谷子苗期耐低氮相关性状的QTL分析

doi:10.3864/j.issn.0578-1752.2023.20.002

摘要/Abstract

摘要：

【目的】分析谷子（Setaria italica L.）耐低氮性状相关的QTL，为耐低氮基因的精细定位、克隆及功能研究奠定基础，同时，也为揭示谷子耐低氮遗传机理和培育耐低氮谷子品种提供技术支撑。【方法】以耐低氮品种豫谷28和低氮敏感品种七叶黄为亲本构建的包含120个家系的重组自交系（RIL）群体为试验材料，在谷子苗期进行低氮和正常氮处理，并对处理21 d的水培幼苗的苗长、主根长、根干质量、苗干质量、总干质量、叶绿素相对含量、植株含氮量7个性状进行分析。同时，采用复合区间作图法（composite interval mapping，CIM）对耐低氮相关性状进行QTL定位与分析，并对QTL置信区间内的候选基因进行预测。【结果】在低氮和正常氮水平下，RIL群体的耐低氮相关性状均表现为连续正态分布，且呈双向超亲分离现象，符合典型的数量性状遗传特点，适于QTL分析。相关分析表明，苗长与主根长、根干质量、苗干质量、总干质量、叶绿素相对含量呈极显著正相关，主根长与植株含氮量呈显著负相关。共定位到低氮和正常氮水平下与苗长、主根长、根干质量、苗干质量、总干质量、叶绿素相对含量及植株含氮量相关的34个QTL，分布于第1—9染色体，单个QTL表型贡献率为5.15%—52.42%。其中，2种氮水平下共同定位到10个QTL，低氮和正常氮单一环境下分别定位到11和13个QTL。15个为主效QTL，qRDW3、qMRL1.1、qMRL1.2、qSL5和qSPAD1 5个主效QTL在2种氮水平下均被定位到。共检测到5个QTL重叠区，聚集了2种氮水平下多个性状的QTL，通过基因预测与功能注释，筛选出5个QTL置信区间内6个与氮代谢相关的候选基因，表明氮同化、吸收和利用相关基因极有可能控制了这些基因的表达。【结论】34个QTL分别聚集于9条染色体上的16个QTL簇，基于基因注释，共筛选了6个与谷子氮代谢相关的候选基因，表明不同性状参与到了共同遗传机制，并可通过分子标记辅助选择进行耐低氮有利等位基因的聚合育种。

关键词: 谷子, 重组自交系（RIL）, 耐低氮, QTL

Abstract:

【Objective】The analysis of quantitative trait loci (QTL) related to low nitrogen tolerance traits of millet (Setaria italica L.) laid a foundation for fine mapping, cloning and functional research of low nitrogen tolerance genes. At the same time, it also provided technical support for revealing the genetic mechanism of low nitrogen tolerance of millet and breeding low nitrogen tolerance varieties. 【Method】The recombinant inbred line (RIL) population consisting of 120 family lines was used as experimental materials, that was constructed from parents Yugu 28, a low nitrogen tolerant variety, and Qiyehuang, a low nitrogen sensitive variety. The RIL populations were treated with low nitrogen and normal nitrogen at seedling stage, and seven traits were analyzed of hydroponic for 21 days, which inculding seedling length, maximum root length, root dry weight, seedling dry weight, plant dry weight, relative chlorophyll content and plant nitrogen content. At the same time, we used composite interval mapping (CIM) to locate and analyze QTLs for traits related to low nitrogen tolerance, and predicted the candidate genes in the confidence intervals of QTL_S. 【Result】The traits associated with low nitrogen tolerance of RIL populations exhibited continuous distribution with apparent transgressive segregation both under low nitrogen and normal nitrogen levels, which conformed to the typical genetic characteristics of quantitative traits and were suitable for QTL genetic analysis. Correlation analysis showed that seeding length was positively correlated with maximum root length, root dry weight, seeding dry weight, plant dry weight and relative chlorophyll content, and maximum root length was negatively correlated with plant nitrogen content. A total of thirty-four QTLs related to seeding length, maximum root length, root dry weight, seeding dry weight, plant dry weight, relative chlorophyll content and plant nitrogen content were located under low nitrogen and normal nitrogen levels, which distributed on chromosomes from 1 to 9. They explained individually 5.15%-52.42% phenotypic variation. Ten QTLs were simultaneously detected under both two nitrogen levels, eleven and thirteen QTLs were only identified under single low nitrogen and normal nitrogen conditions, respectively. A total of fifteen QTLs were major QTL, and five major QTLs were repeatedly detected under both two nitrogen levels, which including qRDW3, qMRL1.1, qMRL1.2, qSL5 and qSPAD1. Five QTL overlaps were detected with gathering multiple QTLs under two nitrogen levels. Six candidate genes related to nitrogen metabolism were identified from the confidence interval of the five QTL overlaps, suggesting that genes related to nitrogen assimilation, absorption and utilization probably control the expression of these genes. 【Conclusion】Thirty-four QTLs were scattered on sixteen clusters of nine chromosomes. Based on gene annotation, a total of 6 candidate genes related to nitrogen metabolism were screened in foxtail millet, indicating the different traits involved in common genetic mechanisms, and the favorable alleles for low nitrogen tolerance can be polymerized by marker-assisted selection.

