番茄5个抗病基因KASP分型技术体系的建立与应用

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

摘要/Abstract

摘要：

竞争性等位基因特异性PCR（Kompetitive allele specific PCR,KASP）是一种基于单核酸多态性（Single nucleotide polymorphism,SNP）的新型基因分型技术,建立高效、经济的番茄抗病基因的KASP分型技术对促进抗病品种的选育具有重要意义。本研究中设计了抗番茄黄花曲叶病毒（tomato yellow leaf curl virus,TYLCV）病基因Ty-1和抗根结线虫（root-knot nematode,RKN）基因Mi-1的KASP引物,结合已报道的抗番茄花叶病毒（tomato mosaic virus,ToMV）病基因Tm-2²、抗番茄斑萎病毒（tomato spotted wilt virus,TSWV）病基因Sw-5和抗镰刀菌颈腐根腐（fusarium crown and root rot,FCRR）病基因Frl的KASP引物,进行了番茄抗病基因KASP分型检测技术研究。利用不同基因型的番茄抗病种质,对影响KASP的反应体积、DNA模板浓度、引物浓度及反应循环数等进行了优化,建立了最优番茄抗病基因的KASP分型检测技术体系。研究结果显示,优化的KASP体系与Ty-1、Mi-1、Tm-2²、Sw-5和Frl基因的PCR分子标记检测结果相一致。所用的KASP反应体系不但能节省试剂,且具有高准确性和强稳定性的优点,因此其可以广泛应用于番茄抗病基因的鉴定、遗传多样性和基因定位等研究,为番茄种质资源分子标记辅助的抗病性鉴定、抗原的快速筛选及利用提供技术支持和理论依据。

关键词: 番茄, 种质资源, KASP技术, 基因分型, Ty-1, Mi-1, Tm-2², Sw-5, Frl

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

中图分类号:

S641.2

王鹏, 田哲娟, 康忱, 李亚栋, 王洪乐, 杨超沙, 邙光伟, 康亮, 范庆杰, 吴志明. 番茄5个抗病基因KASP分型技术体系的建立与应用[J]. 园艺学报, 2021, 48(11): 2211-2226.

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.

