小麦苗期生物量及氮效率相关性状的全基因组关联分析

doi:10.3864/j.issn.0578-1752.2021.21.001

摘要/Abstract

摘要：

【目的】探究不同氮素供应环境下与小麦苗期生物量及氮效率相关性状显著关联的SNP位点,预测相关候选基因,为小麦氮效率的基因克隆及其在育种中的应用提供参考。【方法】以134个小麦品种（系）组成的群体为供试群体,设置低氮、正常氮和高氮3个处理,各处理重复4次,并在2年（2013和2014年）进行了2次完全重复的营养液培养试验。试验对小麦苗期生物量及氮效率相关的14个性状进行了表型鉴定,采用MLM+K+Q混合线性模型,利用90K SNP芯片对小麦生物量及氮效率相关性状进行全基因组关联分析（genome-wide association study,GWAS）,获得显著关联的SNP位点。【结果】与正常氮处理相比,低氮处理条件下,根系、地上部及植株氮含量和氮积累量均显著下降,而根生物量和根、植株氮效率均显著增加,高氮处理下,几乎所有鉴定性状均显著增加;14个性状的广义遗传力均在40%以上,其中,植株总干重的遗传力最高（95.73%）。利用9 329个SNP标记进行关联分析,共检测到838个SNP标记位点与供试材料的14个性状存在显著关联（P≤0.001）,分布在21条染色体上。有435个（51.91%）SNP标记位点仅在一个关联分析环境中被检测到;有403个位点至少在2个处理环境（包含均值环境）中被检测到与同一性状显著关联（稳定关联标记）。其中8个SNP标记位点至少在3个环境中被检测到。在4个环境下（包括均值环境）均检测到的稳定关联位点有2个：Kukri_c65481_121和tplb0025f09_1052,分别与植株总氮利用效率（total nitrogen use efficiency of plant,TNUE）和根系总氮利用效率（root nitrogen use efficiency,RNUE）显著关联;同时与至少6个性状（生物量及养分效率相关性状）显著关联的SNP标记位点共5个,分别位于1A、1B（3）和2A染色体上;根据小麦基因组注释及LD衰减水平,在同时定位了6个性状的5个SNP位点和2个多环境（4个环境）稳定关联的SNP位点的214 kb的基因组区域中共筛选候选基因84个,根据已知克隆氮效率基因的编码蛋白类型、候选基因功能注释信息及利用植物比较基因组学资源库蛋白序列同源比对分析,筛选到3个候选基因与生物量及氮效率相关。【结论】不同氮素处理显著影响小麦苗期生物量、氮效率相关性状及其相关QTL的表达,大多数SNP位点仅在1个氮素检测环境中被检测到,但也存在环境稳定性较强的位点。生物量及氮效率相关性状之间存在显著相关关系,并在一定程度上受到相同的QTL/基因控制。

关键词: 小麦, 全基因组关联分析, 生物量, 氮效率

Abstract:

