甘薯苗期氮高效利用性状的GWAS分析及候选基因的筛选与验证

doi:10.3864/j.issn.0578-1752.2023.18.002

摘要/Abstract

摘要：

【目的】解析甘薯氮高效利用的遗传机制，挖掘氮利用性状的关联位点及氮高效候选基因，为甘薯氮高效型分子育种、品种遗传改良提供支持。【方法】以来自世界各地的129个甘薯栽培种为材料，设置缺氮（0 mmol·L^-1纯氮）和正常氮（14 mmol·L^-1纯氮）处理，采用水培试验对甘薯苗期地上部生物增加量、地下部生物增加量、地上部氮累积量、地下部氮累积量、地上部氮生理利用效率和地下部氮生理利用效率共6个表型性状进行基于混合线性模型（mixed linear model，MLM）的全基因组关联分析（genome-wide association study，GWAS）。根据分析结果确定氮高效候选基因，并对候选基因进行RT-qPCR验证。【结果】6个甘薯苗期氮高效利用性状在正常氮和缺氮处理条件下存在着广泛变异。其中，缺氮处理条件下，地上部生物增加量变异系数最大，为69.5%；地下部氮生理利用效率变异系数最小，为12.1%。除地下部氮生理利用效率外，其他5个性状彼此均显著相关。GWAS定位到与地上部生物增加量、地下部生物增加量、地下部氮累积量和地上部氮生理利用效率4个性状显著关联的134个区段内888个SNP位点。筛选、过滤得到与地上部氮生理利用效率显著关联且可靠性较高的10个区段内的93个SNP标记，基因注释得到6个甘薯氮高效候选基因。RT-qPCR验证认为3个候选基因（itf01g08120.t1、itf01g22030.t1和itf01g22100.t2）分别编码谷氨酸脱氢酶、NPH3蛋白和TIP41-like蛋白，具有进一步研究价值。【结论】在129份甘薯种质资源中共检测到888个与甘薯苗期氮高效利用性状关联的SNP位点，其中，与地上部氮生理利用效率显著相关的SNP位点93个，筛选、鉴定到6个甘薯氮高效利用的候选基因。itf01g08120.t1、itf01g22030.t1和itf01g22100.t2存在进一步研究价值。

关键词: 甘薯, 氮肥利用效率, 氮高效基因, 全基因组关联分析, RT-qPCR

Abstract:

【Objective】The objective of this paper was to analyze the genetic mechanisms of nitrogen use efficiency (NUE), and to explore the loci and candidate genes associated nitrogen (N) efficient traits, to provide support for the N-efficient molecular breeding and genetic improvement of sweetpotato.【Method】A total of 129 sweetpotato cultivars from all over the world were treated with N deficiency (0 mmol·L^-1) and normal N (14 mmol·L^-1). A hydroponic experiment was conducted to facilitate the genome-wide association study (GWAS) of six phenotypic traits (shoot biomass increment, root biomass increment, shoot N accumulation, root N accumulation, shoot N physiological utilization efficiency, and root N physiological utilization efficiency) of sweetpotato at the seedling stage. The N-efficient candidate genes were identified based on the GWAS and subsequently- verified using RT-qPCR.【Result】There were wide variations among the six traits related to NUE in sweetpotato under the normal N and N deficiency treatment conditions. The coefficient of variation (CV) of the shoot biomass increment under the N deficiency treatment condition was the greatest at 69.5%. The CV of the root N physiological utilization efficiency under N deficiency treatment condition was the smallest at 12.1%. All five traits were significantly correlated except for root N physiological utilization efficiency. The MLM model was used to conduct a GWAS of the six phenotypic trait values. A total of 134 QTL and 888 SNP loci were identified as being significantly associated with four out of the six traits, namely, shoot biomass increment, root biomass increment, root N accumulation, and shoot N physiological utilization efficiency. A total of 93 SNP markers across ten regions were significantly associated with shoot N physiological utilization efficiency with a high reliability. Six N efficiency candidate genes were obtained via gene annotation. RT-qPCR verified that the three candidate genes (itf01g08120.t1, itf01g22030.t1 and itf01g221000.t2) encoded glutamate dehydrogenase, NPH3 protein and TIP41-like protein, respectively, which warrants further research.【Conclusion】A total of 888 SNP loci associated with N utilization traits were detected in 129 sweetpotato cultivars. Among these, 93 SNP loci were significantly associated with shoot N physiological utilization efficiency, and six candidate genes were identified. Preliminary verification indicated that the itf01g08120.t1, itf01G2203.t1 and itf01g22100.t2 genes hold promising value for further research.

