河北省130份西瓜品种与种质资源抗病基因KASP检测分析
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摘要
旨在筛选河北地区抗枯萎病、炭疽病、白粉病西瓜品种与资源,分析其所含有的抗病基因,为筛选和培育抗病西瓜新品种提供参考。采用改良后的CTAB法提取西瓜种芽DNA,并用超微量分光光度计检测DNA浓度,将其稀释至2～10 ng/μL后使用许勇团队开发的西瓜抗枯萎病、抗炭疽病、抗白粉病分子标记,利用高通量分型KASP标记技术对西瓜抗枯萎病、抗炭疽病和抗白粉病性状相关的基因进行了检测。试验材料为河北省近年来审(鉴)定西瓜品种和优势组合(星研七号、美佳、美胜、贵妃、17-11等)以及优异种质资源(花早绿、JB-1、JB-3、901新等)共130份材料,抗病对照为3抗302,感病对照为GBZG。结果表明,所有被检测材料中33份材料含抗枯萎病基因(Fon-1),19份材料含抗炭疽病基因(AR1),7份材料含抗白粉病基因(PM1),5份材料同时含Fon-1和AR1,1份材料同时含AR1和PM1,1份材料同时含Fon-1、AR1、PM1 3种抗病基因。根据KASP检测结果对试验材料进行聚类分析,将试验材料分为4类,分别为抗白粉病基因处均无检测信号或杂合抗性12份、抗白粉病或同时感2种病害材料67份、抗枯萎病或抗炭疽病材料38份,抗枯萎病、抗炭疽病或抗白粉病基因方面均无检测信号或杂合抗性13份。
This study aimed to screen the watermelon varieties and germplasms resistant to fusarium wilt, anthracnose and powdery mildew in Hebei Province, and analyze the related resistance genes, which could provide a reference for breeding disease-resistant varieties. The modified CTAB method was used to extract the DNA from watermelon bud, and the DNA concentration was determined by Ultramicro spectrophotometer. After the DNA was diluted to 2-10 ng/μL, they were detected by KASP. The molecular markers of resistance to fusarium wilt, anthracnose and powdery mildew of watermelon were developed by XU Yong's team. The resistant genes related to fusarium wilt, anthracnose and powdery mildew of watermelon were identified by high-throughput KASP markers. Totally 130 watermelon materials were tested, including varieties or advantaging combinations(Xingyan No.7, Meijia, Meisheng, Guifei, 17-11 and so on) that have been approved or will be approved in Hebei Province in recent years, and some excellent germplasms(Huazaolü,JB-1,JB-3,901 xin and so on). 3 Kang302 was used as the disease-resistant control and GBZG as the susceptible control. The result showed that 33 materials contained fusarium wilt resistance gene Fon-1, 19 contained anthracnose resistance gene AR1, 7 contained powdery mildew resistance gene PM1, 5 contained Fon-1 and AR1, 1 contained AR1 and PM1, and 1 contained all three resistance genes(Fon-1, AR1 and PM1). According to the result of KASP, the materials were clustered, and divided into 4 categories: 12 materials resistant to powdery mildew without detection signal or with heterozygous resistance, 67 resistant to powdery mildew or susceptible to at least two diseases, 38 resistant to fusarium wilt or anthracnose, and 13 resistant to fusarium wilt or anthracnose or powdery mildew without detection signal or with heterozygous resistance.