图/表 7

参考文献 44

[1]	Allen A M, Barker G L, Berry S T, Coghill J A, Gwilliam R, Kirby S, Robinson P, Brenchley R C, D’Amore R, McKenzie N, Waite D, Hall A, Bevan M, Hall N, Edwards K J. 2011. Transcript-specific,single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat(Triticum aestivum L.). Plant Biotechnology Journal, 9 (9):1086-1099. doi: 10.1111/pbi.2011.9.issue-9 URL
[2]	Arens P, Mansilla C, Deinum D, Cavellini L, Moretti A, Rolland S, Van D S, Calvache D, Ponz F, Collonnier C, Mathis R, Smilde D, Caranta C, Vosman B. 2011. Development and evaluation of robust molecular markers linked to disease resistance in tomato for distinctness,uniformity and stability testing. Theoretical and Applied Genetics, 120 (3):655-664. doi: 10.1007/s00122-009-1183-2 URL
[3]	Berhanu T E, Veronica O, Mosisa W, Biswanath D, Michael O, Maryke L, Kassa S. 2015. Comparison of Kompetitive Allele Specific PCR(KASP)and genotyping by sequencing(GBS)for quality control analysis in maize. BMC Genomics, 16 (1):908-919. doi: 10.1186/s12864-015-2180-2 URL
[4]	Devran Z, Kahveci E. 2019. Development and validation of a user-friendly KASP marker for the Sw-5 locus in tomato. Australasian Plant Pathology, 48 (5):503-507. doi: 10.1007/s13313-019-00651-1 URL
[5]	Dianese R, Noronha F M, Goldbach R, Kormelink R, Inoue N A, Resende R, Boiteux L. 2010. Development of a locus-specific,co-dominant SCAR marker for assisted-selection of the Sw-5(Tospovirus resistance)gene cluster in a wide range of tomato accessions. Molecular Breeding, 25 (1):133-142. doi: 10.1007/s11032-009-9313-8 URL
[6]	Ge Nai-peng, Cui Long, Li Han-xia, Ye Zhi-biao. 2014. Double SNP marker of Ty-1 alleles in a co-dominant manner in tomato. Acta Horticulturae Sinica, 41 (8):1583-1590. (in Chinese)
	葛乃蓬, 崔龙, 李汉霞, 叶志彪. 2014. 番茄抗黄化曲叶病毒基因Ty-1的双重SNP标记的开发. 园艺学报, 41 (8):1583-1590.
[7]	Grogan S M, Brown-Guedira G, Haley S D, McMaster G S, Reid S D, Smith J, Byrne P F. 2016. Allelic variation in developmental genes and effects on winter wheat heading date in the U. S. great plains. PLoS ONE, 11:e0152852.
[8]	Guo Jia-jin, Zhang Sheng, Cheng Lin, Chen Fu-dong, Zhang Xiao-yan, Dong Tian, Li Xiao-jie, Liu Yue, Wang Ru-fang, Dong Jing, Yu Cai-yu. 2016. High-throughput molecular marker for identifying neck rot and root rot resistance of tomatoes and marking method and application thereof. China:CN105671190A, 2016-06-15. (in Chinese)
	国家进, 张生, 程琳, 陈福东, 张晓燕, 董甜, 李晓杰, 刘岳, 王如芳, 董静, 于彩玉. 2016. 鉴定番茄颈腐根腐病抗性的高通量分子标记及其标记方法与应用. 中国:CN105671190A, 2016-06-15.
[9]	He C, Holme J, Anthony J. 2014. SNP genotyping:the KASP assay. Methods in Molecular Biology, 1145:75-86.
[10]	Hu Xia, Wang Rong, Li Fei-fei, Zhou Tao, Yang Wen-cai. 2014. Development of new marker for tomato TYLCV resistant gene and its application in selection of multi resistant gene pyramiding. China Vegetables,(10):18-23. (in Chinese)
	胡霞, 王蓉, 李菲菲, 周涛, 杨文才. 2014. 番茄抗TYLCV基因新标记的开发及其在多抗聚合选择中的应用. 中国蔬菜,(10):18-23.
[11]	Jia Yu-yan, Chen Hong. 2003. Molecular genetic marker of single nucleotide polymorphism and its application. China Cattle Science, 29 (1):42-45. (in Chinese)
	贾玉艳, 陈宏. 2003. SNP分子标记的研究及应用. 中国牛业科学, 29 (1):42-45.
[12]	Kong Hui-li, Jing Li-jun, He Zong-shun, Zhang Long-yu, Xia Qin-fang, Zhang Wen-li, Sun Ji-qing, Wei Yi, Yu Hui-hui, Zhou Fa-song. 2018. Molecular marker for identifying resistance gene of tomato mosaic virus(TOMV)and application thereof. China:CN108330201A, 2018-07-27. (in Chinese)
