洋葱雄性不育恢复基因Ms位点KASP标记的开发及应用

doi:10.16420/j.issn.0513-353x.2025-0172

摘要/Abstract

摘要：

为了提高洋葱雄性不育恢复基因Ms位点的选择效率，加快育种进程，利用AcSKP1等位基因序列上的SNP差异位点，开发了适用于高通量检测的竞争性等位基因特异性PCR（kompetitive allele specific PCR，KASP）标记。结果表明，KASP标记KMs4可以有效地将F₂分离群体Ms位点的基因型分为MsMs、Msms、msms三类，并将BC₁分离群体Ms位点的基因型分为Msms和msms两类，其分型结果与AcSKP1标记鉴定及表型观察结果完全一致。此外，该标记在不同遗传背景已知基因型的材料中展现出良好的通用性。利用该标记对洋葱开放授粉群体进行育性基因型筛选系统评价，筛选出具有不同育性特征的育种系，为后续杂交选育提供了优质亲本材料。研究表明，KMs4标记是与洋葱雄性不育恢复基因Ms位点紧密连锁的分子标记，能够实现大规模育种材料的高效基因分型。

关键词: 洋葱, 雄性不育, 恢复基因, KASP标记

Abstract:

To improve the selection efficiency of the onion male sterility restorer gene Ms locus and accelerate the breeding process，this study developed a kompetitive allele-specific PCR（KASP）marker suitable for high-throughput detection based on SNP variation sites in the allelic sequence of the AcSKP1 gene. The results showed that the KASP marker KMs4 effectively classified the genotypes（MsMs，Msms，msms）of the Ms locus in an F₂ segregating population into three distinct categories，and the genotypes at the Ms locus in the BC₁ segregating population into two categories（Msms，msms）. Moreover，the genotyping results fully consistent with those obtained using the AcSKP1 marker. The versatility of the KMs4 marker was further confirmed across materials with known genotypes from diverse genetic backgrounds. Fertility trait evaluation was conducted on collected onion open-pollinated populations，from which different breeding lines were screened and selected for hybrid breeding. In summary，the KMs4 marker is a molecular marker tightly linked to the onion male sterility restorer gene Ms locus，enabling high-throughput genotyping of large-scale breeding materials.

Key words: Allium cepa, male sterility, restoration gene, KASP

杨妍妍, 李艳伟, 王振宝, 曹德高, 徐鑫, 刘冰江. 洋葱雄性不育恢复基因Ms位点KASP标记的开发及应用[J]. 园艺学报, 2025, 52(10): 2642-2654.

YANG Yanyan, LI Yanwei, WANG Zhenbao, CAO Degao, XU Xin, LIU Bingjiang. Development and Application of KASP Marker for the Ms Locus of Male Sterility Restoration Gene in Onion[J]. Acta Horticulturae Sinica, 2025, 52(10): 2642-2654.

