QTL-Seq Analysis for Identification of Resistance Locus to Bacterial Leaf Streak in Rice

doi:10.16819/j.1001-7216.2023.220607

Abstract

Abstract:

【Objective】 The aim is to identify new resistance genes to bacterial leaf streak(BLS) and enrich the disease-resistance gene resources in rice, so as to lay a foundation for resistance gene cloning and molecular breeding in rice. 【Method】 A population of 278 recombinant inbred lines (RILs) was developed by crossing WP1 and 9311. WP1 was derived from Guangxi common wild rice(Oryza rufipogon Griff.), which was resistant to BLS, while 9311 was highly susceptible to BLS. QTL-Seq was used to analyze the high resistant and susceptible pools and their parents for quickly locating the genomic regions harboring QTLs resistance to BLS. Subsequently the resistant locus was further verified by QTL mapping using Ici Mapping 4.1 and narrowed down by substitution mapping.【Result】 Three genomic regions harboring resistance QTLs to BLS were identified on chromosomes 4, 8 and 10, respectively. A major resistance locus was verified by ICIM on chromosome 4, designated as qBLS4.1 accounting for 10.65% of the phenotypic variation with a LOD score of 5.03. Finally, the qBLS4.1 locus was delimited in the region of 521 kb from 5.27 to 5.79 Mb by substitution mapping, and there were 41 predicted protein-encoding genes with a total of 252 non-synonymous mutations between the resistant and susceptible parents.【Conclusion】 QTL-Seq combined with ICIM method can locate QTLs in rice more quickly and efficiently. The QTL qBLS4.1, a new locus against BLS will lay a basis and provide a reference for locating and cloning new genes for BLS resistance.

Key words: rice, bacterial leaf streak(BLS), QTL-Seq, mapping

摘要：

【目的】发掘水稻抗细菌性条斑病新基因，丰富抗病基因资源，为水稻抗细菌性条斑病基因克隆及分子育种提供依据。【方法】以抗病材料广西普通野生稻WP1和感病品种9311及其衍生的F_8:9代RIL群体为研究材料，利用QTL-Seq初定位与细菌性条斑病抗性相关的区间，之后利用QTL Ici Mapping 4.1进行复合区间作图以验证结果并精细定位。【结果】分别在第4、8、10染色体上鉴定了一个与细菌性条斑病抗性相关的位点，复合区间作图验证了第4染色体抗细菌性条斑病QTL位点qBLS4.1，表型贡献率和LOD值分别为10.65%和5.03，并进一步将qBLS4.1精细定位在521 kb的范围内。抗感亲本序列比对分析发现，在41个基因的编码区共有252个非同义突变，可能与细菌性条斑病抗性相关。【结论】通过QTL-seq分析结合复合区间作图法可以更快速高效地对水稻QTL进行定位，鉴定了一个抗细菌性条斑病性QTL新位点qBLS4.1，为水稻抗细菌性条斑病新基因鉴定与克隆奠定基础。

关键词: 水稻, 细菌性条斑病, QTL-Seq, 定位

WEI Minyi, MA Zengfeng, HUANG Dahui, QIN Yuanyuan, LIU Chi, LU Yingping, LUO Tongping, LI Zhenjing, ZHANG Yuexiong, QIN Gang. QTL-Seq Analysis for Identification of Resistance Locus to Bacterial Leaf Streak in Rice[J]. Chinese Journal OF Rice Science, 2023, 37(2): 133-141.

韦敏益, 马增凤, 黄大辉, 秦媛媛, 刘驰, 卢颖萍, 罗同平, 李振经, 张月雄, 秦钢. 基于QTL-Seq的水稻抗细菌性条斑病QTL定位[J]. 中国水稻科学, 2023, 37(2): 133-141.

