玉米氮效率QTL定位和候选基因筛选

doi:10.3864/j.issn.0578-1752.2024.21.002

摘要/Abstract

摘要：

【目的】实现玉米养分高效利用的遗传改良是保障国家粮食安全的重要途径。挖掘玉米氮高效QTL与相关候选基因可为提高玉米氮肥使用效率，为培育高产高效玉米品种提供理论基础。【方法】通过对KA105/KB024构建的重组自交系群体在不同氮素处理下的籽粒产量以及氮肥偏生产力、耐低氮系数、氮肥农学利用效率进行QTL定位分析，同时，结合亲本KA105在苗期低氮水平下的转录组数据筛选差异表达基因，利用共表达分析挖掘玉米氮效率候选基因，并利用qRT-PCR对筛选到的候选基因进行验证。【结果】通过定位分析，共检测到36个分布在不同染色体上的QTL，可解释1.63%—17.26%的表型变异。其中，鉴定到8个表型变异解释率超过10%的主效QTL，7个在不同性状或环境下共同被鉴定到的遗传稳定QTL。其中，位于第1染色体的qNNGYP1在前人研究中被多次检测到，其表型解释率可达11.73%，不同环境下多个QTL（qNNGYP1和qPFPN1）在此区间内被检测到，可作为重点区段进行更进一步的研究。结合苗期低氮胁迫下转录组数据，在QTL区间内共筛选到39个差异表达基因，进行共表达网络预测后，鉴定到6个节点基因作为候选基因，qRT-PCR结果显示，在2种氮处理下，候选基因的表达趋势与转录组数据相同。其中，GRMZM2G366873参与生长素稳态的调控，可能通过生长素信号转导参与玉米低氮胁迫、干旱胁迫与硼胁迫的响应，并调控玉米穗长；GRMZM2G414192参与光合系统对低氮胁迫的响应，其蛋白含量受油菜素甾醇调控；GRMZM2G414043与玉米粒长及生物量相关；GRMZM2G040642可能参与氮的远距离信号传导。【结论】检测到36个QTL，分布于第1、4、5、7、8和9染色体上，其中，包含8个主效QTL（PVE>10%）。筛选到GRMZM2G366873、GRMZM2G414192、GRMZM2G414043、GRMZM2G040642可作为玉米氮效率候选基因。

关键词: 玉米, 氮效率, QTL, 转录组, 候选基因

Abstract:

【Objective】Genetic improvement for efficient utilization of maize nutrients represents a crucial method to ensure national food security. Exploring quantitative trait locus (QTL) and related candidate genes of nitrogen use efficiency can provide a theoretical basis for improving the efficiency of nitrogen fertilizer in maize and cultivating high-yield and high-efficiency maize varieties. 【Method】In this study, QTL mapping analysis in one recombinant inbred line (RIL) population constructed by KA105 and KB024 was performed for grain yield under two different nitrogen treatments, including the derived traits partial factor productivity from applied nitrogen (PFPN), low nitrogen tolerance coefficient (LNTC) and nitrogen agronomic efficiency (NAE). Concurrently, integrating the seedling transcriptome data of the parent KA105 under nitrogen treatment, differentially expressed genes were identified, and candidate genes associated with maize nitrogen use efficiency were mined through co-expression analysis. Subsequently, the selected candidate genes were validated using qRT-PCR. 【Result】Through mapping analysis, a total of 36 QTLs distributed across different chromosomes were detected, explaining 1.63% to 17.26% of the phenotypic variation. Among these, eight major QTLs with a phenotypic variation explanation rate exceeding 10% were identified, along with seven genetically stable QTLs commonly identified across different traits or environments. Notably, qNNGYP1 located on chromosome 1 has been repeatedly detected in previous studies, with a phenotypic explanation rate of up to 11.73%. Additionally, other QTLs (qNNGYP1, qPFPN1) co-located in this interval across different environments, suggesting it as a focal region for further investigation. Combining transcriptome data of seedlings under low nitrogen stress, 39 differentially expressed genes within these QTL intervals were identified, and 6 key genes were identified through co-expression network prediction. The result of qRT-PCR indicated that the expression trends of the candidate genes under both nitrogen treatments were consistent with the transcriptome data. Specifically, GRMZM2G366873 was involved in the regulation of auxin homeostasis and may participate in maize responses to low nitrogen stress, drought stress, and boron stress through auxin signal transduction, also regulating ear length. GRMZM2G414192 was involved in the response of the photosynthetic system to low nitrogen stress and was regulated by brassinosteroids. GRMZM2G414043 was associated with maize grain length and biomass, while GRMZM2G040642 may be involved in the long-distance signal transduction of nitrogen. 【Conclusion】In summary, a total of 36 QTLs were identified, distributed across chromosomes 1, 4, 5, 7, 8, and 9, including eight major QTLs (PVE>10%). The candidate genes GRMZM2G366873, GRMZM2G414192, GRMZM2G414043, and GRMZM2G040642 were identified as potential genes for maize nitrogen efficiency.