Key words: foxtail millet, recombinant inbred line (RIL), low nitrogen tolerance, QTL

秦娜, 付森杰, 朱灿灿, 代书桃, 宋迎辉, 魏昕, 王春义, 叶珍言, 李君霞. 谷子苗期耐低氮相关性状的QTL分析[J]. 中国农业科学, 2023, 56(20): 3931-3945.

QIN Na, FU SenJie, ZHU CanCan, DAI ShuTao, SONG YingHui, WEI Xin, WANG ChunYi, YE ZhenYan, LI JunXia. QTL Analysis for Seeding Traits Related to Low Nitrogen Tolerance in Foxtail Millet[J]. Scientia Agricultura Sinica, 2023, 56(20): 3931-3945.

图/表 7

图1

表1

图2

表2

表3

谷子苗期耐低氮相关性状的QTL定位"

性状 Traits	氮水平 N level	染色体 Chromosome	数量性状位点 QTL	标记区间 Marker interval	LOD值 LOD value	加性效应 Additive effect	贡献率 Proportion of the variance explained (%)
苗长 SL	低氮-N	1	qSL1	SICAAS1060—SICAAS1057	3.20	-0.88	5.15
		3	qSL3	SICAAS3043—SICAAS3048	4.70	-1.71	20.39
		5	qSL5	SICAAS5085—SICAAS5049	2.99	1.22	10.37
		6	qSL6	SICAAS6055—SICAAS6008	3.30	1.13	8.72
		7	qSL7	SICAAS7028—SICAAS7038	3.56	1.08	8.08
	正常氮+N	3	qSL3.1	SICAAS3022—SICAAS3029	3.59	1.11	7.14
		3	qSL3.2	SICAAS3029—SICAAS3034	3.37	1.15	7.51
		5	qSL5.1	SICAAS5085—SICAAS5049	3.27	1.16	7.92
		5	qSL5.2	SICAAS5055—SICAAS5083	3.49	-1.14	8.62
		5	qSL5.3	SICAAS5083—SICAAS5034	3.49	-1.02	6.82
		6	qSL6	SICAAS6019—SICAAS6005	2.99	-0.97	5.79
主根长 MRL	低氮-N	1	qMRL1.1	SICAAS1034—SICAAS1039	2.52	-2.82	27.31
		1	qMRL1.2	SICAAS1039—SICAAS1008	4.72	-2.23	13.76
		1	qMRL1.3	SICAAS1008—SICAAS1010	3.64	1.32	6.00
		3	qMRL3	SICAAS3043—SICAAS3048	3.18	1.40	6.78
		5	qMRL5.1	SICAAS5083—SICAAS5034	2.42	1.27	5.19
		5	qMRL5.2	SICAAS5034—SICAAS5035	4.31	1.88	11.07
		7	qMRL7	SICAAS7005—SICAAS7028	3.55	1.71	9.82
		9	qMRL9	SICAAS9033—SICAAS9130	3.60	-1.74	8.31
	正常氮+N	1	qMRL1.1	SICAAS1034—SICAAS1039	5.80	-2.44	28.50
		1	qMRL1.2	SICAAS1039—SICAAS1008	7.35	-2.48	26.56
		2	qMRL2	SICAAS2046—SICAAS2016	2.82	1.09	5.64
		6	qMRL6	SICAAS6005—SICAAS6058	2.28	-1.18	6.56
		8	qMRL8	SICAAS8001—SICAAS8007	3.39	-1.36	8.65
根干质量 RDW	低氮-N	3	qRDW3	SICAAS3043—SICAAS3048	7.51	-0.36	28.54
		6	qRDW6	SICAAS6055—SICAAS6008	2.23	0.19	6.86
		7	qRDW7	SICAAS7005—SICAAS7028	2.30	-0.22	9.06
	正常氮+N	1	qRDW1	SICAAS1057—SICAAS1052	2.95	-0.2	5.58