This study aimed to screen the watermelon varieties and germplasms resistant to fusarium wilt, anthracnose and powdery mildew in Hebei Province, and analyze the related resistance genes, which could provide a reference for breeding disease-resistant varieties. The modified CTAB method was used to extract the DNA from watermelon bud, and the DNA concentration was determined by Ultramicro spectrophotometer. After the DNA was diluted to 2-10 ng/μL, they were detected by KASP. The molecular markers of resistance to fusarium wilt, anthracnose and powdery mildew of watermelon were developed by XU Yong's team. The resistant genes related to fusarium wilt, anthracnose and powdery mildew of watermelon were identified by high-throughput KASP markers. Totally 130 watermelon materials were tested, including varieties or advantaging combinations(Xingyan No.7, Meijia, Meisheng, Guifei, 17-11 and so on) that have been approved or will be approved in Hebei Province in recent years, and some excellent germplasms(Huazaolü,JB-1,JB-3,901 xin and so on). 3 Kang302 was used as the disease-resistant control and GBZG as the susceptible control. The result showed that 33 materials contained fusarium wilt resistance gene Fon-1, 19 contained anthracnose resistance gene AR1, 7 contained powdery mildew resistance gene PM1, 5 contained Fon-1 and AR1, 1 contained AR1 and PM1, and 1 contained all three resistance genes(Fon-1, AR1 and PM1). According to the result of KASP, the materials were clustered, and divided into 4 categories: 12 materials resistant to powdery mildew without detection signal or with heterozygous resistance, 67 resistant to powdery mildew or susceptible to at least two diseases, 38 resistant to fusarium wilt or anthracnose, and 13 resistant to fusarium wilt or anthracnose or powdery mildew without detection signal or with heterozygous resistance.
引文
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[2] 张敬敬,武彦荣,高秀瑞,李冰,潘秀清.河北省西瓜甜瓜产业现状及发展趋势[J].中国瓜菜,2016,29(12):55-57.doi:10.16861/j.cnki.zggc.20161222.014.Zhang J J,Wu Y R,Gao X R,Li B,Pan X Q.Hebei watermelon melon industry present situation and development trend[J].China Cucurbits and Vegetables,2016,29(12):55-57.
[3] 黎裕,王建康,邱丽娟,马有志,李新海,万建民.中国作物分子育种现状与发展前景[J].作物学报,2010,36(9):1425-1430.doi:10.3724/SP.J.1006.2010.01425.Li Y,Wang J K,Qiu L J,Ma Y Z,Li X H,Wan J M.Crop molecular breeding in china:current status and perspectives[J].Acta Agronomica Sinica,2010,36(9):1425-1430.
[4] 邹景伟,贾万利,李立鑫,陈旭,贾丹,闫长生,张秀英,肖世和,孙果忠.120份小麦品种(系)重要性状功能基因的KASP标记检测[J].分子植物育种,2018-08-28.Zou J W,Jia W L,Li L X,Chen X,Jia D,Yan C S,Zhang X Y,Xiao S H,Sun G Z.KASP assays for functional genes of important trait in 120 wheat cultivars (lines)[J].Molecular Plant Breeding,2018-08-28.
[5] 赵勇,刘晓东,赵洪锟,袁翠平,齐广勋,王玉民,董英山.大豆SNP分型方法的比较[J].分子植物育种,2017,15(9):3540-3546.doi:10.13271/j.mpb.015.003540.Zhao Y,Liu X D,Zhao H K,Yuan C P,Qi G X,Wang Y M,Dong Y S.Comparison of methods for SNP genotyping in soybean[J].Molecular Plant Breeding,2017,15(9):3540-3546.doi:10.13271/j.mpb.015.003540.
[6] 杨子博,顾正中,周羊梅,王安邦,高平中,熊正海,刘畅,蒋学祥,沈业松.江苏淮北地区小麦品种资源籽粒硬度基因等位变异的KASP检测[J].麦类作物学报,2017,37(2):153-161.doi:10.7606/j.issn.1009-1041.2017.02.02.Yang Z B,Gu Z Z,Zhou Y M,Wang A B,Gao P Z,Xiong Z H,Liu C,Jiang X X,Shen Y S.Detection of allelic variation for grain hardness in Huaibei region of Jiangsu province by KASP markers[J].Journal of Triticeae Crops,2017,37(2):153-161.
[7] He C,Holme J,Anthony J.SNP genotyping:the KASPassay[J].Methods Mol Biol,2014,1145 :75-86.
[8] Graves H,Rayburn A L,Gonzalez-Hernandez J L,Nah G,Kim D S,Lee D K.Validating DNA polymorphisms using KASP assay in prairie cordgrass(Spartina pectinata Link)Populations in the U.S[J].Front Plant Sci,2015,6 :1271.doi:10.3389/fpls.2015.01271.
