利用高密度Bin图谱定位水稻抽穗期剑叶叶绿素含量QTL

doi:10.3864/j.issn.0578-1752.2022.05.001

摘要/Abstract

摘要：

【目的】挖掘新的控制水稻叶绿素含量的相关位点和基因,为水稻叶绿素含量的遗传机制研究提供理论基础。【方法】利用剑叶叶色存在明显差异的粳稻TD70和籼稻Kasalath杂交构建的包含186个株系的重组自交系群体为供试材料,通过对两亲本及RIL群体重测序,构建了包含12 328个Bin标记的高密度遗传图谱。RIL群体及亲本分别于2011和2020年正季在江苏省农业科学院种植。抽穗后第3天使用叶绿素仪测定剑叶SPAD值。使用IciMappingv3.4软件完备区间作图法,对控制水稻抽穗期剑叶叶绿素含量的QTL进行鉴定。利用便携式光合仪测定RIL群体中20个SPAD极端株系的水分利用效率、蒸腾速率、气孔导度和净光合速率等光合作用参数。【结果】2年共检测到19个抽穗期剑叶叶绿素含量相关QTL,分别分布在除第8、9和10染色体外的其他9个染色体上。单一QTL贡献率为3.09%—13.13%,LOD值为2.74—14.08。通过物理位置比对,发现其中10个QTL与前人定位到的叶绿素含量相关位点在相同或邻近区域。qCHL2-1和qCHL5-1 2年均被检测到,表现出较强的稳定性。qCHL2-1位于第2染色体的7.63—7.71 Mb处,2年LOD值分别为14.08和7.93,贡献率分别为13.13%和7.94%。qCHL5-1位于第5染色体的23.44—23.49 Mb处,2年LOD值分别为4.31和3.76,贡献率分别为3.57%和4.82%。结合功能注释和亲本间序列分析,分别在qCHL2-1和qCHL5-1染色体区间内找到2个与剑叶叶绿素含量相关的基因Os02g0236000和Os05g0476700。这两个基因的核苷酸序列在两亲本间均存在差异。Os02g0236000编码水稻天冬氨酸氨基转移酶（AAT1）,是水稻氮代谢途径中的重要酶,与蛋白质及氨基酸含量有关。Os05g0476700编码叶面斑点相关蛋白,推测与叶片颜色有关。根据AAT1在CDS+273 bp有无突变对RIL群体进行等位型分类。在20个SPAD极端株系中,AAT1不同等位型株系的剑叶SPAD值和水分利用效率、蒸腾速率、气孔导度和净光合速率等光合作用指标均存在显著差异。【结论】共检测到19个控制水稻抽穗期剑叶叶绿素含量QTL,鉴定了2个稳定存在的QTL——qCHL2-1和qCHL5-1,在这两个QTL区间筛选到2个可能调控水稻抽穗期剑叶叶绿素含量的基因。其中1个AAT1（Os02g0236000）不同等位型的光合作用参数在20个极端SPAD株系中存在显著差异,推测其为最可能的候选基因,可用于后续剑叶叶绿素调控基因的功能研究。

关键词: 水稻, 重组自交系, 高密度Bin图谱, 叶绿素含量, QTL

Abstract:

【Objective】Finding new loci and genes related to rice chlorophyll content, and providing new theoretical basis for the research on the genetic mechanism of rice chlorophyll content. 【Method】 A recombinant inbred line (RIL) population containing 186 lines was constructed by crossing the japonica rice TD70 and the indica rice Kasalath with obvious difference in the chlorophyll content of the flag leaf. The two parents and RIL population were re-sequenced to construct a high-density genetic linkage map with 12 328 recombination Bin markers. The RILs and two parents were planted in fields at the Jiangsu Academy of Agricultural Sciences, in Nanjing in 2011 and 2020. The contents of chlorophyll of flag leaves were directly measured using the chlorophyll meter SPAD-502 on the 3^rd day after heading. QTLs that control the chlorophyll content of the flag leaf at the heading stage of rice were detected by IciMappingv3.4 software with inclusive compound interval mapping method. The photosynthesis parameters of 20 SPAD extreme strains in the RIL population were measured with a portable photosynthesis system. 【Result】19 QTLs controlling chlorophyll content of flag leaves were detected on 9 chromosomes except Chr.8, Chr.9 and Chr.10 in two years. The phenotype variation explained (PVE) of single QTL ranged from 3.09% to 13.13%, LOD value ranged from 2.74 to 14.08. After comparing the physical positions, 10 QTLs were found to locate in the same interval or adjacent to previously QTLs. qCHL2-1 and qCHL5-1 were detected every year showing their genetic stability. qCHL2-1 was mapped between the 7.63-7.71 Mb on chromosome 2, and the two-year LOD values are 14.08 and 7.93 with the PVE 13.13% and 7.94%, respectively. qCHL5-1 was mapped between the 23.44-23.49 Mb on chromosome 5, and the two-year LOD values are 4.31 and 3.76, respectively. After the annotation and sequences analysis of genes located in the region of qCHL2-1and qCHL5-1, two genes, Os02g0236000 and Os05g0476700, were found to be associated with chlorophyll content of flag leaves in the rice. There are differences in sequences of the two genes between TD70 and Kasalath. Os02g0236000 is the AAT1 gene encoding the Aspartate Aminotransferase, which is an important enzyme in nitrogen metabolism and related to protein and amino acid content of rice. Os05g0476700 encodes protein relating to spotted leaf, which might associate with leaf color. Based on the mutation of AAT1 at CDS+273 bp, the haplotypes of ATT1 were classified in RIL population. Among the 20 extreme SPAD RIL lines, there were significant differences between different haplotype of ATT1 in SPAD value, chlorophyll content, water use efficiency, transpiration rate, stomatal conductance and net photosynthetic rate of flag leaf. 【Conclusion】19 QTLs associated with chlorophyll content in flag leaf at heading stage of rice were detected and two stable QTL loci, qCHL2-1and qCHL5-1 were identified. Two candidate genes were obtained after annotation and sequence comparison. One of them, ATT1, was considered as the most possible candidate gene after effort analysis of different haplotypes in photosynthetic efficiency. The QTLs and gene we obtained could be used for subsequent functional studies of flag leaf chlorophyll regulation and molecular marker breeding.

Key words: rice (Oryza sativa L.), recombinant inbred lines, high-density bin map, chlorophyll content, QTL

赵凌, 张勇, 魏晓东, 梁文化, 赵春芳, 周丽慧, 姚姝, 王才林, 张亚东. 利用高密度Bin图谱定位水稻抽穗期剑叶叶绿素含量QTL[J]. 中国农业科学, 2022, 55(5): 825-836.

ZHAO Ling, ZHANG Yong, WEI XiaoDong, LIANG WenHua, ZHAO ChunFang, ZHOU LiHui, YAO Shu, WANG CaiLin, ZHANG YaDong. Mapping of QTLs for Chlorophyll Content in Flag Leaves of Rice on High-Density Bin Map[J]. Scientia Agricultura Sinica, 2022, 55(5): 825-836.

图/表 7

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表2

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表4

本研究定位的QTL和已知叶绿素含量相关位点的位置比较"