		3	qRDW3.1	SICAAS3069—SICAAS3022	3.19	-0.26	8.59
		3	qRDW3.2	SICAAS3022—SICAAS3029	4.32	-0.33	13.40
		3	qRDW3.3	SICAAS3043—SICAAS3048	4.13	-0.34	14.62
		4	qRDW4.1	SICAAS4060—SICAAS4033	2.20	0.26	9.04
		4	qRDW4.2	SICAAS4003—SICAAS4062	3.45	0.38	17.53
		9	qRDW9	SICAAS9024—SICAAS9027	2.02	0.28	7.89
苗干质量 SDW	低氮-N	1	qSDW1.1	SICAAS1060—SICAAS1057	2.76	-0.50	8.71
		1	qSDW1.2	SICAAS1057—SICAAS1052	2.25	-0.41	5.69
		3	qSDW3	SICAAS3043—SICAAS3048	5.43	0.78	23.00
	正常氮+N	5	qSDW5.1	SICAAS5083—SICAAS5034	3.17	0.78	10.71
		5	qSDW5.2	SICAAS5035—SICAAS5036	2.20	0.61	5.39
		6	qSDW6.1	SICAAS6055—SICAAS6008	2.60	-0.63	6.86
		6	qSDW6.2	SICAAS6008—SICAAS6081	2.81	-0.79	10.24
总干质量 PDW	低氮-N	1	qPDW1.1	SICAAS1060—SICAAS1057	3.64	-0.92	15.67
		1	qPDW1.2	SICAAS1057—SICAAS1052	2.28	-0.68	8.64
		2	qPDW2	SICAAS2010—SICAAS2068	2.78	0.97	17.74
		3	qPDW3	SICAAS3043—SICAAS3048	6.88	1.7	52.42
		5	qPDW5	SICAAS5036—SICAAS5037	3.64	0.90	15.01
		7	qPDW7	SICAAS7005—SICAAS7028	2.04	-0.59	6.28
	正常氮+N	3	qPDW3	SICAAS3029—SICAAS3034	2.26	1.20	10.25
叶绿素相对含量SPAD	低氮-N	1	qSPAD1	SICAAS1034—SICAAS1039	2.99	0.85	9.41
		2	qSPAD2	SICAAS2035—SICAAS2038	2.40	-0.86	9.77
		5	qSPAD5.1	SICAAS5034—SICAAS5035	3.22	1.05	13.28
		5	qSPAD5.2	SICAAS5035—SICAAS5036	2.94	0.84	8.27
		8	qSPAD8.1	SICAAS8007—SICAAS8014	2.15	0.98	12.44
		8	qSPAD8.2	SICAAS8014—SICAAS8019	2.41	1.02	12.64
	正常氮+N	1	qSPAD1	SICAAS1034—SICAAS1039	3.57	1.11	13.47
		9	qSPAD9.1	SICAAS9001—SICAAS9107	2.77	-0.73	5.67
		9	qSPAD9.2	SICAAS9006—SICAAS9013	2.42	-0.76	5.40
植株含氮量 PNC	低氮-N	1	qPNC1.1	SICAAS1060—SICAAS1057	2.87	0.12	8.77
		1	qPNC1.2	SICAAS1057—SICAAS1052	2.51	0.10	6.78
		2	qPNC2	SICAAS2046—SICAAS2016	2.90	0.09	6.04
		3	qPNC3	SICAAS3043—SICAAS3048	2.40	0.99	6.82
		5	qPNC5.1	SICAAS5083—SICAAS5034	4.44	0.17	16.92
		5	qPNC5.2	SICAAS5034—SICAAS5035	5.03	0.18	20.80
		6	qPNC6.1	SICAAS6019—SICAAS6005	2.50	-0.10	6.96
		6	qPNC6.2	SICAAS6055—SICAAS6008	2.79	-0.12	9.06
	正常氮+N	2	qPNC2	SICAAS2053—SICAAS2020	2.01	-0.16	8.84
	正常氮+N	8	qPNC8	SICAAS8014—SICAAS8019	2.82	0.18	9.62

