, using the GEC software to calculate the number of effective independent SNPS for the determination of threshold value, determine the significance of SNP markers associated with target traits. Based on the peak SNP detected in two years and the LD attenuation distance of each chromosome in rice, the major QTLs for co-localization of biennial number were determined, and the non-synonymous SNPs and promoter regions of all gene exon regions in the QTL interval were further extracted. SNPs were subjected to haplotype analysis, and then combined with gene annotation to screen candidate genes affecting japonica rice tillering number. 【Result】 The tillering number of 295 japonica rice varieties were basically the same in 2018 and 2019, and they all had a large phenotypic distribution. Under the threshold of P < 5.46 × 10^-6, three QTLs (qTiller8, qTiller9 and qTiller10) related to tillering number of Japonica rice were identified on chromosomes 8, 9 and 10 by genome-wide association analysis. qTiller9 jointly detected that the contribution rate of phenotype in 2018 and 2019 was 11.86% and 10.61%, respectively. qtiller8 and qtiller10 were only detected in 2018, and the contribution rate of phenotype was 10.61% The rates were 9.36% and 9.10%, respectively. Haplotype analysis of all 15 genes in qTiller9 interval showed that there were 6 genes in qTiller9 interval (LOC_Os09g25090, LOC_Os09g25100, LOC_Os09g25150, LOC_Os09g25190, LOC_Os09g25200 and LOC_Os09g25220). LOC_Os09g25090 was divided into two haplotypes by promoter SNP, and the tillering number of hap2 (TAA) was significantly higher than that of hap1 (AGG). LOC_Os09g25100 was divided into two haplotypes by non-synonymy mutation SNP, and hap2 (GAGA) had a significantly higher tillering number than hap1 (AGCC). LOC_Os09g25150 was divided into two haplotypes by non-synonymy mutation SNP, and the tillering number of hap2 (ATG) was significantly higher than that of hap1 (GCC). LOC_Os09g25190 was divided into two haplotypes by promoter SNP and the tillering number of hap2 (GCATCGCATCGACGCCGA) was significantly higher than that of hap1 (ATGCTGATGAAGTCATCC). LOC_Os09g25200 was divided into two haplotypes by non-synonymic mutant SNP and the tillering number of hap2 (TAG) was significantly higher than that of hap1 (AGA). LOC_Os09g25220 was divided into two haplotypes by non-synonymic mutant SNP, and the tillering number of hap1 (GG) was significantly higher than that of hap2 (AA).Combined with gene annotation, it was found that LOC_Os09g25090 and LOC_Os09g25100 both predicted the encoding of calcineurin dependent protein kinases and were Ca²⁺ sensors necessary for abscisic acid (ABA) expression. Previous studies have shown that ABA can affect both tillering number and branch number of Arabidopsis. Therefore, LOC_Os09g25090 and LOC_Os09g25100 are candidate genes affecting the tillering number in japonica rice. 【Conclusion】LOC_Os09g25090 and LOC_Os09g25100 were screened as candidate genes affecting the tiller number of japonica rice.

Key words: japonica rice, tillering number, genome-wide association study, haplotype analysis, candidate gene

张继峰,刘华东,王敬国,刘化龙,孙健,杨洛淼,贾琰,吴文申,郑洪亮,邹德堂. 粳稻分蘖数全基因组关联分析及候选基因的挖掘[J]. 中国农业科学, 2020, 53(16): 3205-3213.

ZHANG JiFeng,LIU HuaDong,WANG JingGuo,LIU HuaLong,SUN Jian,YANG LuoMiao,JIA Yan,WU WenShen,ZHENG HongLiang,ZOU DeTang. Genome-Wide Association Study and Candidate Gene Mining of Tillering Number in Japonica Rice[J]. Scientia Agricultura Sinica, 2020, 53(16): 3205-3213.

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年份 Year	均值±标准差 Mean±standard	变幅 Range	变异系数 CV (%)	偏度 Skewness	峰度 Kurtosis
2018	18.35±5.98	7.00—49.00	32.58	1.31	0.49
2019	15.75±4.65	6.00—33.80	29.55	3.09	0.05

QTL	年份 Year	峰值SNP Peak SNP	染色体 Chr.	P值 P value	表型贡献率 R²(%)	已定位QTL QTL located
qTiller8	2018	7527329	8	2.02E-06	9.36
qTiller9	2018	15065450	9	6.99E-08	11.86	qNOT9-1^[29]
qTiller10	2018	15406671	10	2.64E-06	9.10
qTiller9	2019	15060619	9	2.55E-07	10.61	qNOT9-1^[29]

基因 Gene	单倍型1/品种数 hap1/Number	单倍型2/品种数 hap2/Number	单倍型3/品种数 hap3/Number
LOC_Os09g25090	AGG/182	TAA/81
LOC_Os09g25100	AGCC/188	GAGA/89
LOC_Os09g25150	ATG/190	GCC/69	GTC/17
LOC_Os09g25190	ATGCTGATGAAGTCATCC/126	GCATCGCATCGACGCCGA/76
LOC_Os09g25200	AGA/181	TAG/89
LOC_Os09g25220	GG/97	AA/187

基因 Gene	基因功能注释 Gene annotation
LOC_Os09g25090	钙调蛋白依赖性蛋白激酶 Calmodulin depedent protein kinases
LOC_Os09g25100	钙调蛋白依赖性蛋白激酶 Calmodulin depedent protein kinases
LOC_Os09g25150	肉桂酰辅酶A还原酶 Cinnamoyl-CoA reductase
LOC_Os09g25190	蛋白结合蛋白Protein binding protein
LOC_Os09g25200	蛋白结合蛋白Protein binding protein
LOC_Os09g25220	蛋白结合蛋白Protein binding protein