Key words: maize, nitrogen use efficiency, QTL, transcriptome, candidate genes

韩旭东, 杨传奇, 张擎, 李亚伟, 杨夏夏, 何佳甜, 薛吉全, 张兴华, 徐淑兔, 刘建超. 玉米氮效率QTL定位和候选基因筛选[J]. 中国农业科学, 2024, 57(21): 4175-4191.

HAN XuDong, YANG ChuanQi, ZHANG Qing, LI YaWei, YANG XiaXia, HE JiaTian, XUE JiQuan, ZHANG XingHua, XU ShuTu, LIU JianChao. QTL Mapping and Candidate Gene Screening for Nitrogen Use Efficiency in Maize[J]. Scientia Agricultura Sinica, 2024, 57(21): 4175-4191.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.21.002

https://www.chinaagrisci.com/CN/Y2024/V57/I21/4175

图/表 9

表1

表2

表3

表4

图1

图2

表5

氮效率相关性状QTL定位结果"

性状 Trait	位点 QTL	地点 Location	染色体 Chromosome	两侧标记物理位置 Physical position of the beside markers (bp)	LOD	表型解释率 PVE (%)	加性效应 Additive effect
正常施氮下单株产量 NNGWP	qNNGYP1	WN	1	35995804-36424242	5.15	11.73	5.31
	qNNGYP1	BLUP	1	35995804-36424242	6.84	7.56	2.32
	qNNGYP7-1	BLUP	7	131382844-131410325	9.38	10.49	2.74
	qNNGYP7-2	BLUP	7	162437502-162997059	4.69	4.90	-1.87
	qNNGYP7-3	WN	7	165055895-165294994	4.40	9.77	-4.83
	qNNGYP9	BLUP	9	3283059-3562938	4.77	5.02	1.89
低氮下单株产量 LNGWP	qLNGYP1-1	WN	1	34719687-34815083	8.94	4.14	6.05
	qLNGYP1-2	WN	1	253561747-253822941	27.33	17.26	12.35
	qLNGYP1-3	WN	1	246413563-249477487	14.04	7.45	-8.14
	qLNGYP1-4	WN	1	257856595-258579921	15.38	7.91	-8.36
	qLNGYP4	WN	4	225263551-225495039	5.29	2.34	-4.61
	qLNGYP7-1	YL	7	108562462-112964730	11.37	11.64	-6.51
	qLNGYP7-2	YL	7	82754779-108730885	8.09	7.70	5.29
	qLNGYP7-3	YL	7	129914065-129964901	4.30	4.09	3.85
	qLNGYP7-4	BLUP	7	165497988-165833830	6.54	8.51	-1.96
	qLNGYP7-5	WN	7	167024420-167263154	3.89	1.64	-3.82
	qLNGYP8-1	WN	8	142064004-144623033	6.33	2.78	4.97
	qLNGYP8-2	BLUP	8	125629874-126267369	4.78	5.99	1.64
	qLNGYP9	BLUP	9	3283059-3562938	4.35	5.44	1.57
耐低氮系数 LNTC	qLNTC1-1	BLUP	1	4445103-4538029	7.82	5.41	0.03
	qLNTC1-2	BLUP	1	8534228-8452804	3.91	2.63	-0.02
	qLNTC1-3	WN	1	70738546-71034049	4.87	5.98	-0.05
	qLNTC1-4	WN	1	86368227-90237055	9.72	12.92	0.07
	qLNTC5	WN	5	184392272-185547677	4.46	5.48	-0.04
	qLNTC8-1	BLUP	8	120298869-123737924	5.95	4.00	0.02
	qLNTC8-2	WN	8	145442273-147617047	4.61	5.66	0.04
	qLNTC8-3	BLUP	8	125629874-126267369	14.02	10.66	-0.04
氮肥偏生产力 PFPN	qPFPN1	BLUP	1	35995804-36424242	5.15	11.73	1.77
	qPFPN1	WN	1	35995804-36424242	6.84	7.56	0.77
	qPFPN7-1	BLUP	7	131382844-131410325	9.38	10.49	0.91
	qPFPN7-2	BLUP	7	162437502-162997060	4.69	4.91	-0.62
	qPFPN7-3	WN	7	165055895-165294994	4.40	9.77	-1.61
	qPFPN9	BLUP	9	3283059-3562938	4.77	5.02	0.63
氮肥农学利用效率 NAE	qNAE1	BLUP	1	4445103-4538029	6.26	7.51	0.47
	qNAE9-1	YL	9	147632676-148309394	3.85	6.22	-1.02
	qNAE9-2	BLUP	9	148309394-148346218	5.29	6.35	-0.43