	孔会利, 井立军, 何宗顺, 张龙雨, 夏琴芳, 张文丽, 孙吉庆, 韦懿, 喻辉辉, 周发松. 2018. 鉴定番茄花叶病毒抗性基因的分子标记及其应用. 中国:CN108330201A, 2018-07-27.
[13]	Kuang Meng, Wang Yan-qin, Zhou Da-yun, Ma Lei, Fang Dan, Xu Shuang-jiao, Yang Wei-hua, Wei Shou-jun, Ma Zhi-ying. 2016. High-throughput genotyping assay technology for cotton hybrid purity based on single-copy SNP markers. Cotton Science, 28 (3):227-233. (in Chinese)
	匡猛, 王延琴, 周大云, 马磊, 方丹, 徐双娇, 杨伟华, 魏守军, 马峙英. 2016. 基于单拷贝SNP标记的棉花杂交种纯度高通量检测技术. 棉花学报, 28 (3):227-233.
[14]	Li Yong-ping, Kang Jian-ban, Lin Hui. 2016. Optimization and verification of ISSR-PCR reaction system for Lycopersicon esculentum. Journal of Agriculture, 6 (3):52-56. (in Chinese)
	李永平, 康建坂, 林珲. 2016. 番茄ISSR-PCR反应体系的优化与验证. 农学学报, 6 (3):52-56.
[15]	Li Zong-jun, Wang Xian-yu, Liu Meng-jiao, Cui Xin-yue, Zhao Xiong, Chen Peng, Ou Qing-qing. 2019. Auxiliary screening of polymeric multi-resistance tomato materials using KASP molecular marker technology. China Vegetables,(8):42-46. (in Chinese)
	李宗俊, 王先裕, 刘梦姣, 崔馨月, 赵雄, 陈鹏, 欧青青. 2019. 利用KASP分子标记技术辅助筛选多抗番茄材料. 中国蔬菜,(8):42-46.
[16]	Liang Shuang, Yuan Yu-xiang, Zhang Xiao-wei, Xue Yin-ge, Yao Qiu-ju, Jiang Wu-sheng, Tian Bao-ming, Zhang Qiang, Zhao Yan-yan. 2012. Optimization of InDel-PCR reaction system for Chinese cabbage(Brassica rapa L.ssp.pekinensis). Journal of Henan Agricultural Sciences, 41 (9):110-113. (in Chinese)
	梁爽, 原玉香, 张晓伟, 薛银鸽, 姚秋菊, 蒋武生, 田保明, 张强, 赵艳艳. 2012. 大白菜InDel-PCR反应体系的优化. 河南农业科学, 41 (9):110-113.
[17]	Liu Lei, Deng Xuebin, Feng Jinging, Wang Jing, Sun Xiaorong, Shu Jinshuai, Li Junming. 2018. Exploring variant sites associated with Ve-1,I-2,Mi-1 and Pto genes in processing tomato by genome-wide association analysis. Acta Horticulturae Sinica, 45 (6):1089-1100. (in Chinese)
	刘磊, 邓学斌, 冯晶晶, 王静, 孙晓荣, 舒金帅, 李君明. 2018. 加工番茄Ve-1、I-2、Mi-1和Pto基因关联变异位点的挖掘. 园艺学报, 45 (6):1089-1100.
[18]	Liu Li-hua, Pang Bin-shuang, Liu Yang-na, Li Hong-bo, Wang Na, Wang Zheng, Zhao Chang-ping. 2018. High-throughput identification mode for wheat varieties based on SNP markers. Journal of Triticeae Crops, 38 (5):529-534. (in Chinese)
	刘丽华, 庞斌双, 刘阳娜, 李宏博, 王娜, 王拯, 赵昌平. 2018. 基于SNP标记的小麦高通量身份鉴定模式. 麦类作物学报, 38 (5):529-534.
[19]	Liu Z Y, Zhu C S, Jiang Y, Tian Y L, Yu J, An H Z, Tang W J, Sun J, Tang J P, Chen G M, Zhai H Q, Wang C M, Wan J M. 2016. Association mapping and genetic dissection of nitrogen use efficiency- related traits in rice (Oryza sativa L.). Funct Integr Genomics, 16 (3):323-333. doi: 10.1007/s10142-016-0486-z URL
[20]	Lou H J, Dong L L, Zhang K P, Wang D W, Zhao M L, Li Y W, Rong C W, Qin H J, Zhang A M, Dong Z Y, Wang D W. 2017. High-throughput mining of e-genome-specific SNPs for characterizing thinopyrum elongatum introgressions in common wheat. Molecular Ecology Resources, 17 (6):1318-1329. doi: 10.1111/men.2017.17.issue-6 URL
[21]	Nair S K, Babu R, Magorokosho C, Mahuku G, Semagn K, Beyene Y, Das B, Makumbi D, Kumar P L, Olsen M. 2015. Fine mapping of Msv1,a major QTL for resistance to maize streak virus leads to development of production markers for breeding pipelines. Theor Appl Genet, 128:1839-1854. doi: 10.1007/s00122-015-2551-8 URL
[22]	Paux E, Sourdille P, Mackay I, Feuillet C. 2012. Sequence-based marker development in wheat:advances and applications to breeding. Biotechnology Advances, 30 (5):1071-1088. doi: 10.1016/j.biotechadv.2011.09.015 URL