图/表 11

参考文献 40

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洋葱类型 Onion type	品系/杂交种 Line/hybrids	性状 Characteristic	生态型 Ecotype	基因型 Genotype	KASP
不育系 Male-sterile lines	110	EM，Y	中日照 Intermediate day	msms	A︰A
	118	EM，Y	中日照 Intermediate day	msms	A︰A
	502	MEM，Y	中日照 Intermediate day	msms	A︰A
	503	MEM，Y	中日照 Intermediate day	msms	A︰A
	101	MEM，Y	中日照 Intermediate day	msms	A︰A
	117-5	MEM，R	中日照 Intermediate day	msms	A︰A
	117-11-1	MEM，R	中日照 Intermediate day	msms	A︰A
	117-10-1	MEM，R	中日照 Intermediate day	msms	A︰A
	117-10-2	MEM，R	中日照 Intermediate day	msms	A︰A
	117-10-3	MEM，R	中日照 Intermediate day	msms	A︰A
	L701	MEM，R	长日照 Long day	msms	A︰A
	L702	MEM，R	长日照 Long day	msms	A︰A
	L703	MEM，R	长日照 Long day	msms	A︰A
保持系 Maintainer lines	210	EM，Y	中日照 Intermediate day	msms	A︰A
	218	EM，Y	中日照 Intermediate day	msms	A︰A
	602	MEM，Y	中日照 Intermediate day	msms	A︰A
	603	MEM，Y	中日照 Intermediate day	msms	A︰A
	202	MEM，Y	中日照 Intermediate day	msms	A︰A
	217-5	MEM，R	中日照 Intermediate day	msms	A︰A
	217-11-1	MEM，R	中日照 Intermediate day	msms	A︰A
	217-10-1	MEM，R	中日照 Intermediate day	msms	A︰A
	217-10-2	MEM，R	中日照 Intermediate day	msms	A︰A
	217-10-3	MEM，R	中日照 Intermediate day	msms	A︰A
	L801	MEM，R	长日照 Long day	msms	A︰A
父本系 Paternal lines	301	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	302	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	303	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	304	M，Y	中日照 Intermediate day	MsMs	T︰T
	305	M，Y	中日照 Intermediate day	MsMs	T︰T
	306	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	307	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	308	EM，W	短日照 Short day	MsMs	T︰T
	309	M，W	中日照 Intermediate day	MsMs	T︰T
	310	MLM，R	中日照 Intermediate day	MsMs	T︰T
	311	M，Y	中日照 Intermediate day	MsMs	T︰T
	312	LM，Y	中日照 Intermediate day	MsMs	T︰T
	313	EM，Y	中日照 Intermediate day	MsMs	T︰T
	316	MEM，R	中日照 Intermediate day	MsMs	T︰T
	317	MEM，R	中日照 Intermediate day	MsMs	T︰T
	318	EM，R	中日照 Intermediate day	MsMs	T︰T
杂交种 Hybrids	TABAO	M，Y	中日照 Intermediate day	Msms	T︰A
	ATON	MLM，Y	中日照 Intermediate day	Msms	T︰A
	EARTH	MLM，Y	中日照 Intermediate day	Msms	T︰A
	MOMIJI No.3	MLM，Y	中日照 Intermediate day	Msms	T︰A
	AMA70	MEM，Y	中日照 Intermediate day	Msms	T︰A
	ADVANCE	MEM，Y	中日照 Intermediate day	Msms	T︰A
	TASAN	M，Y	中日照 Intermediate day	Msms	T︰A

