Establishment and Application of a Tomato KASP Genotyping System Based on Five Disease Resistance Genes

doi:10.16420/j.issn.0513-353x.2020-0913

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (11): 2211-2226.doi: 10.16420/j.issn.0513-353x.2020-0913

• Original article • Previous Articles Next Articles

Establishment and Application of a Tomato KASP Genotyping System Based on Five Disease Resistance Genes

WANG Peng¹, TIAN Zhejuan¹, KANG Chen¹, LI Yadong¹, WANG Hongle², YANG Chaosha¹, MANG Guangwei², KANG Liang², FAN Qingjie², WU Zhiming¹^,^*()

¹Institute of Cash Crops,Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang 050051,China
²Chengde Academy of Agriculture and Forestry Sciences,Chengde,Hebei 067055,China

Received:2021-05-24 Revised:2021-08-06 Published:2021-12-02
Contact: WU Zhiming E-mail:zhiming71@126.com

Abstract

Abstract:

Kompetitive allele specific PCR（KASP）is a novel genotyping tool based on single nucleotide polymorphism（SNP）. An efficient and economical resistance gene KASP genotyping method is very useful in tomato breeding program. In this study,KASP primer pairs of five tomato disease resistance genes were used to establish a KASP genotyping system,among them the primer sets for tomato yellow leaf curl virus（TYLCV）resistance gene Ty-1 and root-knot nematode（RKN）resistance gene Mi-1 were designed in this study,and KASP primers of tomato mosaic virus（ToMV）resistance gene Tm-2²,tomato spotted wilt virus（TSWV）resistance gene Sw-5 and fusarium crown and root rot（FCRR）resistance gene Frl were from previous publications. By using tomato germplasms containing different disease resistance genes,the KASP genotyping system was optimized for its template reaction volume,DNA concentration,primer concentration and the number of PCR cycles. Detection results using the optimized KASP method were consistent with the PCR detection results of molecular markers of five disease resistance genes. The KASP genotyping system developed in this study not only can save cost,but also possesses high accuracy and stability. Therefore,it can be widely used in identification of tomato disease resistance genes,genetic diversity analysis and gene mapping,which provides technical supports and theoretical basis for the tomato molecular marker-assisted disease resistance identification,rapid screening and utilization of resistance germplasms for tomato breeding.

Key words: tomato, germplasm, KASP, genotyping, Ty-1, Mi-1, Tm-2², Sw-5, Frl

CLC Number:

S641.2

WANG Peng, TIAN Zhejuan, KANG Chen, LI Yadong, WANG Hongle, YANG Chaosha, MANG Guangwei, KANG Liang, FAN Qingjie, WU Zhiming. Establishment and Application of a Tomato KASP Genotyping System Based on Five Disease Resistance Genes[J]. Acta Horticulturae Sinica, 2021, 48(11): 2211-2226.

Figures/Tables 7

References 44

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抗病基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence	抗病标记/bp Resisting marker	感病标记/bp Unresisting marker	参考文献 Reference
Ty-1	NL3-F	TGTATGCGTCGTGTTAGAAGGTC	984	1 434	葛乃蓬等,2014
	NL3-R	AATGGTGACAGAATCAAGATCCAC
	NL2-F	AAACGCTCTTCTTCCCCTCG
	NL2-R	AGTTAGATTCAGGCTCCTCGCA
Mi-1	PMiF3	GGTATGAGCATGCTTAATCAGAGCTCTC	550	350	Arens et al.,2011
Mi-1	PMiR3	CCTACAAGAAATTATTGTGCGTGTGAATG	550	350	Arens et al.,2011
Tm-2²	Tm-2a-A1	GCCTCATTCAACTTCCTTCTGG	472		朱明涛等,2010
Tm-2²	Tm-2a-A2	GCCAGTATATAACGGTCTAAAC	472		朱明涛等,2010
Frl	C2-25-F	ATGGGCGCTGCATGTTTCGTG	750	1 000	Staniaszek et al.,2014
Frl	C2-25-R	ACACCTTTGTTGAAAGCCATCCC	750	1 000	Staniaszek et al.,2014
Sw-5	Sw-5-2F	AATTAGGTTCTTGAAGCCCATCT	574	510	Dianese et al.,2010
Sw-5	Sw-5-2R	TTCCGCATCAGCCAATAGTGT	574	510	Dianese et al.,2010

