Development and Application of KASP Markers for Clubroot Resistance Gene CRs in Chinese Cabbage

doi:10.16420/j.issn.0513-353x.2020-0542

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (6): 1094-1106.doi: 10.16420/j.issn.0513-353x.2020-0542

• Research Papers • Previous Articles Next Articles

Development and Application of KASP Markers for Clubroot Resistance Gene CRs in Chinese Cabbage

YANG Shuangjuan, YUAN Yuxiang, ZHAO Yanyan, WEI Xiaochun, WANG Zhiyong, ZHAO Xiaobin, ZHANG Xiaowei()

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

Received:2020-12-18 Revised:2021-02-17 Online:2021-06-25 Published:2021-07-07
Contact: ZHANG Xiaowei E-mail:xiaowei5737@163.com

Abstract

Abstract:

CRs is an recently discovered clubroot resistance gene in Chinese cabbage. In this study,through PCR amplication and sequencing of four CRs linked markers in eight resistant materials and eight susceptible materials,we found six sequence variations between resistant and susceptible materials. Among which,five variations（9 956 808,9 956 888,9 956 904,10 707 299 and 10 707 385 bp in chromosome A08）were novel,G/A SNP（Single Nucleotide Polymorphisms）variation in 10 539 495 bp of A08 is consistent with previous study. Five KASP（Kompetitive allele specific PCR）markers, Probe55K1,Probe55K2,Probe64K1,Probe64K2 and Probe60K1,were developed in terms of variations in 9 956 808,9 956 888,10 707 299,10 707 385 and 10 539 495 bp. It turned out that all the five KASP markers can successfully differentiate the resistance from the susceptible materials. Probe55K1 and Probe64K1 were further genotyped in 45 resistant and 48 susceptible individuals, which were part of a BC₁P₁ population. The results showed that the consistency rate between genotypes and phenotypes of the above 93 individuals was high to 94.62%. Thus,the KASP markers developed in this study can be efficently used for marker-assisted selection（MAS）of clubroot resistant gene in breeding.

Key words: Chinese cabbage, clubroot, CRs gene, KASP marker

CLC Number:

S634.1

YANG Shuangjuan, YUAN Yuxiang, ZHAO Yanyan, WEI Xiaochun, WANG Zhiyong, ZHAO Xiaobin, ZHANG Xiaowei. Development and Application of KASP Markers for Clubroot Resistance Gene CRs in Chinese Cabbage[J]. Acta Horticulturae Sinica, 2021, 48(6): 1094-1106.

Figures/Tables 10

Fig. 1 Eight resistant（R1-R8）and susceptible（S1-S8）Chinese cabbage DH lines

Table 1 Primer information of four HRM markers linked to CRs gene

标记 Marker	SNP位置 SNP position	正向引物序列 Forward primer	探针序列 Probe	反向引物序列 Reverse primer
Probe55	9 956 858	ATTCAGATGACCTTCTTCCGAC	ACTTTAGTGCGTAAGTTCTCT	GATATGTTACCACGGCGAAAAC
Probe59	10 497 705	ACAGCAAAACCTCTCTTCTCTC	TTTGTCGTCGTAGCTGGTGGC	ATGTCAAACAAGCGTCTAAAGC
Probe60	10 539 495	ACACTTAGGTGAAGAAGCAACA	TTCGATGTTAGACTGCAGATA	GCATGCATCAAGTAACTTGCAA
Probe 64	10 707 385	GTTGGAGAACACACTGAGATCA	CGTTCCAGGAGTTGAGGACAA	TCTCTCGAGGCTTCTGTTACTA

Fig. 2 Comparison of nucleotide sequences of Probe55 in resistant（R1~R8）and susceptible（S1~S8）DH lines

Fig. 3 Comparison of nucleotide sequences of Probe64 in resistant（R1-R8）and susceptible（S1-S8）DH lines

Table 2 Physical position of variation locus in this study

标记 Marker	变异类型 Variation type	变异位点 Variation locus	物理位置/bp Physical position
Probe55	SNP	T/A	9 956 808
Probe55	InDel	AAACTAGTTCA	9 956 888
	InDel	TATA	9 956 904
Probe60	SNP	G/A	10 539 495
Probe64	SNP	G/A	10 707 299
	SNP	T/C	10 707 385

Table 3 Information of KASP markers in this study

KASP标记 KASP marker	变异位点 Variation locus	引物序列（5′-3′） Primer sequence
Probe55K1	T/A	Fa：GAAGGTGACCAAGTTCATGCTATTCTCCACCGCTCTTTTCT
		Fb：GAAGGTCGGAGTCAACGGATTATTCTCCACCGCTCTTTTCA
		R：ATATCTGCCGAGAGGGCTGACC
Probe55K2	AAACTAGTTCA	Fa：GAAGGTGACCAAGTTCATGCTTTGGTTCCTTTTCAATCTAAAC
		Fb：GAAGGTCGGAGTCAACGGATTTTGGTTCCTTTTCAATCTAATAAAG
		R：CAGGCTACAAAGAAGATGATGACG
Probe60K1	G/A	Fa: GAAGGTGACCAAGTTCATGCTGTTTCTTCATCTTTTCGATGTTAG
		Fb: GAAGGTCGGAGTCAACGGATTTCTTCATCTTTTCGATGTTAA
		R: GGTATATAGATATAGATAGGTATCTGC
Probe64K1	G/A	Ra: GAAGGTGACCAAGTTCATGCTCGTGGCTGCTGTTGTTTTGGAC
		Rb: GAAGGTCGGAGTCAACGGATTTGGCTGCTGTTGTTTTGGAT
		F: GAGATCAGCATCTGCAGACAAGCTC
Probe64K2	T/C	Ra: GAAGGTGACCAAGTTCATGCTTCCTCAACTCCTGGAACGA
		Rb: GAAGGTCGGAGTCAACGGATTGTCCTCAACTCCTGGAACGG
		F: GGACAGCCGTTGAAGTCGATGGAC

Fig. 4 KASP genotyping of 8 resistant（R1-R8）and 8 susceptible（S1-S8）DH lines by primers Probe55K1,Probe60K1,Probe64K1 and Probe64K2 These spots within the axis represent different single plant,and data spots might be overlapped.

Fig. 5 KASP genotyping of BC1P1 population by primer Probe55K1 A. The result of KASP genotyping in 96-well plate format;B. The result of KASP genotyping in X-Y coordinates.

Fig. 6 KASP genotyping of BC1P1 population by primers Probe60K1（A）和Probe64K2（B） A. Genotypes of BC1P1by marker Probe60K1 were all same with susceptible DH line S5;B. Genotypes of BC1P1by marker Probe64K2 were all same with resistant DH line R4.

Fig. 7 Variation of Probe60K1 and Probe64K2 in Chinese cabbage materials R4,S5,1e177-25-140 and YR16-11 A. PCR amplification results with primers Probe60F and Probe60R;B. The KASP genotyping results of marker Probe60K1;C. The KASP genotyping results of marker Probe64K2. R4 and 1e177-25-140 are resistant materials,S5 and YR16-11 are susceptible materials.

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Development and Application of KASP Markers for Clubroot Resistance Gene CRs in Chinese Cabbage

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