表5

图3

表6

QTL区间内差异表达基因"

候选基因ID Candidate gene ID	差异倍数 log₂FC	QTL名称 QTL name	基因注释 Gene annotation
GRMZM2G172758★	-5.11	qLNTC1-2	类核仁素Nucleolin-like
GRMZM2G017011	-11.14	qLNTC1-4	花粉Ole e 1过敏原和扩展蛋白家族蛋白Pollen Ole e 1 allergen and extensin family protein
GRMZM2G469371	1.61	qLNTC1-4	环-H2指蛋白ATL57 RING-H2 finger protein ATL57
GRMZM2G090029	2.20	qLNTC1-4	类细胞周期蛋白依赖性蛋白激酶抑制剂SMR1 Cyclin-dependent protein kinase inhibitor SMR1-like
GRMZM2G046848	3.19	qLNTC1-4	含U-box结构域的蛋白质70 U-box domain-containing protein 70
GRMZM2G021055	-4.15	qLNGYP1-2	排毒蛋白29 Protein DETOXIFICATION 29
GRMZM2G414192★	-1.52	qLNGYP1-3	叶绿素a-b结合蛋白Chlorophyll a-b binding protein
GRMZM2G171118	2.53	qLNGYP1-3	类环戊二烯C2-羟化酶Ent-cassadiene C2-hydroxylase-like
GRMZM2G046101	-3.978	qLNGYP1-4	葡聚糖内-1,3-beta-葡萄糖苷酶7 Glucan endo-1,3-beta-glucosidase 7
GRMZM2G055037	2.09	qLNGYP1-4	泛素蛋白配体/锌离子结合蛋白Ubiquitin-protein ligase/ zinc ion binding protein
GRMZM2G474119	1.78	qLNGYP4	-
GRMZM2G357371	-12.12	qLNGYP7-2	类严格甲素合成酶蛋白10 Protein STRICTOSIDINE SYNTHASE-LIKE 10
GRMZM2G057208	-6.09	qLNGYP7-2	细胞色素P450 Cytochrome P450
GRMZM2G129453	-3.77	qLNGYP7-2	δ(8)-脂肪酸去饱和酶2 Delta(8)-fatty-acid desaturase 2
GRMZM2G141679	-2.91	qLNGYP7-2	乙烯反应性转录因子RAP2-4 Ethylene-responsive transcription factor RAP2-4
GRMZM2G149295	-2.11	qLNGYP7-2	杯状蛋白，RmlC型Cupin, RmlC-type
GRMZM2G414813	-1.91	qLNGYP7-2	核碱基-抗坏血酸转运体6 Nucleobase-ascorbate transporter 6
GRMZM2G473001	-1.60	qLNGYP7-2	磷酸烯醇丙酮酸羧化酶4 Phosphoenolpyruvate carboxylase 4
GRMZM2G162396	-1.51	qLNGYP7-2	-
GRMZM2G074781	1.47	qLNGYP7-2	α-淀粉酶1 Alpha-amylase 1
GRMZM2G176721	2.08	qLNGYP7-2	主要协同转运蛋白超家族Major facilitator superfamily protein
GRMZM2G056875	2.20	qLNGYP7-2	富脯氨酸受体样蛋白激酶PERK7 Proline-rich receptor-like protein kinase PERK7
GRMZM2G479906	1.57	qLNGYP7-2	ABC转运体G家族成员53 ABC transporter G family member 53
GRMZM2G008665▲	9.97	qNNGYP7-2 qPFPN7-2	锌指SWIM结构域家族蛋白Zinc finger SWIM domain family protein
GRMZM2G116426▲	-1.80	qNNGYP7-3 qPFPN7-3	α/β-水解酶超家族蛋白Alpha/beta-Hydrolases superfamily protein
GRMZM2G414043★	-2.91	qLNGYP7-4	-
GRMZM2G081857	-1.85	qLNGYP7-5	可能不活跃的受体激酶At5g10020 Probable inactive receptor kinase At5g10020
GRMZM2G366873★	-12.20	qLNTC8-1	生长素酰胺合成酶12 Auxin amido synthetase12
GRMZM2G083016	-1.61	qLNTC8-1	Ⅱ型偏半胱天冬酶Metacaspase type Ⅱ
GRMZM2G034197	-11.51	qLNGYP8-1	叶绿体磷脂酶A1 PLIP1 Phospholipase A1 PLIP1, chloroplastic
GRMZM2G052844	-3.35	qLNGYP8-1	可能的聚半乳糖醛酸酶Probable polygalacturonase
GRMZM2G040642★	-1.41	qLNGYP8-1	可能的蛋白磷酸酶2C7 Probable protein phosphatase 2C7
GRMZM2G025387	-1.39	qLNGYP8-1	钙依赖蛋白激酶Calcium-dependent protein kinase
GRMZM2G091358	2.89	qLNGYP8-1	推定DUF1221域含蛋白激酶家族蛋白 Putative DUF1221-domain containing protein kinase family protein
GRMZM2G094356	-3.15	qLNTC8-2	CASP样蛋白4U1 CASP-like protein 4U1
GRMZM2G179696★	-1.23	qLNTC8-2	聚半乳糖醛酸酶Polygalacturonase
GRMZM2G094165	-1.06	qLNTC8-2	碳酸酐酶6 Carbonic anhydrase6
GRMZM2G432128	1.09	qLNTC8-2	细胞质异柠檬酸脱氢酶Cytosolic isocitrate dehydrogenase
GRMZM2G157334	1.66	qLNTC8-2	GTP结合核蛋白Ran-2 GTP-binding nuclear protein Ran-2

