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作物杂志,2023, 第6期: 69–78 doi: 10.16035/j.issn.1001-7283.2023.06.010

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

基于BSA-Seq技术初步鉴定油用亚麻抗白粉病候选基因

叶春雷1(), 王炜1(), 陈军1, 陈琛1, 罗俊杰1, 王一2, 张建平3()   

  1. 1甘肃省农业科学院生物技术研究所，730070，甘肃兰州
    2福建农林大学植物保护学院，350002，福建福州
    3甘肃省农业科学院作物研究所，730070，甘肃兰州
  • 收稿日期:2022-05-20 修回日期:2022-09-08 出版日期:2023-12-15 发布日期:2023-12-15
  • 通讯作者: 王炜，主要从事作物细胞工程及诱变育种工作，E-mail：sjswangwei@gsagr.ac.cn；张建平为共同通信作者，研究方向为胡麻育种及推广，E-mail：401101917@qq.com
  • 作者简介:叶春雷，主要从事胡麻育种、栽培及病虫害防治工作，E-mail：ycl-80@163.com
  • 基金资助:
    甘肃省农业科学院农业科技创新专项(2020GAAS08);甘肃省农业科学院农业科技创新专项(2020GAAS43);甘肃省农业科学院农业科技创新专项(2021GAAS25);甘肃省自然科学基金项目(21JR7RA726);国家特色油料产业技术体系胡麻兰州综合试验站(GARS-17-SYZ-6)

Preliminary Identification of Candidate Genes with Resistance to Powdery Mildew in Oil Flax Based on BSA-Seq

Ye Chunlei1(), Wang Wei1(), Chen Jun1, Chen Chen1, Luo Junjie1, Wang Yi2, Zhang Jianping3()   

  1. 1Bio-Technology Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
    2College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3Institute of Crops, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
  • Received:2022-05-20 Revised:2022-09-08 Online:2023-12-15 Published:2023-12-15

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摘要：

白粉病是油用亚麻的主要病害之一，但由于抗病资源缺乏及相关基因挖掘与利用目前还较为欠缺，严重制约了油用亚麻抗白粉病育种。鉴于此，以前期EMS诱变所获得高抗白粉病材料H98-1与极感白粉病材料D367为亲本，采用混池分组分析法（bulked segreant analysis，BSA）对亲本和抗感F2混池开展全基因组重测序，通过关联分析方法确定了抗病基因的候选区域；进一步采用BLAST软件对候选区间内的编码基因进行深度注释，鉴定出抗白粉病候选基因。结果表明，H98-1抗白粉病性状相关的候选区域主要位于油用亚麻2号染色体12 436 438~ 12 438 246、12 475 667~12 476 462、12 478 211~12 479 109区间及4号染色体上14 120 916~18 541 859区间内，总长度4.43Mb，包含844个基因，这些基因主要参与次生代谢物生物合成、运输和分解代谢、蛋白质转运、信号转导等过程。经基因注释、代谢通路分析及文献分析，筛选出11个可能与油用亚麻抗白粉病相关基因。

关键词: 油用亚麻, 白粉病, BSA, 候选基因

Abstract:

Powdery mildew is one of the main diseases of oil flax. However, due to the lack of disease resistant germplasm resources and the lack of relevant gene mining and utilization, breeding of oil flax with powdery mildew resistance has been seriously restricted. In view of this, oil flax germplasm H98-1 with high resistance to powdery mildew and another oil flax germplasm D367 with high susceptibility to powdery mildew obtained by EMS mutation were used as parents, and used mixed pool grouping analysis (BSA) to perform whole genome resequencing on the parents and the resistant and susceptible F2 mixed pool. Candidate regions for disease resistance genes were identified through association analysis. BLAST software was applied to deeply annotate the coding genes within the candidate interval and identify candidate genes for resistance to powdery mildew. The results showed that the candidate regions related to the resistance to powdery mildew trait of H98-1 were mainly located in the intervals of 12 436 438-12 438 246, 12 475 667-12 476 462, 12 478 211-12 479 109 on chromosome 2, and 14 120 916-18 541 859 on chromosome 4 of oil flax, with a total length of 4.43Mb and containing 844 genes which are mainly involved in secondary metabolite biosynthesis, transport and catabolism, protein transport, signal transduction and other biological processes. The 11 genes may be related to the resistance of oil flax to powdery mildew which screened out by gene annotation, metabolic pathway analysis and literature analysis.