抗病基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence	抗病标记/bp Resisting marker	感病标记/bp Unresisting marker	参考文献 Reference
Ty-1	NL3-F	TGTATGCGTCGTGTTAGAAGGTC	984	1 434	葛乃蓬等,2014
	NL3-R	AATGGTGACAGAATCAAGATCCAC
	NL2-F	AAACGCTCTTCTTCCCCTCG
	NL2-R	AGTTAGATTCAGGCTCCTCGCA
Mi-1	PMiF3	GGTATGAGCATGCTTAATCAGAGCTCTC	550	350	Arens et al.,2011
Mi-1	PMiR3	CCTACAAGAAATTATTGTGCGTGTGAATG	550	350	Arens et al.,2011
Tm-2²	Tm-2a-A1	GCCTCATTCAACTTCCTTCTGG	472		朱明涛等,2010
Tm-2²	Tm-2a-A2	GCCAGTATATAACGGTCTAAAC	472		朱明涛等,2010
Frl	C2-25-F	ATGGGCGCTGCATGTTTCGTG	750	1 000	Staniaszek et al.,2014
Frl	C2-25-R	ACACCTTTGTTGAAAGCCATCCC	750	1 000	Staniaszek et al.,2014
Sw-5	Sw-5-2F	AATTAGGTTCTTGAAGCCCATCT	574	510	Dianese et al.,2010
Sw-5	Sw-5-2R	TTCCGCATCAGCCAATAGTGT	574	510	Dianese et al.,2010

抗病基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence	等位基因 Allele	参考文献 Reference
Ty-1	Ty-1-Allele-F1	GAAGGTCGGAGTCAACGGATTGAATATGGTAAAGGTGCATCCAGA	A︰A	胡霞等,2014
	Ty-1-Allele-F2	GAAGGTGACCAAGTTCATGCTGAATATGGTAAAGGTGCATCCAGT	T︰T
	Ty-1-R	CCGTTGATTGAAGAAATTGATG
Mi-1	Mi-1-Allele-R1	GAAGGTGACCAAGTTCATGCTGATGTTTACAAGTATTTGTGGCG	G︰G	Zübeyir et al.,2016
	Mi-1-Allele-R2	GAAGGTCGGAGTCAACGGATTGATGTTTACAAGTATTTGTGGCA	A︰A
	Mi-1-F	TTCAGACGAAGTTAGACATAAAGGA
Tm-2²	Tm-2-Allele-F1	GAAGGTCGGAGTCAACGGATTCAATATGCTACAAGCCAAATTACTC	C︰C	孔会利等,2018
	Tm-2-Allele-F2	GAAGGTGACCAAGTTCATGCTCAATATGCTACAAGCCAAATTACTT	T︰T
	Tm-2-R	GAAACAACTCCATAATGCAAATC
Frl	Frl-Allele-F1	GAAGGTGACCAAGTTCATGCTTAAAGGACCATTTTTCGTAGACCATT	T︰T	国家进等,2016
	Frl-Allele-F2	GAAGGTCGGAGTCAACGGATTTAAAGGACCATTTTTCGTAGACCATA	A︰A
	Frl-R	ATTTCTTACCATATGTGAAGAGAA
Sw-5	FP1SNP724	GAAGGTCGGAGTCAACGGATTCATGGACACAACTGGAGTTATTA	A︰A	Devran & Kahveci,2019
	FP2SNP724	GAAGGTGACCAAGTTCATGCTCATGGACACAACTGGAGTTATTG	G︰G
	RPSNP724	CAGTCATTCTTCAAATTTTACTTGT