Figures/Tables 8

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标记	物理位置	正向引物	反向引物
Marker	Position / bp	Forward primer(5’-3’)	Reverse primer(5’-3’)
C4M1	557 006	TATACACGCAACTCCCCCTC	TGCCAATACCACAGTCCTGA
C4M2	1 332 530	TTTCTCCAAACCGAACAAGC	CAGTGCAGCAGGGAAGTACA
C4M3	2 519 322	CTCCCACCTGACAAGTTCGT	CAACCAGAGAAAAGCAAGCA
C4M5	4 314 270	CGAAAGGTCAAAACGTTGGT	GTGCATCCGCCTTAATTTGT
C4M6	5 006 279	CGTCATGGTCATCGTACGTC	CACGTAACGCAACGGATATG
C4M8	5 534 085	AATCACCGAGAGCATTTTGG	GTGCAACTTTTACGTGGCCT
C4M9	6 070 782	AGAGACTGTCGCTCCCAAAA	GGGAAAGCTCTGAAATGATCC
C4M10	6 888 374	GAGAGCCCGTAAACATTCCA	ACCATCAACCGGATTATTCA
C4M13	11 782 373	CCCTGGAAAGAACAAACCAA	ATGACCGAGCCTGATATTGC
C4M14	13 911 219	AGCATGTTAAATCATCATCCCA	TGGCTTAACGAAAGAAGAGAGAA
C4MJ1	5 269 417	GGTCTCTCTCTCATGCGTCC	ACCAGACCAGGCAGCTAAGA
C4MJ5	5 364 530	TTCCCCTAAACCATTTTTCTCTC	AGGAGGAAGGGAGGTGCTAC
C4MJ2	5 691 595	CGTCAAGGAACACAGCGATA	TGGGACGGATGGATTAGTTT
C4MJ3	5 790 460	GCCTGAATCACGAAGCTCAT	GAGAAGGTTTGCTCCTGCAC

标记	物理位置	正向引物	反向引物
Marker	Position / bp	Forward primer(5’-3’)	Reverse primer(5’-3’)
C4M1	557 006	TATACACGCAACTCCCCCTC	TGCCAATACCACAGTCCTGA
C4M2	1 332 530	TTTCTCCAAACCGAACAAGC	CAGTGCAGCAGGGAAGTACA
C4M3	2 519 322	CTCCCACCTGACAAGTTCGT	CAACCAGAGAAAAGCAAGCA
C4M5	4 314 270	CGAAAGGTCAAAACGTTGGT	GTGCATCCGCCTTAATTTGT
C4M6	5 006 279	CGTCATGGTCATCGTACGTC	CACGTAACGCAACGGATATG
C4M8	5 534 085	AATCACCGAGAGCATTTTGG	GTGCAACTTTTACGTGGCCT
C4M9	6 070 782	AGAGACTGTCGCTCCCAAAA	GGGAAAGCTCTGAAATGATCC
C4M10	6 888 374	GAGAGCCCGTAAACATTCCA	ACCATCAACCGGATTATTCA
C4M13	11 782 373	CCCTGGAAAGAACAAACCAA	ATGACCGAGCCTGATATTGC
C4M14	13 911 219	AGCATGTTAAATCATCATCCCA	TGGCTTAACGAAAGAAGAGAGAA
C4MJ1	5 269 417	GGTCTCTCTCTCATGCGTCC	ACCAGACCAGGCAGCTAAGA
C4MJ5	5 364 530	TTCCCCTAAACCATTTTTCTCTC	AGGAGGAAGGGAGGTGCTAC
C4MJ2	5 691 595	CGTCAAGGAACACAGCGATA	TGGGACGGATGGATTAGTTT
C4MJ3	5 790 460	GCCTGAATCACGAAGCTCAT	GAGAAGGTTTGCTCCTGCAC

关联分析方法 Association analysis method	染色体 Chromosome	起始Start	终止End	片段大小 Size/Mb	基因数量 Gene number
基于SNP的ED方法关联结果 Results of ED method based on SNP	10	16,070,000	21,090,000	5.02	925
	4	6,170,000	6,840,000	0.67	99
	8	20,840,000	24,160,000	3.32	547
基于SNP的ΔSNP-index方法关联结果 Results of ΔSNP-index method based on SNP	10	17,060,000	20,860,000	3.80	697
基于InDel的ED方法关联结果 Results of ED method based on InDel	10	16,180,000	21,160,000	4.98	919
	4	5,640,000	7,520,000	1.88	244
	8	20,650,000	24,170,000	3.52	575
基于InDel的ΔSNP-index方法关联结果 Results of SNP-index method based on InDel	10	17,280,000	21,000,000	3.72	693