【Objective】 To identify and locate the molecular markers which were stable and significantly correlated to the traits of biomass and N efficiency under different N nutrition levels will help to provide reference for cloning and characterization of the related genes.【Method】A group of 134 wheat varieties (or lines) were used in a two-years (2013 and 2014) hydroponic experiments, in which three treatments applying normal level N, low level N and high level N were set up. Fourteen traits related to biomass and N efficiency were measured, as the respective average values of each treatment in one year and two years. Genome-wide association analysis using 90K SNP molecular markers was carried out for the tested traits by MLM+K+Q mixed linear model. 【Result】 Compared with normal nitrogen treatments, roots, shoots, and plant nitrogen content and nitrogen accumulation were significantly reduced in low nitrogen treatments, while root biomass and root and plant nitrogen efficiency were significantly increased. In high nitrogen treatments, almost all traits are significantly increased. The heritability of all the tested traits were above 40%. According to genome-wide association analysis on the 9 329 SNPs, a total of 838 molecular marker sites were identified associating with 14 traits significantly (P≤0.001). These markers located on 21 chromosomes, among which 435 (51.91%) molecular marker sites were detected in only one environment, 403 and 8 environment stable sites were identified in at least two or three environments, two environmental stable SNP marker sites were identified in at least four environments. The two stable markers (Kukri_c65481_121 and tplb0025f09_1052) were significantly related to total nitrogen use efficiency of plant (TNUE) and root nitrogen use efficiency (RNUE), respectively. Five multi-trait co-location SNP marker sites which simultaneously associated with at least six traits were located on chromosomes 1A, 1B(2), and 2A(2). Furtherly, candidate gene prediction was conducted in the 214 kb genomic region of 5 SNP sites co-located with 6 traits (biomass and N efficiency traits) and 2 SNP sites associated with multiple environments (4 environments). According to the genome annotation and LD attenuation level, a total of 84 candidate genes were determined. Gene function annotations of these genes were performed using the coding protein types of known cloned nitrogen efficiency genes, candidate gene function annotation information and the use of plant comparative genomics resource library protein sequence homology analysis, 3 candidate genes were initially determined. 【Conclusion】 Different N treatments significantly affected the phenotypic traits of biomass, N efficiency and the expression of related QTLs at seedling stage of wheat. Most SNPs were detected in only one N environment, but there were some locations with relatively strong environmental stability. There was a significant correlation between biomass and N efficiency related traits, and they might be partly controlled by the same QTL/gene. The functions of related candidate genes related to N efficiency and biomass of wheat selected in this paper needed to be further verified.

Key words: wheat, GWAS, biomass, nitrogen efficiency

张鹏霞,周秀文,梁雪,郭营,赵岩,李斯深,孔凡美. 小麦苗期生物量及氮效率相关性状的全基因组关联分析[J]. 中国农业科学, 2021, 54(21): 4487-4499.

ZHANG PengXia,ZHOU XiuWen,LIANG Xue,GUO Ying,ZHAO Yan,LI SiShen,KONG FanMei. Genome-Wide Association Analysis for Yield and Nitrogen Efficiency Related Traits of Wheat at Seedling Stage[J]. Scientia Agricultura Sinica, 2021, 54(21): 4487-4499.

图/表 8

表1

表2

小麦苗期生物量和氮效率相关性状的统计参数和遗传力"