抗病基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence	等位基因 Allele	参考文献 Reference
Ty-1	Ty-1-Allele-F1	GAAGGTCGGAGTCAACGGATTGAATATGGTAAAGGTGCATCCAGA	A︰A	胡霞等,2014
	Ty-1-Allele-F2	GAAGGTGACCAAGTTCATGCTGAATATGGTAAAGGTGCATCCAGT	T︰T
	Ty-1-R	CCGTTGATTGAAGAAATTGATG
Mi-1	Mi-1-Allele-R1	GAAGGTGACCAAGTTCATGCTGATGTTTACAAGTATTTGTGGCG	G︰G	Zübeyir et al.,2016
	Mi-1-Allele-R2	GAAGGTCGGAGTCAACGGATTGATGTTTACAAGTATTTGTGGCA	A︰A
	Mi-1-F	TTCAGACGAAGTTAGACATAAAGGA
Tm-2²	Tm-2-Allele-F1	GAAGGTCGGAGTCAACGGATTCAATATGCTACAAGCCAAATTACTC	C︰C	孔会利等,2018
	Tm-2-Allele-F2	GAAGGTGACCAAGTTCATGCTCAATATGCTACAAGCCAAATTACTT	T︰T
	Tm-2-R	GAAACAACTCCATAATGCAAATC
Frl	Frl-Allele-F1	GAAGGTGACCAAGTTCATGCTTAAAGGACCATTTTTCGTAGACCATT	T︰T	国家进等,2016
	Frl-Allele-F2	GAAGGTCGGAGTCAACGGATTTAAAGGACCATTTTTCGTAGACCATA	A︰A
	Frl-R	ATTTCTTACCATATGTGAAGAGAA
Sw-5	FP1SNP724	GAAGGTCGGAGTCAACGGATTCATGGACACAACTGGAGTTATTA	A︰A	Devran & Kahveci,2019
	FP2SNP724	GAAGGTGACCAAGTTCATGCTCATGGACACAACTGGAGTTATTG	G︰G
	RPSNP724	CAGTCATTCTTCAAATTTTACTTGT

编号 No.	Ty-1			Mi-1			Tm-2²			Frl			Sw-5
编号 No.	PCR	KASP	基因型 Genotype	PCR	KASP	基因型 Genotype	PCR	KASP	基因型 Genotype	PCR	KASP	基因型 Genotype	PCR	KASP	基因型 Genotype
1	++	A︰A	Ty-1/Ty-1	+-	G︰A	Mi-1/mi-1	+	T︰C	Tm-2²/tm-2²	+ -	A︰T	Frl/frl	--	A︰A	sw-5/sw-5
2	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	+ +	G︰G	Sw-5/Sw-5
3	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
4	+ -	A︰T	Ty-1/ty-1	++	G︰G	Mi-1/Mi-1	+	T︰C	Tm-2²/tm-2²	+ +	A︰A	Frl/Frl	+-	G︰A	Sw-5/sw-5
5	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
6	++	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
7	++	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
8	++	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
9	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
10	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
11	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
12	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
13	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
14	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
15	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
16	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
17	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
18	+ -	A︰T	Ty-1/ty-1	+-	G︰A	Mi-1/mi-1	+	T︰C	Tm-2²/tm-2²	+ -	A︰T	Frl/frl	--	A︰A	sw-5/sw-5
19	+ -	A︰T	Ty-1/ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰C	Tm-2²/tm-2²	+ -	A︰T	Frl/frl	--	A︰A	sw-5/sw-5
20	+ -	A︰T	Ty-1/ty-1	+-	G︰A	Mi-1/mi-1	+	T︰C	Tm-2²/tm-2²	+ -	A︰T	Frl/frl	--	A︰A	sw-5/sw-5
21	+ -	A︰T	Ty-1/ty-1	+-	G︰A	Mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
22	- -	T︰T	ty-1/ty-1	+-	G︰A	Mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
23	+ +	A︰A	Ty-1/Ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
24	+ +	A︰A	Ty-1/Ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
25	+ -	A︰T	Ty-1/ty-1	--	A︰A	mi-1/mi-1	+	T︰C	Tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
26	+ -	A︰T	Ty-1/ty-1	+-	G︰A	Mi-1/mi-1	+	T︰C	Tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
27	+ -	A︰T	Ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
28	+ -	A︰T	Ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
29	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
30	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
31	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
32	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
33	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
34	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
35	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
36	+ +	A︰A	Ty-1/Ty-1	+ +	G︰G	Mi-1/Mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
37	- -	T︰T	ty-1/ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
38	- -	T︰T	ty-1/ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
39	- -	T︰T	ty-1/ty-1	+ +	G︰G	Mi-1/Mi-1	+	T︰T	Tm-2²/Tm-2²	+ +	A︰A	Frl/Frl	--	A︰A	sw-5/sw-5
40	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
41	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
42	+ +	A︰A	Ty-1/Ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
43	+ +	A︰A	Ty-1/Ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
44	+ +	A︰A	Ty-1/Ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
45	+ +	A︰A	Ty-1/Ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
46	+ +	A︰A	Ty-1/Ty-1	--	A︰A	mi-1/mi-1	+	T︰T	Tm-2²/Tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
47	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5
48	- -	T︰T	ty-1/ty-1	--	A︰A	mi-1/mi-1	- -	C︰C	tm-2²/tm-2²	- -	T︰T	frl/frl	--	A︰A	sw-5/sw-5

Establishment and Application of a Tomato KASP Genotyping System Based on Five Disease Resistance Genes

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