关联分析方法 Association analysis method	染色体 Chromosome	起始Start	终止End	片段大小 Size/Mb	基因数量 Gene number
基于SNP的ED方法关联结果 Results of ED method based on SNP	10	16,070,000	21,090,000	5.02	925
	4	6,170,000	6,840,000	0.67	99
	8	20,840,000	24,160,000	3.32	547
基于SNP的ΔSNP-index方法关联结果 Results of ΔSNP-index method based on SNP	10	17,060,000	20,860,000	3.80	697
基于InDel的ED方法关联结果 Results of ED method based on InDel	10	16,180,000	21,160,000	4.98	919
	4	5,640,000	7,520,000	1.88	244
	8	20,650,000	24,170,000	3.52	575
基于InDel的ΔSNP-index方法关联结果 Results of SNP-index method based on InDel	10	17,280,000	21,000,000	3.72	693

ORF编号	基因	突变类型	基因功能注释
Accession	Gene	Mutation type	Putative function
1	LOC_Os04g09870	SNP	假设蛋白 Hypothetical protein
2	LOC_Os04g09880	SNP	表达蛋白 Expressed protein
3	LOC_Os04g09900	SNP	贝壳杉烯合酶，叶绿体前体 Ent-kaurene synthase, chloroplast precursor
4	LOC_Os04g09910	SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
5	LOC_Os04g09920	InDel/SNP	细胞色素P450 Cytochrome P450
6	LOC_Os04g09950	SNP	假设蛋白 Hypothetical protein
7	LOC_Os04g09960	InDel	表达蛋白 Expressed protein
8	LOC_Os04g09990	InDel	假设蛋白 Hypothetical protein
9	LOC_Os04g10010	InDel/SNP	表达蛋白 Expressed protein
10	LOC_Os04g10070	SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
11	LOC_Os04g10100	SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
12	LOC_Os04g10110	SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
13	LOC_Os04g10120	SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
14	LOC_Os04g10140	SNP	假设蛋白 Hypothetical protein
15	LOC_Os04g10160	SNP	细胞色素P450 Cytochrome P450
16	LOC_Os04g10170	SNP	假设蛋白 Hypothetical protein
17	LOC_Os04g10180	InDel/SNP	表达蛋白 Expressed protein
18	LOC_Os04g10190	InDel/SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
19	LOC_Os04g10200	SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
20	LOC_Os04g10214	SNP	表达蛋白 Expressed protein
21	LOC_Os04g10219	InDel	表达蛋白 Expressed protein
22	LOC_Os04g10230	SNP	表达蛋白 Expressed protein
23	LOC_Os04g10240	SNP	表达蛋白 Expressed protein
24	LOC_Os04g10260	SNP	含碱性亮氨酸拉链结构域的蛋白 Basic region leucine zipper domain containing protein
25	LOC_Os04g10270	InDel/SNP	反转录转座子蛋白 Retrotransposon protein, unclassified
26	LOC_Os04g10290	SNP	表达蛋白 Expressed protein
27	LOC_Os04g10300	SNP	假设蛋白 Hypothetical protein
28	LOC_Os04g10320	SNP	表达蛋白 Expressed protein
29	LOC_Os04g10330	SNP	表达蛋白 Expressed protein
30	LOC_Os04g10390	InDel/SNP	反转录转座子蛋白 Retrotransposon protein, unclassified
31	LOC_Os04g10400	SNP	电子转运黄素蛋白亚单位β Electron transfer flavoprotein subunit beta
32	LOC_Os04g10410	SNP	酰胺酶 Amidase
33	LOC_Os04g10420	InDel/SNP	CW7
34	LOC_Os04g10450	SNP	假设蛋白 Hypothetical protein
35	LOC_Os04g10500	SNP	表达蛋白 Expressed protein
36	LOC_Os04g10550	InDel/SNP	表达蛋白 Expressed protein
37	LOC_Os04g10569	SNP	谷氨酰-tRNA酰胺转移酶 Glutamyl-tRNA amidotransferase
38	LOC_Os04g10590	SNP	反转录转座子蛋白 Retrotransposon protein, unclassified
39	LOC_Os04g10620	SNP	反转录转座子蛋白，Ty3-gypsy亚类 Retrotransposon protein, Ty3-gypsy subclass
40	LOC_Os04g10630	InDel/SNP	表达蛋白 Expressed protein
41	LOC_Os04g10650	SNP	CDT1A-DNA复制起始蛋白 CDT1A-DNA replication initiation protein