洋葱类型 Onion type	品系/杂交种 Line/hybrids	性状 Characteristic	生态型 Ecotype	基因型 Genotype	KASP
不育系 Male-sterile lines	110	EM，Y	中日照 Intermediate day	msms	A︰A
	118	EM，Y	中日照 Intermediate day	msms	A︰A
	502	MEM，Y	中日照 Intermediate day	msms	A︰A
	503	MEM，Y	中日照 Intermediate day	msms	A︰A
	101	MEM，Y	中日照 Intermediate day	msms	A︰A
	117-5	MEM，R	中日照 Intermediate day	msms	A︰A
	117-11-1	MEM，R	中日照 Intermediate day	msms	A︰A
	117-10-1	MEM，R	中日照 Intermediate day	msms	A︰A
	117-10-2	MEM，R	中日照 Intermediate day	msms	A︰A
	117-10-3	MEM，R	中日照 Intermediate day	msms	A︰A
	L701	MEM，R	长日照 Long day	msms	A︰A
	L702	MEM，R	长日照 Long day	msms	A︰A
	L703	MEM，R	长日照 Long day	msms	A︰A
保持系 Maintainer lines	210	EM，Y	中日照 Intermediate day	msms	A︰A
	218	EM，Y	中日照 Intermediate day	msms	A︰A
	602	MEM，Y	中日照 Intermediate day	msms	A︰A
	603	MEM，Y	中日照 Intermediate day	msms	A︰A
	202	MEM，Y	中日照 Intermediate day	msms	A︰A
	217-5	MEM，R	中日照 Intermediate day	msms	A︰A
	217-11-1	MEM，R	中日照 Intermediate day	msms	A︰A
	217-10-1	MEM，R	中日照 Intermediate day	msms	A︰A
	217-10-2	MEM，R	中日照 Intermediate day	msms	A︰A
	217-10-3	MEM，R	中日照 Intermediate day	msms	A︰A
	L801	MEM，R	长日照 Long day	msms	A︰A
父本系 Paternal lines	301	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	302	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	303	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	304	M，Y	中日照 Intermediate day	MsMs	T︰T
	305	M，Y	中日照 Intermediate day	MsMs	T︰T
	306	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	307	MLM，Y	中日照 Intermediate day	MsMs	T︰T
	308	EM，W	短日照 Short day	MsMs	T︰T
	309	M，W	中日照 Intermediate day	MsMs	T︰T
	310	MLM，R	中日照 Intermediate day	MsMs	T︰T
	311	M，Y	中日照 Intermediate day	MsMs	T︰T
	312	LM，Y	中日照 Intermediate day	MsMs	T︰T
	313	EM，Y	中日照 Intermediate day	MsMs	T︰T
	316	MEM，R	中日照 Intermediate day	MsMs	T︰T
	317	MEM，R	中日照 Intermediate day	MsMs	T︰T
	318	EM，R	中日照 Intermediate day	MsMs	T︰T
杂交种 Hybrids	TABAO	M，Y	中日照 Intermediate day	Msms	T︰A
	ATON	MLM，Y	中日照 Intermediate day	Msms	T︰A
	EARTH	MLM，Y	中日照 Intermediate day	Msms	T︰A
	MOMIJI No.3	MLM，Y	中日照 Intermediate day	Msms	T︰A
	AMA70	MEM，Y	中日照 Intermediate day	Msms	T︰A
	ADVANCE	MEM，Y	中日照 Intermediate day	Msms	T︰A
	TASAN	M，Y	中日照 Intermediate day	Msms	T︰A

引物名称 Primer name	引物序列（5′-3′） Primer sequence	引物名称 Primer name	引物序列（5′-3′） Primer sequence
KMs3	F_P1：ACTTGAACAGGATATAAAATCATTTATATGGA F_P2：CTTGAACAGGATATAAAATCATTTATATGGG R：TACTTGTTGTATAGWGCCATCAGCAGTTT	KMs25	F_P1：AAGAAAAGAAAAGAGCTGGAGGAGG F_P2：CAAGAAAAGAAAAGAGCTGGAGGAGA R：GACATRGATCATCACCTTCGACAGTTT
KMs4	F_P1：GCAACATCATCTTCTTCTTGTTGTATAGA F_P2：GCAACATCATCTTCTTCTTGTTGTATAGT R：TCATTTATATGGRTAAAAACTGCTGATGGC	KMs28	F_P1：TATATATTTATTCAGAATGCAGAAACTGAAGA F_P2：ATATATTTATTCAGAATGCAGAAACTGAAGT R：ACTGAACGTTTATCACCGCCCACAT
KMs5	F_P1：TGAGAGAAGCTTCATTGACTCTCG F_P2：GAGAGAAGCTTCATTGACTCTCT R：GCATGGGATCTTCAAGAAATTATCCCATT	KMs33	F_P1：GTTTTCTTTTTATTCTTTTTAACCTTTTTGCA F_P2：GTTTTCTTTTTATTCTTTTTAACCTTTTTGCC R：GCTRCAGTTTATTACTAATATATGCTGAAA
KMs12	F_P1：AGCTCTTTTTATTATTCTAGGAGCGAAAAA F_P2：AGCTCTTTTTATTATTCTAGGAGCGAAAAT R：CCAACAATCTTTCRGTGTCCATTTTAACAA	KMs35	F_P1：CACCAAAAGATAATTCTAGTGATGGCT F_P2：ACCAAAAGATAATTCTAGTGATGGCC R：ATGCACWCTGGATAAGGCAAATAACCTTT
KMs15	F_P1：CGAACCTCTTCAGGAGATTTTTCA F_P2：CTCGAACCTCTTCAGGAGATTTTTCT R：CACATGTACAGCMCTTGCTCGTAAAAT	KMs38	F_P1：CATCATCATCATCTAACTCGATATTTGC F_P2：ATCATCATCATCATCTAACTCGATATTTGA R：CCACTAATCCAAGTACAAGTTCAATGCTA
KMs24	F_P1：CAGCTCTTTTCTTTTCTTGGCAT F_P2：CCAGCTCTTTTCTTTTCTTGGCAC R：GCGACCCACGGRTTCGATTGTTAA