表3

图3

表4

候选基因注释"

性状 Traits	数量性状位点 QTL	染色体 Chromosome	候选基因 Candidate genes	KO注释 KO annotation	GO注释 GO annotation	功能注释 Functional annotation
主根长 MRL	qMRL1.1	1	Seita.1G121100	K18482	GO:0004084	D-氨基酸转氨酶 D-amino-acid transaminase
主根长 MRL	qMRL3	3	Seita.3G051900		GO:0006808	氮利用调控蛋白P-Ⅱ Nitrogen regulatory protein P-Ⅱ
主根长 MRL	qMRL5.1	5	Seita.5G068000	K14638	GO:0015333	蛋白NRT1/PTR6.1家族 Protein NRT1/PTR FAMILY 6.1
主根长 MRL	qMRL5.2	5	Seita.5G400900	K14209	GO:0003333	氨基酸透性酶第8亚家族 Amino acid permease 8
叶绿素相对含量 SPAD	qSPAD1	1	Seita.1G121100	K18482	GO:0004084	D-氨基酸转氨酶 D-amino-acid transaminase
叶绿素相对含量 SPAD	qSPAD5.1	5	Seita.5G400900	K14209	GO:0003333	氨基酸透性酶第8亚家族 Amino acid permease 8
根干质量 RDW	qRDW3	3	Seita.3G051900		GO:0006808	氮利用调控蛋白P-Ⅱ Nitrogen regulatory protein P-Ⅱ
根干质量 RDW	qRDW6	6	Seita.6G078400	K19476	GO:0016021	赖氨酸组氨酸转运子1 Lysine histidine transporter 1
根干质量 RDW	qRDW9	9	Seita.9G477200	K14638	GO:0022857	蛋白NRT1/PTR8.3家族 Protein NRT1/PTR FAMILY 8.3
苗干质量 SDW	qSDW3	3	Seita.3G051900		GO:0006808	氮利用调控蛋白P-Ⅱ Nitrogen regulatory protein P-Ⅱ
苗干质量 SDW	qSDW5.1	5	Seita.5G068000	K14638	GO:0015333	蛋白NRT1/PTR6.1家族 Protein NRT1/PTR FAMILY 6.1
苗干质量 SDW	qSDW6.1	6	Seita.6G078400	K19476	GO:0016021	赖氨酸组氨酸转运子1 Lysine histidine transporter 1
总干质量 PDW	qPDW3	3	Seita.3G051900		GO:0006808	氮利用调控蛋白P-Ⅱ Nitrogen regulatory protein P-Ⅱ
植株含氮量 PNC	qPNC3	3	Seita.3G051900		GO:0006808	氮利用调控蛋白P-Ⅱ Nitrogen regulatory protein P-Ⅱ
植株含氮量 PNC	qPNC5.1	5	Seita.5G068000	K14638	GO:0015333	蛋白NRT1/PTR6.1家族 Protein NRT1/PTR FAMILY 6.1
植株含氮量 PNC	qPNC5.2	5	Seita.5G400900	K14209	GO:0003333	氨基酸透性酶第8亚家族 Amino acid permease 8
植株含氮量 PNC	qPNC6.2	6	Seita.6G078400	K19476	GO:0016021	赖氨酸组氨酸转运子1 Lysine histidine transporter 1
苗长 SL	qPH5.3	5	Seita.5G068000	K14638	GO:0015333	蛋白NRT1/PTR6.1家族 Protein NRT1/PTR FAMILY 6.1
苗长 SL	qPH6	6	Seita.6G078400	K19476	GO:0016021	赖氨酸组氨酸转运子1 Lysine histidine transporter 1

