大白菜抗根肿病基因位点CRs的KASP标记开发及应用

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

摘要/Abstract

摘要：

CRs为最近发现的1个抗根肿病基因。以连锁的4个标记作为引物,以8个抗病和8个感病大白菜材料为模板,进行PCR扩增和测序。结果发现,抗病和感病材料中存在6个序列变异位点,其中5个（A08染色体9 956 808、9 956 888、9 956 904、10 707 299和10 707 385 bp）为新的变异位点,1个（10 539 495 bp处的G/A SNP变异）与前人研究结果一致。针对4个新的变异位点（因9 956 888 bp与9 956 904 bp处变异距离很近,故后者未分析）和10 539 495 bp的变异位点设计开发出5个竞争性等位基因特异性PCR（Kompetitive allele specific PCR,KASP）标记Probe55K1、Probe55K2、Probe64K1、Probe64K2和Probe60K1。结果表明,设计的5个KASP标记均可有效将抗病材料和感病材料分为2组。利用标记Probe55K1和Probe64K1在包含45个抗病单株和48个感病单株的BC₁P₁群体中进行验证,结果发现,这2个标记在各个单株的基因型和抗病性表型一致率高达94.62%。因此,本研究中开发的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, CRs基因, KASP标记

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

中图分类号:

S634.1

杨双娟, 原玉香, 赵艳艳, 魏小春, 王志勇, 赵肖斌, 张晓伟. 大白菜抗根肿病基因位点CRs的KASP标记开发及应用[J]. 园艺学报, 2021, 48(6): 1094-1106.

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.

图/表 10

图1 大白菜8个抗病DH系（R1 ~ R8）和8个感病DH系（S1 ~ S8）

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

表1 CRs的4个HRM标记引物信息

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

图2 Probe55在大白菜抗病DH系（R1 ~ R8）和感病DH系（S1 ~ S8）中的序列比对结果

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

图3 Probe64在大白菜抗病DH系（R1 ~ R8）和感病DH系（S1 ~ S8）中的序列比对结果

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

表2 本研究中发现的变异位点的物理位置信息

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

表3 KASP标记信息

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

图4 利用引物Probe55K1、Probe60K1、Probe64K1、Probe64K2对8个抗病（R1 ~ R8）和8个感病DH系（S1 ~ S8）进行KASP分型坐标轴内的每个点代表1个单株,数据点可能存在重叠。

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.

图5 利用标记Probe55K1对BC1P1群体进行KASP基因分型 A. 96孔板下KASP基因分型结果;B. X-Y坐标轴下KASP基因分型结果。

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.

图6 利用标记Probe60K1（A）和Probe64K2（B）对BC1P1群体进行KASP基因分型 A. Probe60K1在BC1P1群体各单株的基因型均和感病DH系S5一致;B. Probe64K2在BC1P1群体各单株的基因型均和抗病DH系R4一致。

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.

图7 标记Probe60K1和Probe64K2在大白菜材料R4、S5、1e177-25-140和YR16-11中的验证 A. 引物Probe60F和Probe60R的PCR扩增结果;B. 标记Probe60K1的KASP分型结果;C. 标记Probe64K2的KASP分型结果。R4和1e177-25-140为根肿病抗性材料,S5和YR16-11为感病材料。

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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