[23]	Rasheed A, Wen W E, Gao F M, Zhai S N, Jin H, Liu J D, Guo Q, Zhang Y J, Dreisigacker S, Xia X C, He Z H. 2016. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theoretical and Applied Genetics, 129 (10):1843-1860. doi: 10.1007/s00122-016-2743-x pmid: 27306516
[24]	Semagn K, Babu R, Hearne S, Olsen M. 2014. Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR(KASP):overview of the technology and its application in crop improvement. Molecular Breeding, 33 (1):1-14. doi: 10.1007/s11032-013-9917-x URL
[25]	Shi Z, Liu S M, James N, Prakash A, Khalid M, Li Z L. 2015. SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance. BMC Genomics, 16 (1):314. doi: 10.1186/s12864-015-1531-3 URL
[26]	Song Jian-jun, Wang Lin-shan, Tian Peng, Wang Rui-fei. 2012. Research progress on molecular marker for disease resistance genes in tomato. Journal of Northeast Agricultural University, 43 (4):1-10. (in Chinese)
	宋建军, 王琳珊, 田鹏, 王瑞飞. 2012. 番茄主要病害抗病基因分子标记的研究进展. 东北农业大学学报, 43 (4):1-10.
[27]	Staniaszek M, Szczechura W, Marczewski W. 2014. Identification of a new molecular marker C2- 25 linked to the Fusarium oxysporum f.sp radicis-lycopersici resistance Frl gene in tomato. Czech Journal of Genetics and Plant Breeding, 50 (4):285-287. doi: 10.17221/CJGPB URL
[28]	Steele K, Quinton M, Amgai R, Dhakal R, Khatiwada S, Vyas D, Heine M, Witcombe J. 2018. Accelerating public sector rice breeding with high-density KASP markers derived from whole genome sequencing of indica rice. Molecular Breeding, 38:38. doi: 10.1007/s11032-018-0777-2 URL
[29]	Su X M, Zhu G T, Huang Z J, Wang X X, Guo Y M, Li B J, Du Y C, Yang W C, Gao J C. 2019. Fine mapping and molecular marker development of the Sm gene conferring resistance to gray leaf spot(Stemphylium spp.)in tomato. Theoretical and Applied Genetics, 132 (4):871-882. doi: 10.1007/s00122-018-3242-z URL
[30]	Tan C T, Yu H J, Yang Y, Xu X Y, Chen M S, Rudd J C, Xue Q W, Ibrahim A M H, Garza L, Wang S C, Liu S Y. 2017. Development and validation of KASP markers for the greenbug resistance gene Gb7 and the Hessian fly resistance gene H32in wheat. Theor Appl Genet, 130:1867-1884. doi: 10.1007/s00122-017-2930-4 URL
[31]	Trick M, Adamski N, Mugford S G, iang C C, Febrer M, Uauy C. 2012. Combining SNP discovery from next-generation sequencing data with bulked segregant analysis(BSA)to fine-map genes in polyploid wheat. BMC Plant Biology, 12:14. doi: 10.1186/1471-2229-12-14 URL
[32]	Wang D G, Fan J B, Siao C J, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris M S, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson T J, Lipshutz R, Chee M, Lander E S. 1998. Large-scale identification,mapping,and genotyping of single-nucleotide polymorphisms in the human genome. Science, 280 (5366):1077-1082. pmid: 9582121
[33]	Wang Hui, Wang Shuang, Jiang Tong, Yang Yan-jie, Lin Duo. 2018. Construction and application of InDel-PCR system of dry pepper. Jiangsu Agricultural Sciences, 46 (10):28-30. (in Chinese)
	王辉, 王爽, 姜童, 杨延杰, 林多. 2018. 干制辣椒InDel-PCR反应体系的构建及应用. 江苏农业科学, 46 (10):28-30.
[34]	Wen C L, Mao A J, Dong C J, Liu H Y, Yu S C, Guo Y D, Weng Y Q, Xu Y. 2015. Fine genetic mapping of target leaf spot resistance gene cca-3 in cucumber,Cucumis sativus L. Theor Appl Genet, 128 (12):2495-2506. doi: 10.1007/s00122-015-2604-z URL
