水稻籽粒γ-氨基丁酸含量的全基因组关联分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0058

摘要/Abstract

摘要：

【目的】挖掘与水稻籽粒γ-氨基丁酸含量显著相关的SNP位点及候选基因，为揭示水稻籽粒γ-氨基丁酸合成与积累的遗传基础和分子机制提供理论依据，为高γ-氨基丁酸含量的水稻品种选育奠定基础。【方法】以139份西北早粳稻核心种质为实验材料，测定其籽粒γ-氨基丁酸含量，结合重测序基因型数据，进行全基因组关联（GWAS）分析，根据单倍型分析和基因注释筛选候选基因，通过表达谱数据库和荧光定量 PCR 对预测基因进行表达模式分析。【结果】水稻籽粒γ-氨基丁酸含量表现出丰富的变异，变异系数为31.25%。全基因组关联分析检测到分别位于第6、8、9、11和12号染色体上共6个有代表性的SNP位点，筛选出已克隆与γ-氨基丁酸含量有关的OsBADH2和OsGABA-T2基因，预测出6个与γ-氨基丁酸含量有关的候选基因，将LOC_Os09g10720基因列为重点分析基因，并进一步验证了其参与水稻籽粒中GABA分解的可能性。【结论】检测到6个与水稻籽粒γ-氨基丁酸含量显著关联的 SNP 位点。筛出6个可能与水稻籽粒γ-氨基丁酸含量相关的候选基因，分别是LOC_Os09g09750、LOC_Os09g09760、LOC_Os09g10210、LOC_Os09g09820、LOC_Os09g10280和LOC_Os09g10720，其中LOC_Os09g10720作为重点候选基因。

关键词: 水稻, γ-氨基丁酸, 全基因组关联分析, 候选基因

Abstract:

【Objective】Identification of single nucleotide polymorphisms（SNPs）and candidate genes significantly associated with the content of γ-aminobutyric acid（GABA）in rice grains may provide a theoretical basis for further exploring the molecular mechanism of GABA enrichment in rice grains, and lay the foundation for cultivating high-GABA content varieties.【Method】This study performed genome-wide association studies（GWAS）for GABA content with re-sequencing in 139 early japonica rice core germplasm resources in Northwest China. The candidate genes were screened based on haplotype analysis and gene annotation, and expression pattern of candidate genes were analyzed by expression profile database and fluorescence quantitative PCR.【Result】GABA content in grains showed abundant variation with a coefficient of variation of 31.25%. Genome-wide association analysis showed a total of 6 significant and representative SNPs on chromosomes 6, 8, 9, 11 and 12 were detected, and OsBADH2 and OsGABA-T2, which have been cloned in relation to GABA content. In addition, six candidate genes were predicted for GABA content in the rice grains, among which LOC_Os09g10720 was identified as highlight candidate gene, and the feasibility of its involvement in the decomposition of GABA in rice grains was further verified.【Conclusion】A total of 6 SNPs significantly associated with GABA content in rice grain are detected. And 6 candidate genes that might be associated with GABA content are screened out, they are LOC_Os09g09750, LOC_Os09g09760, LOC_Os09g10210, LOC_Os09g09820, LOC_Os09g10280 and LOC_ Os09g10720. Among them, LOC_Os09g10720 was used as a focus candidate gene.

Key words: rice, γ-aminobutyric acid, genome-wide association analysis, candidate gene

孙志勇, 杜怀东, 刘阳, 马嘉欣, 于雪然, 马伟, 姚鑫杰, 王敏, 李培富. 水稻籽粒γ-氨基丁酸含量的全基因组关联分析[J]. 生物技术通报, 2024, 40(8): 53-62.

SUN Zhi-yong, DU Huai-dong, LIU Yang, MA Jia-xin, YU Xue-ran, MA Wei, YAO Xin-jie, WANG Min, LI Pei-fu. Genome-wide Association Analysis of γ-aminobutyric Acid in Rice Grains[J]. Biotechnology Bulletin, 2024, 40(8): 53-62.

图/表 9

表1 139份种质资源GABA含量方差分析

Table 1 Variance analysis of GABA content in 139 varieties of germplasm resources

变异来源Source of variation	自由度Degree of freedom	平方和Sum of squares	均方Mean square	F值F value	P 值P value
群组间Between-group	138	133 214.515	965.323	213.578	0.000
群组内Within-group	278	1 256.493	4.520
总计Total	416	134 471.008

图1 139份种质资源籽粒GABA含量的频率分布图

Fig. 1 Frequency distribution of GABA content in the grains of 139 germplasm resources

图2 139份种质资源籽粒GABA含量的全基因组关联分析曼哈顿图和Q-Q图

Fig. 2 Genome-wide association analysis of GABA content in 139 germplasm resources Manhattan plot and Q-Q plot