表6

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环境 Environment	总氮 Total-N (g·kg^-1)	速效磷 Available-P (mg·kg^-1)	速效钾 Available-K (mg·kg^-1)	有机质 Organic (g·kg^-1)	pH
杨凌 YL	0.93	20.98	114.00	16.70	8.46
渭南 WN	0.89	17.87	148.50	16.48	9.04

引物名称Primer name	正向引物Forward primers (5′-3′)	反向引物Reverse primers (5′-3′)
GRMZM2G172758	GGCTCGATGAGTGTCGGAT	GAGGTTCAGGCTCTCTACGG
GRMZM2G414192	ACCAGACCACCAGCTTCCTC	TCGGAGAACGGGCCAAGATA
GRMZM2G414043	TGACGTTAGTTTGCAGCGGA	GGTGTGCTTTCAACCTGCAC
GRMZM2G366873	AGCATCGACTCCGACAAGA	CCAGAACAGCACGTAGTGG
GRMZM2G040642	GGAGTATTTGATGGTCATGGAGG	GACCCAACCCCAACATGAAT
GRMZM2G179696	ACTGTCAAGGAGTGATGGTCC	CAATCGTCACCACAAGCCAG

来源 Source of variation	平方和 Sum of squares	自由度 DF	均方 Mean squares	F值F-value
地点Environment (E)	25183.24	2	12591.62	747.07**
处理Treatment (T)	20072.27	1	20072.27	1190.90**
材料Genotype (G)	82811.52	196	422.51	25.07**
地点×处理E×T	1677.65	2	838.82	49.77**
处理×材料T×G	7854.66	193	40.70	2.47**
地点×材料E×G	55993.96	383	146.20	8.68**
误差Error	5713.75	339	16.86
总计Sum	2928126.42	1117

性状 Trait	地点 Site	亲本Parents		重组自交系群体RILs
性状 Trait	地点 Site	KA105	KB024	平均值 Average	标准差 SD	变异范围 Range	变异系数 CV (%)	峰度 Kurt	偏度 Skew	遗传力 H² (%)
正常氮下单株产量 NNGYP (g)	WN	82.94	58.64	59.40	15.13	22.93-93.51	25.47	-0.47	-0.34	76.92
	YL	58.97	42.15	48.17	12.57	17.44-79.67	26.10	0.01	0.14
	BLUP	61.73	50.10	51.76	6.14	38.10-69.40	11.86	-0.33	0.06
低氮下单株产量 LNGYP (g)	WN	78.77	57.33	49.90	16.44	13.40-85.93	32.95	-0.55	-0.24	68.29
	YL	51.66	29.12	36.88	10.99	13.95-65.01	29.80	-0.31	0.04
	BLUP	58.48	46.04	46.34	5.78	31.70-60.76	12.47	-0.20	0.07
耐低氮系数 LNTC	WN	0.95	0.98	0.83	0.14	0.32-1.00	16.87	0.75	-1.09	65.50
	YL	0.88	0.69	0.78	0.16	0.43-1.00	20.51	-1.10	-0.52
	BLUP	0.95	0.92	0.90	0.08	0.72-1.13	8.89	0.14	0.15
氮肥偏生产力 PFPN	WN	27.65	19.55	19.80	5.04	7.64-31.17	25.45	-0.47	-0.34	76.92
	YL	19.66	14.05	16.06	4.19	5.81-26.56	26.09	0.01	0.14
	BLUP	20.58	16.70	17.25	2.05	12.70-23.13	11.88	-0.33	0.06
氮肥农学利用效率 NAE	WN	1.39	0.44	3.17	2.59	0.01-10.40	81.70	0.04	0.95	69.70
	YL	2.43	1.34	3.76	3.04	0.02-9.93	80.85	-1.02	0.63
	BLUP	1.08	1.35	1.90	1.22	0.15-5.05	64.21	-0.54	0.52