Genome-wide Association Analysis of γ-aminobutyric Acid in Rice Grains

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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

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

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

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

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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