中国水稻科学 ›› 2023, Vol. 37 ›› Issue (2): 133-141.DOI: 10.16819/j.1001-7216.2023.220607
韦敏益1, 马增凤1, 黄大辉1, 秦媛媛2, 刘驰1, 卢颖萍1,3, 罗同平1, 李振经1, 张月雄1,*(), 秦钢1,*()
收稿日期:
2022-06-14
修回日期:
2022-08-05
出版日期:
2023-03-10
发布日期:
2023-03-10
通讯作者:
张月雄,秦钢
基金资助:
WEI Minyi1, MA Zengfeng1, HUANG Dahui1, QIN Yuanyuan2, LIU Chi1, LU Yingping1,3, LUO Tongping1, LI Zhenjing1, ZHANG Yuexiong1,*(), QIN Gang1,*()
Received:
2022-06-14
Revised:
2022-08-05
Online:
2023-03-10
Published:
2023-03-10
Contact:
ZHANG Yuexiong, QIN Gang
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F8:9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。
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.
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.
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 |
表1 用于本研究基因定位的InDel标记
Table 1. InDel markers for gene mapping in this study.
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 |
图2 亲本对细菌性条斑病的抗性鉴定结果(A)和F8:9群体(B)病斑长度分布频率 **亲本间的差异达0.01显著水平。
Fig. 2. Resistance of parents to bacterial leaf streak(A) and distribution frequency of lesion length in F8:9 population(B). ** Difference between the parents was significant at 0.01 level.
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 |
表2 QTL-seq结果统计
Table 2. Results of QTL-seq.
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 |
图3 BSA-seq 基于SNP和InDel的ED和△(SNP-index)关联分析 A图和B图分别是基于SNP和InDel的ED关联分析;C图和D图分别是基于SNP和InDel的△(SNP-index)关联分析,红色的虚线为99%的阈值线。
Fig. 3. ED and △(SNP-index) correlation analysis based on SNP and InDel of BSA-seq. A and B, ED association analysis figures based on SNP and InDel, respectively. C and D, △ (SNP-index) correlation analysis diagram based on SNP and InDel, respectively. The red dotted line is the 99% threshold line.
图4 第4染色体上抗病池、感病池的SNP和InDel指数分析 A图和B图分别为抗、感病混池的SNP指数的分布图;C图为Δ(SNP-index)值的分布图,红色的虚线为99%的阈值线。R03为抗病池,R04为感病池。
Fig. 4. SNP-index and InDel -index of the resistant pool and the susceptible pool on chromosome 4. A, B are the distribution of SNP-index values in resistant and susceptible mixed pools, respectively; C shows the distribution of Δ(SNP-index) value. The red dotted line is the 99% threshold line. R03, Resistant pool; R04, Susceptible pool.
图5 抗细条病基因qBLS4.1验证和精细定位 A图为qBLS4.1在染色体上的位置与遗传效应;B图为重组单株的基因型和表型。
Fig. 5. Verification and fine mapping of the resistance locus qBLS4.1. A, Location and genetic effect of qBLS4.1; B, Graphical genotypes and resistance phenotypes of the recombinants.
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 |
表3 定位区间内的候选基因注释
Table 3. Candidate gene annotation in the location interval.
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 |
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