Mapping and KASP Markers Development for Clubroot Resistance Gene BraA.Pb.8.4 in Chinese Cabbage

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (7): 1317-1328.doi: 10.16420/j.issn.0513-353x.2020-0656

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Mapping and KASP Markers Development for Clubroot Resistance Gene BraA.Pb.8.4 in Chinese Cabbage

YANG Shuangjuan, Zhang Xiaowei, WEI Xiaochun, ZHAO Yanyan, WANG Zhiyong, ZHAO Xiaobin, LI Lin, YUAN Yuxiang^**()

Institute of Horticulture,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China

Received:2020-12-24 Revised:2021-02-19 Online:2021-07-25 Published:2021-08-10
Contact: YUAN Yuxiang E-mail:yuanyuxiang@126.com

Abstract

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

CLC Number:

S634.1

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.

Figures/Tables 14

References 26

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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

Mapping and KASP Markers Development for Clubroot Resistance Gene BraA.Pb.8.4 in Chinese Cabbage

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群体 Population	根肿病抗性 Clubroot resistance	单株数 Number of individual	基因型 Genotype			一致率/% Concordance rate
群体 Population	根肿病抗性 Clubroot resistance	单株数 Number of individual	a	b	h	一致率/% Concordance rate
1E177BC₂F₂-5	抗病Resistant	19	0	1	18	94.74
	感病Susceptible	19	0	18	1
1E177BC₂F₂-24	抗病Resistant	28	9	0	19	100.00
	感病Susceptible	11	0	11	0
1E177BC₂F₂-25	抗病Resistant	34	9	2	23	91.30
	感病Susceptible	12	0	10	2
1E177BC₂F₂-26	抗病Resistant	29	6	0	23	100.00
	感病Susceptible	12	0	12	0
CR1E-10	抗病Resistant	45	0	5	40	94.62
	感病Susceptible	48	0	48	0

基因 Gene	标记或候选基因 Marker or candidate gene	A08物理位置/bp Physical position on A08
BraA.Pb.8.4	BraA.Pb.8.4-K1	7 953 439
	BraA.Pb.8.4-K2	7 961 681
	BraA.Pb.8.4-K3	7 970 088
CRs	Probe55	9 956 858
	Probe59	10 497 705
	Probe60	10 539 495
	Probe64	10 707 395
Rcr9	Bra020936	10 295 068 ~ 10 295 637
Crr1	Bra020861	10 809 433 ~ 10 825 238

标记 Marker	正向引物序列（5′-3′） Forward primer	反向引物序列（5′-3′） Reverse primer	文献来源 Reference
Probe55	ATTCAGATGACCTTCTTCCGAC	GATATGTTACCACGGCGAAAAC	Laila et al.,2019
Probe59	ACAGCAAAACCTCTCTTCTCTC	ATGTCAAACAAGCGTCTAAAGC	Laila et al.,2019
Probe60	ACACTTAGGTGAAGAAGCAACA	GCATGCATCAAGTAACTTGCAA	Laila et al.,2019
Probe 64	GTTGGAGAACACACTGAGATCA	TCTCTCGAGGCTTCTGTTACTA	Laila et al.,2019
Rcr9_specific	AGTGGAGAACCAAAGCCAAA	CAATTGTGGCCACCTTCTTC	Yu et al.,2017
BAS2	TGTTAGACATCAAAGAGGGCTTGAG	TCTCAAAGACGATAATGAACCCAAA	Suwabe et al.,2012
BAS7	AAGCAGAAGGGTTCTTGTCTGAACT	TCACTCTGAGACATGAGGACACTTG	Suwabe et al.,2012

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