本研究This study			已发表的相关位点/基因 Known QTLs/Genes
QTL	染色体 Chr.	物理位置 Position (Mb)	性状/基因 Character/Gene	定位群体/基因登录号 Population /Acc. No.	物理位置 Position (Mb)	参考文献 Reference
qCHL1-1	1	25.69—25.72	抽穗后7 d叶绿素含量 Chlorophyll content at 7d after heading	日本晴/Kasalath//日本晴BILs Nipponbare/Kasalath//Nipponbare BILs	25.13—26.19	[29]
qCHL1-2	1	31.76—31.83	干旱胁迫下的剑叶或倒2叶叶绿素含量 Chlorophyll content in drought stress	珍汕97B/IRAT109 RILs Zhenshan 97B/IRAT109 RILs	30.1—33.86	[26]
			分蘖期上部展开叶叶绿素含量 Chlorophyll content of up most fully expanded leaf at tillering period	ZYQ8/JX17 DHs	30.06—32.06	[27]
			拔节期剑叶叶绿素含量 Chlorophyll content of flag leaf at jointing stage	沈农0530-9/北陆129 F₂（F_2:3） Shennong0530-9/Habataki F₂ (F_2:3)	30.17—34.1	[28]
qCHL2-1	2	7.63—7.71	成熟期剑叶叶绿素含量 Chlorophyll content of flag leaf at mature stage	沈农265/丽江新团黑谷RILs Shennong 265/Lijiangxintuanheigu RILs	2.88—9.47	[11]
			分蘖期剑叶叶绿素含量 Chlorophyll content of flag leaf at tillering stage	岗46B/A232 RILs Gang 46B/A232 RILs	5.20—8.76	[30]
			发育期剑叶叶绿素b含量 Leaf chlorophyll b content at developmental stage	ZS97/WY2 DHs	7.43—11.41	[31]
qCHL3-1	3	25.41—25.44	叶绿素b还原酶 Chlorophyll b reductase	LOC_Os03g45194	25.52	[37]
qCHL4	4	23.30—23.38	分蘖期剑叶叶绿素含量 Chlorophyll content of flag leaf at tillering stage	岗46B/A232 RILs Gang 46B/A232 RILs	23.17—31.27	[30]
			苗期叶绿素含量 Chlorophyll content at seedling stage	珍汕97A/明恢63 RILs Zhenshan 97A/Minghui 63 RILs	22.28—26.86	[36]
			抽穗5 和25 d叶绿素含量的降低 Decreased chlorophyll content between leaves at 5 and 25 days after heading	Nipponbare/Kasalath BC₁F₁	22.27—26.38	[38]
qCHL5-1	5	23.44—23.49	抽穗7d剑叶叶绿素b含量 Chlorophyll b content of flag leaf at 7 days after heading	窄叶青8号/京系17 DH Zhaiyeqing 8/Jingxi 17 DH	19.27—31.45	[32]
			齐穗期剑叶叶绿素含量 Chlorophyll b content of flag leaf at full-heading stage	Dular/Lemont RILs	3.89—24.09	[33]
			开花期剑叶叶绿素含量 Degree of greenness of flag leaf at heading stage	珍汕97B/IRAT109 RILs Zhenshan 97/IRAT109 RILs	20.17—26.84	[34]
			孕穗期剑叶叶绿素b含量 Chlorophyll b contents of flag leaf at booting stage	十和田/丽江新团黑谷RILs Towada/Lijiangxintuanheigu RILs	0.46—23.95	[35]
			谷氨酸-1-半醛转氨酶基因GSA Glutamate -1-semialdehyde aminotransferase	LOC_Os05g39770	23.35	[11, 24]
qCHL5-2	5	0.19—0.26	干旱胁迫下的剑叶或倒2叶叶绿素含量 Chlorophyll content of leaves in drought stress	珍汕97B/IRAT109 RILs Zhenshan 97B/IRAT109 RILs	0.1—0.18	[26]
qCHL6	6	24.40—24.50	干旱胁迫下的剑叶或倒2叶叶绿素含量 Chlorophyll content of leaves in drought stress	珍汕97B/IRAT109 RILs Zhenshan 97B/IRAT109 RILs	24.03—28.13	[26]
qCHL6	6	24.40—24.50	抽穗后5d和25 d叶绿素含量的降低 Decreased chlorophy content between leaves at 5 and 25 days after heading	Nipponbare/Kasalath BC₁F₁	27.61—31.17	[38]
qCH7	7	24.35—24.37	羟甲基后胆色素原合酶 Hydroxymethylbilane synthase	LOC_Os07g40250	24.13	[11]
qCH7	7	24.35—24.37	硝酸盐转运蛋白基因OsNPF7.1 Nitrate and di/tripeptide transporter OsNPF7.1	LOC_Os07g41250	24.72	[12]
qCHL11	11	20.63—20.70	成熟期剑叶叶绿素含量 Chlorophyll content of flag leaf at mature stage	沈农265/丽江新团黑谷RILs Shennong 265/Lijiangxintuanheigu RILs	18.13—28.28	[11]