性状 Trait	处理 Treatment	均值 Average	变异系数 CV (%)	遗传力 hB² (%)	性状 Trait	处理 Treatment	均值 Average	变异系数 CV (%)	遗传力 hB² (%)
地上部干重 SDW (mg)	T1E1	107.95cd	21.40	94.95	地上部氮累积量 SNA (mg/plant)	T1E1	5.00d	12.77	93.53
	T2E1	104.08d	23.99			T2E1	5.98c	6.93
	T3E1	124.71b	23.51			T3E1	7.64b	10.36
	T1E2	112.33c	19.16			T1E2	4.32e	8.64
	T2E2	129.49b	22.28			T2E2	7.79b	10.23
	T3E2	148.77a	22.62			T3E2	9.68a	4.76
根系干重 RDW (mg)	T1E1	27.80c	35.87	92.76	根系氮累积量 RNA(mg/plant)	T1E1	0.99c	22.54	93.49
	T2E1	19.50e	21.49			T2E1	0.77d	24.18
	T3E1	22.40d	22.99			T3E1	0.98c	24.21
	T1E2	50.10a	27.03			T1E2	1.14b	19.78
	T2E2	27.60c	22.30			T2E2	1.00c	23.44
	T3E2	32.20b	22.89			T3E2	1.27a	23.85
植株总干重 TDW (mg)	T1E1	136.06d	21.00	95.37	植株总氮累积量 TNA (mg/plant)	T1E1	5.97d	26.93	94.31
	T2E1	123.08e	21.99			T2E1	6.75c	24.20
	T3E1	147.10c	22.81			T3E1	8.62b	25.48
	T1E2	162.52b	20.53			T1E2	5.46d	21.53
	T2E2	157.18b	21.77			T2E2	8.79b	25.94
	T3E2	180.66a	22.03			T3E2	10.95a	23.66
生物量根冠比 RSDW	T1E1	0.27b	32.68	89.72	氮累积量根冠比 RSNA	T1E1	0.20b	37.13	84.25
	T2E1	0.19d	16.80			T2E1	0.13c	23.66
	T3E1	0.18d	17.48			T3E1	0.13c	26.83
	T1E2	0.44a	16.12			T1E2	0.27a	28.16
	T2E2	0.22c	13.16			T2E2	0.13c	26.20
	T3E2	0.22c	14.00			T3E2	0.13c	27.63
地上部氮含量 SNC (g·kg^-1)	T1E1	46.31d	6.42	86.39	地上部氮利用效率 SNUE (g·g^-1kg^-1)	T1E1	2.35bc	26.93	94.79
	T2E1	57.55c	3.86			T2E1	1.82e	24.20
	T3E1	61.21b	3.85			T3E1	2.04d	25.48
	T1E2	38.66e	10.41			T1E2	3.00a	21.53
	T2E2	59.79b	4.75			T2E2	2.18cd	25.94
	T3E2	64.96a	4.47			T3E2	2.29b	23.66
根系氮含量 RNC (g·kg^-1)	T1E1	35.32c	8.21	85.71	根系氮利用效率 RNUE (g·g^-1kg^-1)	T1E1	0.80b	27.23	89.44
	T2E1	40.48b	8.09			T2E1	0.47c	22.32
	T3E1	43.74a	12.5			T3E1	0.51c	18.75
	T1E2	22.64d	7.25			T1E2	2.26a	19.18
	T2E2	35.51c	10.1			T2E2	0.78b	16.92
	T3E2	39.44b	9.37			T3E2	0.81b	17.52
植株总氮含量 TNC (g·kg^-1)	T1E1	44.07d	5.26	23.68	植株总氮利用效率 TNUE (g·g^-1kg^-1)	T1E1	3.09b	22.66	41.34
	T2E1	54.87c	6.12			T2E1	2.25e	24.62
	T3E1	58.56b	4.25			T3E1	2.51d	23.62
	T1E2	33.75e	5.10			T1E2	4.92a	22.15
	T2E2	55.50c	11.20			T2E2	2.84c	21.40
	T3E2	60.44a	5.26			T3E2	2.99b	22.11

表2

图1

图2

表3

表4

图3

表5

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大量元素 Macronutrients	浓度 Concentration (mmol·L^-1)	微量元素 Trace elements	浓度 Concentration (μmol·L^-1)
KH₂PO₄	0.2	H₃BO₃	1.0
MgSO₄·7H₂O	0.5	(NH₄)₆Mo₇O₂₄·4H₂O	0.1
KCl	1.8	CuSO₄·5H₂O	0.5
CaCl₂	1.5	ZnSO₄·7H₂O	1.0
(NH₄)₂SO₄·H₂O	1.0	MnSO₄·H₂O	1.0
Ca(NO₃)₂·4H₂O	1.0	Fe·EDTA	100.0

性状 Trait	环境 Environment	标记 Marker	染色体 Chr.	位置 Position (bp)	P		贡献率 R²(%)
性状 Trait	环境 Environment	标记 Marker	染色体 Chr.	位置 Position (bp)	最大值 Max	最小值 Min	贡献率 R²(%)
RNUE	T1E1, T3AV, T3E2	RAC875_c29540_391	1A	8290703	9.95E-04	1.39E-05	11.05—18.34
根系干重RDW	T2E1, T3AV, T3E2	BobWhite_c9881_1312	1B	10256166	3.58E-04	3.49E-04	10.09—10.13
根系氮利用效率RNUE	T2AV, T2E1, T3E1	BobWhite_c9881_1312	1B	10256166	7.45E-04	3.75E-05	9.04—13.69
地上部氮含量SNC	T1E1, T3AV, T3E2	GENE-0427_442	1B	5688119	5.79E-04	8.62E-05	9.35—12.35
植株总干重TDW	T1E1, T3AV, T3E2	GENE-0427_442	1B	5688119	6.54E-04	1.88E-04	9.17—11.11
植株总氮含量TNC	T1E1, T3AV, T3E2	GENE-0427_442	1B	5688119	6.25E-04	1.08E-04	9.23—11.98
地上部氮含量SNC	T2AV, T2E1, T1E2	wsnp_RFL_Contig3060_2967139	2A	9928033	8.05E-04	1.87E-04	11.41—13.91
植株总氮含量TNC	T2AV, T2E1, T1E2	wsnp_RFL_Contig3060_2967139	2A	9928033	2.95E-04	2.05E-04	12.57—13.74
植株总氮利用效率TNUE	T2AV, T2E1, T1AV, T1E2	Kukri_c65481_121	3B	13483435	7.19E-04	2.08E-04	9.09—11.40
根系氮利用效率RNUE	T1AV, T1E1, T3AV, T3E2	tplb0025f09_1052	4B	1591267	7.25E-04	1.32E-09	11.49—36.09
地上部干重SDW	T2E2, T1AV, T1E1	wsnp_Ku_c1765_3452586	6A	14086572	6.09E-04	1.39E-04	9.07—11.58
生物量根冠比RSDW	T2AV, T2E2, T3E2	BS00022498_51	7B	6251008	5.32E-04	3.25E-04	12.01—12.85