引物名称 Primer name	引物序列（5′-3′） Primer sequence	引物名称 Primer name	引物序列（5′-3′） Primer sequence
KMs3	F_P1：ACTTGAACAGGATATAAAATCATTTATATGGA F_P2：CTTGAACAGGATATAAAATCATTTATATGGG R：TACTTGTTGTATAGWGCCATCAGCAGTTT	KMs25	F_P1：AAGAAAAGAAAAGAGCTGGAGGAGG F_P2：CAAGAAAAGAAAAGAGCTGGAGGAGA R：GACATRGATCATCACCTTCGACAGTTT
KMs4	F_P1：GCAACATCATCTTCTTCTTGTTGTATAGA F_P2：GCAACATCATCTTCTTCTTGTTGTATAGT R：TCATTTATATGGRTAAAAACTGCTGATGGC	KMs28	F_P1：TATATATTTATTCAGAATGCAGAAACTGAAGA F_P2：ATATATTTATTCAGAATGCAGAAACTGAAGT R：ACTGAACGTTTATCACCGCCCACAT
KMs5	F_P1：TGAGAGAAGCTTCATTGACTCTCG F_P2：GAGAGAAGCTTCATTGACTCTCT R：GCATGGGATCTTCAAGAAATTATCCCATT	KMs33	F_P1：GTTTTCTTTTTATTCTTTTTAACCTTTTTGCA F_P2：GTTTTCTTTTTATTCTTTTTAACCTTTTTGCC R：GCTRCAGTTTATTACTAATATATGCTGAAA
KMs12	F_P1：AGCTCTTTTTATTATTCTAGGAGCGAAAAA F_P2：AGCTCTTTTTATTATTCTAGGAGCGAAAAT R：CCAACAATCTTTCRGTGTCCATTTTAACAA	KMs35	F_P1：CACCAAAAGATAATTCTAGTGATGGCT F_P2：ACCAAAAGATAATTCTAGTGATGGCC R：ATGCACWCTGGATAAGGCAAATAACCTTT
KMs15	F_P1：CGAACCTCTTCAGGAGATTTTTCA F_P2：CTCGAACCTCTTCAGGAGATTTTTCT R：CACATGTACAGCMCTTGCTCGTAAAAT	KMs38	F_P1：CATCATCATCATCTAACTCGATATTTGC F_P2：ATCATCATCATCATCTAACTCGATATTTGA R：CCACTAATCCAAGTACAAGTTCAATGCTA
KMs24	F_P1：CAGCTCTTTTCTTTTCTTGGCAT F_P2：CCAGCTCTTTTCTTTTCTTGGCAC R：GCGACCCACGGRTTCGATTGTTAA

群体 Population	可育 Fertile	不育 Sterile	期望比例 Expected ratio	χ²	p
175DH17F2	106	34	3:1	0.038	0.845
175DH17BC1	210	197	1:1	0.415	0.519