大白菜抗根肿病基因BraA.Pb.8.4的定位及KASP标记开发

doi:10.16420/j.issn.0513-353x.2020-0656

摘要/Abstract

摘要：

以大白菜1E519BC₁F₂和4E511BC₁F₂两个群体为试材,分别构建根肿病抗病和感病池,进行集团分离群体测序（Bulked Segregant Analysis Sequencing,BSA-seq）和分析。结果发现,两个群体中检测到1个共同的根肿病抗性基因候选区间,位于A08染色体7.40 ~ 8.85 Mb,命名为BraA.Pb.8.4。该基因在物理位置上和已经报道的根肿病抗性基因Crr1、Rcr9和CRs均不同,并且Crr1和Rcr9连锁的标记在抗病和感病材料中不具有多态性,CRs连锁的4个标记中只有1个标记（Probe60）在抗病和感病材料中存在多态性,表明BraA.Pb.8.4不同于已经报道的基因,可能为1个新的根肿病抗性基因。在BraA.Pb.8.4候选区间开发了3个KASP标记（BraA.Pb.8.4-K1、BraA.Pb.8.4-K2和BraA.Pb.8.4-K3）,均可以有效将纯合抗病、纯合感病和杂合抗病材料区分开,为共显性标记。利用标记BraA.Pb.8.4-K1在5个群体共257个单株中进行验证,发现各单株的基因型和抗病性表型平均一致率高达96.13%。因此,本研究鉴定的BraA.Pb.8.4基因及开发的KASP标记可以高效用于根肿病的分子标记辅助育种。

关键词: 大白菜, 根肿病, BraA.Pb.8.4基因, 基因定位, KASP标记

Abstract:

In this study,Bulked Segregant Analysis Sequencing（BSA-seq）was conducted on two Chinese cabbage populations,named as 1E519BC₁F₂ and 4E511BC₁F₂. We identified a common candidate region from the above two populations,which located on 7.40-8.85 Mb of chromosome A08. Here,we named this region as BraA.Pb.8.4. BraA.Pb.8.4 might be a novel clubroot resistance gene for two reasons. Firstly,in terms of physical position,BraA.Pb.8.4 is distinct from previous reported genes,Crr1,Rcr9 and CRs. Secondly,markers linked to Crr1 and Rcr9 amplified the same product without length polymorphysims between resistant and susceptible materials,and only one of the four CRs linked markers （Probe60）produced polymorphysims. Among the candidate region of BraA.Pb.8.4,three KASP markers,BraA.Pb.8.4-K1,BraA.Pb.8.4-K2 and BraA.Pb.8.4-K3,were developed. These three KASP markers are co-dominant markers which can not only effectively separate homozygous resistant genotypes from the homozygous susceptible genotypes,but also distinguish the heterozygous genotypes. Marker BraA.Pb.8.4-K1 were further genotyped in a total of 257 individuals,which belongs to five populations. The results showed that the average consistency rate between genotypes and phynotypes of the above 257 individuals was high to 96.13%. Thus,the BraA.Pb.8.4 gene and the KASP markers developed here can be efficently used for marker-assisted selection（MAS）of clubroot resistant gene in breeding.

Key words: Chinese cabbage, clubroot, BraA.Pb.8.4 gene, gene mapping, KASP marker

中图分类号:

S634.1

杨双娟, 张晓伟, 魏小春, 赵艳艳, 王志勇, 赵肖斌, 李林, 原玉香. 大白菜抗根肿病基因BraA.Pb.8.4的定位及KASP标记开发[J]. 园艺学报, 2021, 48(7): 1317-1328.

YANG Shuangjuan, Zhang Xiaowei, WEI Xiaochun, ZHAO Yanyan, WANG Zhiyong, ZHAO Xiaobin, LI Lin, YUAN Yuxiang. Mapping and KASP Markers Development for Clubroot Resistance Gene BraA.Pb.8.4 in Chinese Cabbage[J]. Acta Horticulturae Sinica, 2021, 48(7): 1317-1328.

图/表 14

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样品名称 Sample	高质量读长数 Clean reads	高质量碱基数 Clean bases	比对的读长数 Mapped reads	比对率/% Mapping rate	基因组覆盖倍数 Coverage of B. rapa genome
1E519BC₁F₂-Rp	146 359 698	21 953 954 700	139 582 489	95.37	45.27
1E519BC₁F₂-Sp	130 400 096	19 560 014 400	124 262 099	95.29	40.33
4E511BC₁F₂-Rp	171 165 594	25 674 839 100	163 000 999	95.23	52.94
4E511BC₁F₂-Sp	125 843 472	18 876 520 800	119 448 979	94.92	38.92

样品名称 Sample	高质量读长数 Clean reads	高质量碱基数 Clean bases	比对的读长数 Mapped reads	比对率/% Mapping rate	基因组覆盖倍数 Coverage of B. rapa genome
1E519BC₁F₂-Rp	146 359 698	21 953 954 700	139 582 489	95.37	45.27
1E519BC₁F₂-Sp	130 400 096	19 560 014 400	124 262 099	95.29	40.33
4E511BC₁F₂-Rp	171 165 594	25 674 839 100	163 000 999	95.23	52.94
4E511BC₁F₂-Sp	125 843 472	18 876 520 800	119 448 979	94.92	38.92

群体 Population	位置/Mb Location	抗病混池SNP index Rp_SNP_index	感病混池SNP index Sp_SNP_index	∆SNP-index	P值 P-value
1E519BC₁F₂	A08：7.55 ~ 8.75	0.48	0.14	0.34	< 0.05
4E511BC₁F₂	A08：7.40 ~ 8.85	0.45	0.10	0.35	< 0.05

群体 Population	位置/Mb Location	抗病混池SNP index Rp_SNP_index	感病混池SNP index Sp_SNP_index	∆SNP-index	P值 P-value
1E519BC₁F₂	A08：7.55 ~ 8.75	0.48	0.14	0.34	< 0.05
4E511BC₁F₂	A08：7.40 ~ 8.85	0.45	0.10	0.35	< 0.05

KASP标记 KASP marker	物理位置 Physical position	变异位点 Variation locus	引物序列（5′-3′） Primer sequence
BraA.Pb.8.4-K1	A08：7 953 439	T/C	Fa：GAAGGTGACCAAGTTCATGCTCGTGGTGGAAAGAAATGGTT
			Fb：GAAGGTCGGAGTCAACGGATTCGTGGTGGAAAGAAATGGTC
			R：CGTCCTCCGAGAAGCACACACC
BraA.Pb.8.4-K2	A08：7 961 681	T/G	Fa：GAAGGTCGGAGTCAACGGATTCGTCCTGCATTTCTTAGATGGT
			Fb：GAAGGTGACCAAGTTCATGCTCGTCCTGCATTTCTTAGATGGG
			R：CGAGCACTGATCTGCAAACACA
BraA.Pb.8.4-K3	A08：7 970 088	C/T	Fa：GAAGGTGACCAAGTTCATGCTGTTTAAGTAGAGACTGTGGGAC
			Fb：GAAGGTCGGAGTCAACGGATTGTTTAAGTAGAGACTGTGGGAT
			R：CTTCAAACGCAGCTTTCTTTGACTT