Key words: Oil flax, Powdery mildew, BSA, Candidate genes

图1

参试样品SNP及InDel数量韦恩图 (a) SNP数量；(b) InDel数量

表1

参试样品变异位点注释

变异类型
Variation type		 变异位点信息
Information of variation site		 H98-1 D367 抗病池
Disease-resistance pool		 感病池
Disease-susceptible pool
SNP 编码序列 63 580 51 467 65 069 61 267
基因上游 213 290 165 255 218 843 207 330
基因下游 165 650 130 082 168 902 159 594
基因区间 160 364 129 773 163 657 165 461
内含子 87 418 70 699 90 075 85 010
5'非翻译区 1528 1123 1469 1387
5'非翻译区 3715 2692 3616 3388
剪切供体突变 108 91 110 104
剪切受体突变 111 101 130 125
剪切位点区域突变 2932 2396 2959 2819
同义编码突变 31 290 25 525 31 942 30 032
非同义编码突变 31 283 25 102 32 085 30 258
起始密码子获得 50 32 43 43
起始密码子丢失 50 40 53 50
终止密码子获得 543 436 562 526
终止密码子丢失 334 295 344 325
InDel 编码序列 3479 2961 3675 3520
基因上游 41 329 34 072 44 359 42 494
基因下游 32 005 26 545 34 201 32 639
基因区间 21 841 18 153 23 327 23 461
内含子 20 219 17 296 21 277 20 449
5'非翻译区 521 404 545 513
5'非翻译区 1085 842 1082 1054
剪切供体突变 78 74 89 86
剪切受体突变 74 50 69 66
剪切位点区域突变 691 624 733 713
起始密码子丢失 23 17 21 20
终止密码子获得 42 40 41 40
终止密码子丢失 39 34 38 41
密码子插入 176 154 181 187
密码子删除 356 317 391 367

表2

油用亚麻种质H98-1抗白粉病基因的候选区域

染色体
Chromosome		 关联区域起始位置
Starting position of associated region		 关联区域终止位置
Ending position of associated region		 大小
Size (Mb)		 基因数量
Gene number
chr2 12433995 12433995 0.00 0
chr2 12436438 12438246 0.00 1
chr2 12439649 12440010 0.00 0
chr2 12442385 12442531 0.00 0
chr2 12475667 12476462 0.00 1
chr2 12478211 12479109 0.00 1
chr2 13270712 13279515 0.01 0
chr2 13388502 13388502 0.00 0
chr4 14120916 18541859 4.42 841
合计Total － － 4.43 844

表3

候选区域内SNP及InDel中基因功能注释

功能注释数据库
Annotated-databases		 候选区域基因数
Number of candidate region genes		 非同义突变基因数
Number of nonsynonymous mutant genes		 移码突变基因数
Number of frameshift mutation genes
COG 292 116 36
GO 673 261 67
KEGG 586 221 58
NR 804 303 76
NT 815 306 77
SwissProt 627 239 63
trEMBL 805 303 76
合计Total 815 306 77

图2

候选区域内基因COG注释分类图

表4

候选区域内基因GO注释

基因功能分类
Gene function classfication		 基因功能
Gene function		 基因数
Number of genes
细胞成分
Cellular component		 细胞外区域 12
细胞 86
细胞膜 75
细胞连接 3
膜封闭腔 6
大分子复合体 15
细胞器 64
胞外区部分 4
细胞器部分 20
细胞膜部分 61
细胞部分 86
突触 1
共质体 1
分子功能
Molecular function		 核酸结合转
录因子活性		 7
催化活性 127
受体活性 2
结构分子活性 3
转运子活性 8
结合 142
电子载体活性 2
抗氧化物活性 2
酶调节子活性 3
分子转导活性 2
生物过程
Biological process		 复制 8
免疫系统过程 1
代谢过程 141
细胞过程 120
复制过程 10
信号 7
多细胞生物过程 13
发育过程 13
生长 2
单组织过程 83
应激反应 44
定位 19
多组织过程 9
生物调控 52
细胞成分组织
或生物发生		 19

图3

候选区域内基因通路分布

表5

候选区域内候选基因信息

序号
Number		 候选基因ID
Candidate gene ID		 染色体位置
Chromosome
position		 起始位点
Starting
position		 终止位点
Ending
position		 基因长度
Gene
length (bp)		 基因功能
Gene function
1 L.us.o.g.scaffold187.45 Chr4 16526088 16541454 15366 编码抗病蛋白L6
2 L.us.o.g.scaffold187.47 Chr4 16552817 16557562 4745 为拟南芥HOS1的同源基因，编码E3泛素蛋白连接酶
3 L.us.o.g.scaffold187.55 Chr4 16589848 16591005 1157 为拟南芥At5g62660的同源基因，推测编码F-box蛋白
4 L.us.o.g.scaffold187.89 Chr4 16705005 16708737 3732 编码抗病蛋白L6
5 L.us.o.g.scaffold187.98 Chr4 16754356 16759010 4654 编码抗病蛋白L6
6 L.us.o.g.scaffold187.99 Chr4 16762357 16763315 958 编码抗病蛋白L6
7 L.us.o.g.scaffold187.100 Chr4 16763361 16766387 3026 编码抗病蛋白L6
8 L.us.o.g.scaffold187.121 Chr4 16871587 16875166 3579 编码抗病蛋白L6
9 L.us.o.g.scaffold214.52 Chr4 17370672 17372100 1428 为拟南芥At3g58860的同源基因，推测编码F-box蛋白
10 L.us.o.g.scaffold318.36 Chr4 17211936 17222836 10900 编码抗病蛋白L6
11 L.us.o.g.scaffold322.22 Chr4 16970278 16972939 2661 编码抗病蛋白L6
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