[9] 姬万丽,朱红菊,赵胜杰,路绪强,何楠,刘文革.四倍体与二倍体西瓜苗期接种枯萎病菌生理小种 1 的抗性差异[J].中国瓜菜,2018,31(6):7-10.doi:10.16861/j.cnki.zggc.2018.0103.Ji W L,Zhu H J,Zhao S J,Lu X Q,He N,Liu W G.The difference of disease resistance in tetraploid and diploid watermelon seedlings after inoculated by Fusarium oxysporum f.sp.niveum race 1[J].China Cucurbits and Vegetables,2018,31(6):7-10.
[10] 崔召明,陈泉生.西瓜炭疽病苗期不同抗性鉴定方法的研究[J].安徽农学通报,2009,15(15):135-136.doi:10.16377/j.cnki.issn1007-7731.2009.15.087.Cui Z M,Chen Q S.Study on different identification methods of anthracnose resistance of watermelon at seeding stage[J].Anhui Agricultural Science Bulletin,2009,15(15):135-136.
[11] 徐向丽,宫国义,郭绍贵,许勇,卢向阳,彭振兴,张海英.西瓜对白粉病的抗性鉴定及遗传分析[J].华北农学报,2008,23(4):37-40.doi:10.7668/hbnxb.2008.04.009.Xu X L,Gong G Y,Guo S G,Xu Y,Lu X Y,Peng Z X,Zhang H Y.Identfication and inheritance of resistance to powdery mildew in watermelon[J].Acta Agriculturae Boreali-Sinica,2008,23(4):37-40.
[12] 曹月霞,凌健,谢丙炎,杨宇红.西瓜枯萎病菌的快速检测与鉴定[J].生物技术通报,2015,31(7):58-63.doi:10.13560/j.cnki.biotech.bull.1985.2015.07.009.Cao Y X,Ling J,Xie B Y,Yang Y H.Rapid detection and identification of Fusarium oxysporum f.sp.niveum[J].Biotechnology Bulletin,2015,31(7):58-63.
[13] 唐建辉,王伟,王源超.西瓜炭疽病菌Colletotrichum orbiculare的分子检测[J].中国农业科学,2006,39(10):2028-2035.doi:10.3321/j.issn:0578-1752.2006.10.012.Tang J H,Wang W,Wang Y C.Molecular detection of Colletotrichum orbiculare[J].Scientia Agricultura Sinica,2006,39(10):2028-2035.
[14] 刘易科,朱展望,佟汉文,宁富豪,冯承官,陈泠,邹娟,张宇庆,杨立军,高春保.湖北省主要小麦品种抗病基因分析[J].分子植物育种,2018,16(4):1040-1049.doi:10.13271/j.mpb.016.001040.Liu Y K,Zhu Z W,Tong H W,Ning F H,Feng C G,Chen L,Zou J,Zhang Y Q,Yang L J,Gao C B.Analysis of the resistance genes in the main wheat varieties in Hubei province[J].Molecular Plant Breeding,2018,16(4):1040-1049.
[15] Lv G Y,Guo S G,Zhang H Y,Geng L H,Raymond D Martyn,Xu Y.Colonization of fusarium wilt-resistant and susceptible watermelon roots by a green-fluorescent-protein-taggedisolate of Fusarium oxysporum f.sp.niveum[J].Journal of Phytopathology,2014:228-237.doi:10.1111/jph.12174.
[16] Zhang C Q,Ren Y,Guo S G,Zhang H Y,Gong G Y,Du Y C,Xu Y.Application of comparative genomics in developing markers tightly linked to the Pm-2F gene for powdery mildew resistance in melon (Cucumis melo L.)[J].Euphytica,2013,190(2):157-168.doi:10.1007/s10681-012-0828-4.
[17] 徐平丽,唐桂英,付春,柳展基,鲁成凯,姜言生,单雷.高通量分子标记技术辅助回交选育高油酸花生新种质[J].山东农业科学,2018,50(6):46-51,64.doi:10.14083/j.issn.1001-4942.2018.06.007.Xu P L,Tang G Y,Fu C,Liu Z J,Lu C K,Jiang Y S,Shan L.Breeding of new peanut germplasm with high oleic acid by successive backcross and high-throughput marker assisted selection[J].Shandong Agricultural Sciences,2018,50(6):46-51,64.
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