[35]	Wu J H, Liu S J, Wang Q L, Zeng Q D, Mu J M, Huang S, Yu S Z, Han D J, Kang Z S. 2018. Rapid identification of an adult plant stripe rust resistance gene in hexaploid wheat by high-throughput SNP array genotyping of pooled extremes. Theoretical and Applied Genetics, 131 (1):43-58. doi: 10.1007/s00122-017-2984-3 URL
[36]	Wu Yuan-yuan, Li Hai-tao, Zhang Zi-jun, Zou Qing-dao. 2010. Research progress in molecular marker of disease resistant genes in tomato. Guizhou Agricultural Sciences, 38 (2):27-31. (in Chinese)
	吴媛媛, 李海涛, 张子君, 邹庆道. 2010. 番茄抗病基因分子标记研究进展. 贵州农业科学, 38 (2):27-31.
[37]	Yang Chao-sha, Wang Guo-hua, Wu Zhi-ming, Yin Qing-zhen, Yin Wei-ping. 2018. Detection of resistant genes to tomato yellow leaf curl virus disease and root-knot nematode in tomato. Journal of Hebei Agricultural University, 41 (2):77-80. (in Chinese)
	杨超沙, 王国华, 吴志明, 尹庆珍, 尹伟平. 2018. 番茄黄化曲叶病毒及根结线虫抗性基因的检测. 河北农业大学学报, 41 (2):77-80.
[38]	Yang Shuangjuan, Yuan Yuxiang, Wei Xiaochun, Wang Zhiyong, Zhao Yanyan, Yao Qiuju, Zhang Xiaowei. 2018. Optimization and establishment of KASP reaction system for Chinese cabbage(Brassica rapa L. ssp. Pekinensis). Acta Horticulturae Sinica, 48 (12):2442-2452. (in Chinese)
	杨双娟, 原玉香, 魏小春, 王志勇, 赵艳艳, 姚秋菊, 张晓伟. 2018. 大白菜KASP反应体系的优化与建立. 园艺学报, 48 (12):2442-2452.
[39]	Yang Shuang-juan, Wang Zhi-Yong, Zhao Yan-yan, Wei Xiao-chun, Yuan Yu-xiang, Zhang Xiao-wei. 2020. Development of KASP marker for bolting related gene BrFLC1 in Chinese cabbage(Brassica rapa L.ssppekinensis). Journal of Nuclear Agricultural Sciences, 34 (2):265-272. (in Chinese)
	杨双娟, 王志勇, 赵艳艳, 魏小春, 原玉香, 张晓伟. 2020. 大白菜抽薹相关基因BrFLC1的KASP标记开发. 核农学报, 34 (2):265-272.
[40]	Yuan Yu-xiang, Sun Ri-fei, Zhang Xiao-wei, Wu Jian, Xu Dong-hui, Zhang Hui, He Jiang-ming, Zhang Yan-guo, Wang Xiao-wu. 2008. A CAPS marker linked to bolting related gene BrFLC1 in Brassica rapa. Acta Horticulturae Sinica, 35 (11):1635-1640. (in Chinese)
	原玉香, 孙日飞, 张晓伟, 武剑, 徐东辉, 张慧, 和江明, 张延国, 王晓武. 2008. 芸薹种作物抽薹相关基因BrFLC1的CAPS标记. 园艺学报, 35 (11):1635-1640.
[41]	Zhang Qiang, Zhang Tao, Chang Xiao-ke, Han Ya-nan, Cheng Zhi-fang, Liu Wei, Wang Bin, Yao Qiu-ju. 2019. Transformation and application of one molecular marker from SCAR to KASP in pepper CMS. cta Agriculturae Boreali-Sinica., 34 (5):93-98. (in Chinese)
	张强, 张涛, 常晓轲, 韩娅楠, 程志芳, 刘卫, 王彬, 姚秋菊. 2019. 一个辣椒胞质雄性不育SCAR标记的KASP转化及其应用. 华北农学报, 34 (5):93-98.
[42]	Zhao Yong, Liu Xiao-dong, Zhao Hong-kun, Yuan Cui-ping, Qi Guang-xun, Wang Yu-min, Dong Ying-shan. 2017. Comparison of methods for SNP genotyping in soybean. Molecular Plant Breeding, 15 (9):3540-3546. (in Chinese)
	赵勇, 刘晓冬, 赵洪锟, 袁翠平, 齐广勋, 王玉民, 董英山. 2017大豆SNP分型方法的比较. 分子植物育种, 15 (9):3540-3546.
[43]	Zhu Ming-tao, Sun Ya-lin, Zheng Sha, Zhang Xiao-li, Wang Tao-tao, Ye Zhi-biao, Li Han-xia. 2010. Pyramiding disease resistance genes by molecular marker-assisted selection in tomato. Acta Horticulturae Sinica, 37 (9):1416-1422. (in Chinese)
	朱明涛, 孙亚林, 郑莎, 张晓黎, 王涛涛, 叶志彪, 李汉霞. 2010. 分子标记辅助聚合番茄抗病基因育种. 园艺学报, 37 (9):1416-1422.
[44]	Zübeyir D, Atilla G, Lütfiye M. 2016. Development of molecular markers for the Mi-1 gene in tomato using the KASP genotyping assay. Horticulture Environment and Biotechnology, 57 (2):156-160. doi: 10.1007/s13580-016-0028-6 URL

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