表2 全基因组关联分析显著SNPs位点

Table 2 Genome-wide association analysis of significant SNPs loci

SNP	染色体 Chromosome	位置 Location/bp	置信区间起点 Starting point of confidence interval	置信区间终点 End point of confidence interval	参考碱基 Reference base	变异碱基 Variant base	-log₁₀（P）
S6-3357208	6	3357208	3057208	3657208	G	A	7.07
S8-6442742	8	6442742	6142742	6742742	A	C	6.51
S8-20231522	8	20231522	19931522	20531522	T	C	7.23
S9-5559515	9	5559515	5259515	5859515	C	T	6.45
S11-4162881	11	4162881	3862881	4462881	C	T	6.80
S12-12473570	12	12473570	12173570	12773570	T	C	6.26

图3 SNP S9-5559515的LD热图（A）及候选基因的单倍型分析（B-G） a、b 表示 0.05 水平差异

Fig. 3 LD heatmap of SNP S9-5559515 (A) and haplotype analysis of candidate genes (B-G) a and b indicate significant differences at the 0.05 level

图4 水稻籽粒GABA含量关键基因表达图谱 SR：胚根；ML：成熟叶；YL：幼叶；SAM：顶端分生组织和轴分生组织的萌发期；YI：花过渡与花器官发育期；P2-P3：减数分裂阶段；P4：幼年小孢子期；P5：液泡化花粉期；P6：成熟花粉期；S1：球形胚早期；S2：球形胚中晚期；S3：种子形态发生期；S4：种子成熟期；S5：种子耐休眠和脱水期，下同

Fig. 4 Expression patterns of key genes for GABA content in rice grains SR: Radicle. ML: Mature leaf.YL: Young leaf. SAM: Germination stage of apical meristem and axial meristem. YI: Flower transition and floral organ development stage. P2-P3: Meiosis stage. P4: Young microspore stage. P5: Vacuolated pollen stage. P6: Mature pollen stage. S1: Early globular embryo stage. S2: Mid-late globular embryo stage. S3: Seed morphogenesis stage. S4: Seed maturity stage.S5: Seed dormancy and dehydration tolerance stage, the same below

图5 水稻籽粒GABA含量候选基因表达图谱

Fig. 5 Expression profile of candidate genes for GABA content in rice grains

图6 LOC_Os09g10720基因在花后不同时期相对表达水平 * 与 *** 分别表示0.05 和0.001 的显著水平

Fig. 6 Relative expressions of LOC_Os09g10720 genes at different stages after flowering * and *** indicates significant at 0.05 and 0.001 probability levels respectively

图7 GABA代谢通路 Glu:谷氨酸；2-OG:α-酮戊二酸；Succ-CoA: 琥珀酰辅酶A；Fuma: 延胡索酸；Succ: 琥珀酸；SSA: 琥珀酸半醛；Arg: 精氨酸；P5C: 1-吡咯啉-5-羧酸；Orn: 鸟氨酸；Agm: 鲱精胺；1-Pyrroline: 1-吡咯啉；Put: 腐胺；ABAL: 4-氨基丁醛；Spd: 亚精胺；Spm: 精胺；GDH: 谷氨酸脱氢酶；SSADH: 琥珀酸半醛脱氢酶；GABA-T:γ- 氨基丁酸转氨酶；GAD:谷氨酸脱羧酶；P5CDH:吡咯啉-5-羧酸脱氢酶：P5CS: 1-吡咯啉-5-羧酸合成酶；BADH2:甜菜碱醛脱氢酶2；OAT:鸟氨酸转氨酶；ARG:精氨酸酶；ADC: 精氨酸脱羧酶；ODC:鸟氨酸脱羧酶；AgmAH:鲱精胺酶；DAO: 二胺氧化酶；Spms: 精胺合成酶；PAO:多胺氧化酶；SpdS:亚精胺合成酶

Fig. 7 GABA metabolic pathway Glu: Glutamic acid; 2-OG: α-ketoglutaric acid; Succ-CoA: Succinyl-CoA; Fuma: Fumaric acid; Succ: Succinic acid; SSA: Succinic semialdehyde; Arg: Arginine; P5C: 1-pyrroline-5-carboxylic acid; Orn: Ornithine; Agm: Agmatine; 1-pyrroline: 1-pyrroline; Put: Putrescine; ABAL: 4-aminobutyraldehyde; Spd: Spermidine; Spm: Spermine; GDH: Glutamate dehydrogenase; SSADH: Succinic semialdehyde dehydrogenase; GABA-T: γ-aminobutyric acid transaminase; GAD: Glutamic acid decarboxylase; P5CDH: Pyrroline-5-carboxylic acid dehydrogenase; P5CS: 1-pyrroline-5-carboxylic acid synthetase; BADH2: Betaine aldehyde dehydrogenase 2; OAT: ornithine aminotransferase; ARG: Arginase; ADC: Arginine decarboxylase; ODC: Ornithine decarboxylase; AgmAH: Agmatine amidohydrolase; DAO: Diamine oxidase; Spms: Spermine synthetase; PAO: Polyamine oxidase; SpdS: Spermidine synthetase

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