表4

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年份 Years	亲本Parents		重组自交系RIL population
年份 Years	TD70	Kasalath	平均值Average	变异范围 Range	变异系数 CV (%)	峰度 Kurtosis	偏度 Skewness
2011	48.20±0.70	34.50±0.78	42.08	30.51—49.00	9.29	-0.42	-0.42
2020	46.20±1.37	38.10±0.72	40.58	27.53—50.83	9.36	0.13	-0.11

年份 Year	QTL	染色体 Chr.	标记区间 Marker interval	置信区间 Confidence interval (Mb)	LOD	贡献率 PVE (%)	加性效应 Additive effect
2011	qCHL1-1	1	RBN0894—RBN0895	25.69—25.72	6.46	6.47	-1.43
	qCHL1-2	1	RBN1045—RBN1046	31.76—31.83	6.45	5.55	-1.18
	qCHL2-1*	2	RBN1570—RBN1571	7.63—7.71	14.08	13.13	-1.81
	qCHL2-2	2	RBN2335—RBN2336	35.12—35.15	2.74	3.76	-1.14
	qCHL3-1	3	RBN3011—RBN3012	25.41—25.44	4.85	4.04	0.99
	qCHL5-1*	5	RBN5254—RBN5255	23.44—23.49	4.31	3.57	0.91
	qCH6-1	6	RBN6496—RBN6497	29.42—29.48	4.67	3.92	-1.11
	qCH7	7	RBN7384—RBN7385	24.35—24.37	5.09	7.79	2.37
2020	qCHL1-3	1	RBN0126—RBN0127	3.16—3.22	8.22	10.95	1.42
	qCHL1-4	1	RBN0145—RBN0146	3.93—3.95	9.19	9.64	1.32
	qCHL2-1*	2	RBN1570—RBN1571	7.63—7.71	7.93	7.94	-1.24
	qCHL2-3	2	RBN2330—RBN2331	35.03—35.10	3.27	8.01	-1.28
	qCHL3-2	3	RBN2365—RBN2366	0.33—0.36	4.52	3.54	0.93
	qCHL4	4	RBN4072—RBN4073	23.30—23.38	7.47	7.71	-1.17
	qCHL5-2	5	RBN4489—RBN4490	0.19—0.26	4.72	4.68	1.11
	qCHL5-1*	5	RBN5254—RBN5255	23.44—23.49	3.76	4.82	0.86
	qCH6-2	6	RBN6322—RBN6323	24.40—24.50	5.63	5.68	-1.01
	qCHL11	11	RBN10911—RBN10912	20.63—20.70	4.00	3.89	0.84
	qCHL12-1	12	RBN11570—RBN11571	8.13—8.45	2.84	3.09	1.73
	qCHL12-2	12	RBN12254—RBN12255	25.50—25.54	6.10	4.98	-1.03
	qCHL12-3	12	RBN12262—RBN12263	25.70—25.74	3.84	5.31	-0.98

QTL	染色体 Chr.	物理距离 Interval (Mb)	基因 Gene	基因功能注释 Annotation
qCHL2-1	2	7.63—7.71	Os02g0235000	肌动蛋白相关蛋白2/3复合亚基3类似蛋白 Similar to Actin-related protein 2/3 complex subunit 3
			Os02g0235600	60S核糖体蛋白L11-2 (L16)类似蛋白 Similar to 60S ribosomal protein L11-2 (L16)
			Os02g0235900	包含氧氧化还原酶共价FAD结合位点结构域的蛋白 Oxygen oxidoreductase covalent FAD-binding site domain containing protein
			Os02g0236000	天冬氨酸氨基转移酶 Aspartate aminotransferase (EC 2.6.1.1)
qCHL5-1	5	23.44—23.49	Os05g0476466	CBL互作蛋白激酶28类似蛋白 Similar to CBL-interacting protein kinase 28
			Os05g0476700	U-box E3泛素连接酶,斑点叶 U-box E3 ubiquitin ligase, spotted leaf
			Os05g0477300	含有核糖体蛋白S26e结构域蛋白 Ribosomal protein S26e domain containing protein
			Os05g0477500	含DUF3615结构域未知功能蛋白 Protein of unknown function DUF3615 domain containing protein
			Os05g0477600	α-扩展蛋白OsEXPA4 Alpha-expansion OsEXPA4