Key words: sweetpotato, nitrogen use efficiency, nitrogen efficient gene, GWAS, RT-qPCR

于永超, 范文静, 刘明, 张强强, 赵鹏, 靳容, 王静, 朱晓亚, 唐忠厚. 甘薯苗期氮高效利用性状的GWAS分析及候选基因的筛选与验证[J]. 中国农业科学, 2023, 56(18): 3500-3510.

YU YongChao, FAN WenJing, LIU Ming, ZHANG QiangQiang, ZHAO Peng, JIN Rong, WANG Jing, ZHU XiaoYa, TANG ZhongHou. Genome-Wide Association Study of Nitrogen Use Efficient Traits in Sweetpotato Seeding Stage and Screening and Validation of Candidate Genes[J]. Scientia Agricultura Sinica, 2023, 56(18): 3500-3510.

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基因 Gene	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
Actin	AGCAGCATGAAGATTAAGGTTGTAGCAC	TGGAAAATTAGAAGCACTTCCTGTGAAC
itf01g08120.t1	GGTGGATCTCTAGGCAGGGA	TATCGCACCAGTGGCATCAC
itf01g22030.t1	AGACTCTGATGAAACTAGCGGT	CGGGGAAAGCTTGAGACCAT
itf01g22080.t2	CTTCTGATCAGCTCCTCCGC	GTCCCAGAAGGCACACACTT
itf01g22100.t2	GGAGGGCGACCAGAAAGAAT	GGAGCACTTGCGAGACTCAA
itf01g22400.t1	ATGCTTCCTTCTTAACTGCCG	ACTCTATCCATTTTGGCTGGC
itf01g25900.t1	TCAAGAGCATTACCCGCCAG	TGGTCCACCCTCTGATGAAC

性状 Trait	正常氮处理CK				低氮处理N0
性状 Trait	最大值 Max	最小值 Min	平均数 Mean	变异系数 CV (%)	最大值 Max	最小值 Min	平均值 Mean	变异系数 CV (%)
地上部生物增加量 Shoot biomass increment (g)	3.39	0.16	1.43	46.6	2.75	0.01	0.82	69.5
地下部生物增加量 Root biomass increment (g)	1.57	0.08	0.65	46.9	1.61	0.12	0.61	51.7
地上部氮累积量 Shoot N accumulation (g/plant)	0.247	0.030	0.108	36.2	0.120	0.012	0.043	53.6
地下部氮累积量 Root N accumulation (g/plant)	0.061	0.002	0.023	50.6	0.032	0.003	0.012	51.4
地上部氮生理利用效率 Shoot N physiological utilization efficiency (%)	21.18	2.17	12.87	21.8	40.10	0.70	18.52	42.4
地下部氮生理利用效率 Root N physiological utilization efficiency (%)	58.97	22.33	29.81	15.9	74.63	38.11	52.45	12.1

性状 Trait	地上部生物增加量 Shoot biomass increment	地下部生物增加量 Root biomass increment	地上部氮累积量 Shoot N accumulation	地下部氮累积量 Root N accumulation	地上部氮生理利用效率 Shoot N physiological utilization efficiency	地下部氮生理利用效率 Root N physiological utilization efficiency
地上部生物增加量 Shoot biomass increment	1.000
地下部生物增加量 Root biomass increment	0.620**	1.000
地上部氮累积量 Shoot N accumulation	0.686**	0.711**	1.000
地下部氮累积量 Root N accumulation	0.643**	0.986**	0.753**	1.000
地上部氮生理利用效率 Shoot N physiological utilization efficiency	0.806**	0.316**	0.226**	0.312**	1.000
地下部氮生理利用效率 Root N physiological utilization efficiency	-0.230**	-0.129	-0.356**	-0.261**	-0.050	1.000

染色体 Chromosome	物理位置 Position (Mb)	SNP个数 No. of SNPs	P
Chr.1	6.6806—6.8006	1	8.95904493533599
	22.0211—22.0411	2	9.25247697926448
	22.5536—22.7536	11
	22.8065—23.1636	48
	23.7800—24.0800	6
	24.6486—24.6686	1
	25.5962—25.8837	3
Chr.8	4.5428—4.7648	7	10.16845708524990
Chr.13	20.3201—20.3592	3	8.80255904945633
Chr.13	20.8716—20.9597	11