表4

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性状 Trait	氮水平 N level	亲本Parents		t测验 t test	RIL群体RIL population
性状 Trait	氮水平 N level	豫谷28 Yugu 28	七叶黄Qiyehuang	t测验 t test	均值Mean	变异范围Range
苗长 SL (cm)	+N	29.10±1.60	24.40±0.90	*	27.90±1.10	15.20—39.50
苗长 SL (cm)	-N	21.10±0.90	17.50±0.60	*	20.40±1.01	8.00—31.00
差异Difference (%)		-27.50*	-28.30*		-26.88*
主根长 MRL (cm)	+N	16.10±1.10	11.20±0.70	ns	17.20±0.40	6.50—35.50
主根长 MRL (cm)	-N	18.10±0.30	14.20±0.40	*	22.50±1.10	8.20—40.50
差异Difference (%)		12.50*	17.86*		30.81**
根干质量 RDW (g)	+N	0.040±0.0010	0.030±0.0010	*	0.030±0.010	0.010—0.050
根干质量 RDW (g)	-N	0.030±0.0020	0.020±0.0010	ns	0.020±0.0020	0.0010—0.040
差异Difference (%)		-25.00*	-33.30 ns		-33.33**
苗干质量 SDW (g)	+N	0.10 ±0.020	0.060±0.0010	*	0.080±0.0020	0.040—0.160
苗干质量 SDW (g)	-N	0.060±0.0020	0.010±0.0010	*	0.040±0.0010	0.010—0.10
差异Difference (%)		-40.00**	-83.30**		-50.00**
总干质量 PDW (g)	+N	0.15±0.030	0.080±0.0020	*	0.11±0.010	0.040—0.22
总干质量 PDW (g)	-N	0.080 ±0.0020	0.040±0.0010	*	0.060±0.0020	0.010—0.12
差异Difference (%)		-46.67*	-50.00*		-45.45**
叶绿素相对含量 SPAD	+N	31.70±1.90	25.20±2.01	*	31.30±2.20	23.05—36.57
叶绿素相对含量 SPAD	-N	28.50±2.10	21.20±0.80	*	22.50±1.90	16.75—28.13
差异Difference (%)		-10.10 ns	-15.87*		-28.11*
植株氮含量 PNC (mg·100 mg^-1)	+N	2.60±0.10	2.20±0.090	ns	2.70±0.10	1.89—4.82
植株氮含量 PNC (mg·100 mg^-1)	-N	2.20±0.080	1.60±0.10	*	1.90±0.30	1.21—2.97
差异Difference (%)		-15.38 ns	-27.27*		-29.63*

氮水平 N level	性状 Trait	苗长 SL	主根长 RL	根干质量 RDW	苗干质量 SDW	总干质量 PDW	叶绿素相对含量 SPAD	植株含氮量 PNC
低氮-N	苗长SL		0.653**	0.584*	0.814**	0.798**	0.692**	0.142
	主根长MRL			0.701**	0.568*	0.684**	0.583*	-0.223*
	根干质量RDW				0.646**	0.708**	0.561*	0.285*
	苗干质量SDW					0.825**	0.675**	0.305*
	总干质量PDW						0.794**	0.313*
	叶绿素相对含量SPAD							0.724**
正常氮+N	苗长SL	0.795**	0.817**	0.792**	0.884**	0.830**	0.751**	0.256*
	主根长MRL		0.806**	0.726**	0.806**	0.784**	0.696**	-0.348**
	根干质量RDW			0.652**	0.734**	0.805**	0.265*	0.217*
	苗干质量SDW				0.633**	0.794**	0.852**	0.324*
	总干质量PDW					0.749**	0.631**	0.352*
	叶绿素相对含量SPAD						0.760**	0.554*
	植株含氮量PNC							0.670**