标记和染色体 Marker and Chr.	性状 Trait	分析环境 Analysis environment	贡献率 R² (%)
BS00077492_51 1A	地上部干重SDW	T1E1	12.63
	植株总干重TDW	T1E1	11.31
	地上部氮含量SNC	T1E1	11.73
	植株总氮含量TNC	T1E1	11.76
	地上部氮利用效率SNUE	T1E1	12.42
	植株总氮利用效率TNUE	T1E1	11.80
BS00065737_51 1B	地上部干重SDW	T1E1, T3E2	8.93—11.37
	植株总干重TDW	T1E1	8.60
	地上部氮含量SNC	T1E1	9.80
	植株总氮含量TNC	T1E1	8.87
	地上部氮利用效率SNUE	T1E1, T3E2	10.11—10.75
	植株总氮利用效率TNUE	T3E2	9.01
GENE-0427_442 1B	地上部干重SDW	T1E1, T3E2	10.86—11.65
	植株总干重TDW	T1E1, T3AV, T3E2	9.17—11.11
	地上部氮含量SNC	T1E1, T3AV, T3E2	9.35—12.35
	植株总氮含量TNC	T1E1, T3AV, T3E2	9.23—11.98
	地上部氮利用效率SNUE	T3E2	10.44
	植株总氮利用效率TNUE	T3AV, T3E2	9.65—11.33
IACX16577 1B	地上部干重SDW	T1E1, T3E2	9.49—11.83
	植株总干重TDW	T1E1, T3E2	8.82—8.97
	地上部氮含量SNC	T1E1, T3E2	8.92—9.96
	植株总氮含量TNC	T1E1, T3E2	8.72—9.30
	地上部氮利用效率SNUE	T1E1, T3E2	10.82—11.02
	植株总氮利用效率TNUE	T1E1, T3E2	9.19—9.81
wsnp_RFL_Contig3060_2967139 2A	地上部干重SDW	T2AV, T2E1	13.39—13.77
	植株总干重TDW	T2AV, T2E1	12.85—13.57
	地上部氮含量SNC	T2AV, T2E1, T1E2	13.25—13.91
	植株总氮含量TNC	T2AV, T2E1, T1E2	13.33—13.74
	地上部氮利用效率SNUE	T2AV, T2E1	12.99—13.69
	植株总氮利用效率TNUE	T2AV, T2E1	12.04—12.73

染色体 Chr.	基因 Gene	位置 Position (Mb)	标记 Marker	基因注释或编码蛋白 Gene annotation or coding protein
1B	TraesCS1B02G058300	183.39	BS00065737_51	无性的MADS盒子转录因子 Agamous MADS-box transcription factor
2A	TraesCS2A02G122000	332.13	wsnp_RFL_Contig3060_2967139	与膜相关的30 kD蛋白 Membrane-associated 30 kD protein
4B	TraesCS4B02G007100	46.43	tplb0025f09_1052	含R3h结构域的蛋白质 R3h domain containing protein