利用花药培养技术创制加工型辣椒抗疫病新种质
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摘要
为培育抗疫病加工型辣椒品种,本研究利用花药培养与分子标记辅助选择(molecular assisted selection,MAS)相结合的方法将抗疫病材料PPTC54和PI201234的抗性基因导入优良自交系939、812、83-3、968和花培双单倍体(doubled haploid, DH) H12-11,进行抗疫病新种质创制的研究。结果表明,在5个加工型辣椒基因型中以(PPTC54×83-3) F1诱导出的胚状体成苗率最高,可达87.1%;流式细胞仪测定36个花培再生株的DNA含量分布,其中单倍体株数占80.6%;经PCR检测,筛选出14个抗疫病DH系,且聚合抗疫病基因的各株系间农艺性状存在差异。本研究说明结合花药培养与MAS可快速创制特异育种新种质,加速育种进程。
In order to breeding processing type pepper varieties with Phytophthora blight resistance, diseaseresistant gene of PPTC54 and PI201234 was introduced to excellent inbred line 939, 812, 83-3, 968 and doubled haploid line H12-11 by combination of anther culture and marker assisted selection(MAS) technology for studies on the creation of new germplasm with Phytophthora blight resistance. The results showed that the seedling rate of embryoid induced by(PPTC54×83-3) F1 was the highest in five processing type pepper genotypes, which reached87.1%. The DNA content distribution of 36 regenerated plantlets was detected by FCM(flow cytometry), among which the haploid plants accounted for 80.6%. 14 plants resistant to Phytophthora capsici were screened through PCR detection, and there was difference among plant agronomic traits of each polymeric gene with resistance to Phytophthora capsici. This study indicated that anther culture and MAS might rapidly create new germplasm for specific breeding and accelerate the progress of breeding.
In order to breeding processing type pepper varieties with Phytophthora blight resistance, diseaseresistant gene of PPTC54 and PI201234 was introduced to excellent inbred line 939, 812, 83-3, 968 and doubled haploid line H12-11 by combination of anther culture and marker assisted selection(MAS) technology for studies on the creation of new germplasm with Phytophthora blight resistance. The results showed that the seedling rate of embryoid induced by(PPTC54×83-3) F1 was the highest in five processing type pepper genotypes, which reached87.1%. The DNA content distribution of 36 regenerated plantlets was detected by FCM(flow cytometry), among which the haploid plants accounted for 80.6%. 14 plants resistant to Phytophthora capsici were screened through PCR detection, and there was difference among plant agronomic traits of each polymeric gene with resistance to Phytophthora capsici. This study indicated that anther culture and MAS might rapidly create new germplasm for specific breeding and accelerate the progress of breeding.
引文
Chen X.,Zhan Y.S.,Xu X.L.,Qi W.Q.,Chang G.Z.,Zhi Y.B.,Yi M.L.,and Lv S.Y.,2003,Preliminary studies on establishing doubled haploid lines of pepper(Capsicum annuum L.)by anther culture,Henan Nongye Kexue(Journal of Henan A-gricultural Sciences),33(9):52-55(陈晓,詹玉丝,徐小利,齐卫强,常高正,郅玉宝,以明林,吕淑玉,2003,花药培养建立辣椒DH纯系的初步研究,河南农业科学,33(9):52-55)
Chen B.,Zhao H.,Geng S.S.,Zhang B.X.,Zhang Y.Y.,and Liu F.,2007,Studies on ploidy composing in regenerated plants from anther culture of pepper(Capsicum annuum L.),Huabei Nongxuebao(Acta Agriculture Boreali-Sinica),22(1):123-128(陈斌,赵泓,耿三省,张宝玺,张月云,刘凡,2007,辣椒花药培养再生株群体染色体倍性构成的多样性,华北农学报,22(1):123-128)
Chen B.,Geng S.S.,Zhao H.,Zhang X.F.,Zhang B.X.,and Liu F.,2010,Studies on the main agronomic traits and virus resistance of three DH population derived from anther culture of Capsicum annuum L.,Huabei Nongxuebao(Acta Agriculture Boreali-Sinica),25(1):231-238(陈斌,耿三省,赵泓,张晓芬,张宝玺,刘凡,2010,3个辣椒花药培养再生株群体主要农艺性状及苗期抗病性研究,华北农学报,25(1):231-238)
Guo Z.C.,Wang Y.Y.,Qian N.F.,Gu S.R.,Gong M.L.,and Xu H.J.,1973,Investigations on the anther culture in vitro of Nicotiana tabacum L.and Capsicum annuum L.,Zhiwu Xuebao(Acta Botanica Sinica),15(1):37-52(郭仲琛,王玉英,钱南芬,顾淑荣,龚明良,徐惠君,1973,菸草(Nicotiana tabacum)和辣椒(Capsicum annuum)花药离体培养的研究,植物学报,15(1):37-52)
Kim B.S.,and Hur J.M.,1991,Inheritance of resistance to bacterial spot and phytophthora blight in peppers,Journal of Korean Society for Horticultural Science,31:350-357
Li Y.F.,Zhang S.C.,Jiang X.Y.,Huang R.Z.,Lei K.R.,Huang Q.Z.,and Lin Q.,2017,The creation of processing type pepper cytoplasmic male sterility restoring line by anther culture,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2317-2321(李怡斐,张世才,蒋晓英,黄任中,雷开荣,黄启中,林清,2017,利用花药培养技术创制加工型辣椒细胞质雄性不育恢复系,分子植物育种,15(6):2317-2321)
Quirin E.A.,Ogundiwin E.A.,Prince J.P.,Mazourek M.O.,Briggs M.,Chlanda T.S.,Kim K.T.,Falise M.,and Kang B.C.,2005,Development of sequence characterized amplified region(SCAR)primers for the detection of Phyto.5.2,a major QTL for resistance to Phytophthora capsici Leon.in pepper,Theor.Appl.Genet.,110(4):605-612
Ristaino J.B.,and Johnston S.A.,1999,Ecologically based approaches to management of phytophthora blight on bell pepper,Plant Disease,83(12):1080-1089
Song D.D.,Yuan L.,Gao G.J.,and He Y.Q.,2011,Improving blast resistance and brown planthopper resistance of 29Bthrough MAS and anther culture,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),9(4):418-424(宋丁丁,袁丽,高冠军,何予卿,2011,利用花药培养和分子标记选择相结合改良水稻稻瘟病和褐飞虱抗性,分子植物育种,9(4):418-424)
Sun H.B.,Zou M.Z.,Ren H.Y.,Wang J.Y.,Yan S.Y.,Li Y.P.,and Feng R.G.,2015,Study of quick cultivation of new seeds with three diseases resistance by anther culture method,Huabei Nongxuebao(Acta Agriculture Boreali-Sinica),30(2):116-123(孙海波,邹美智,任洪岩,王景余,闫双勇,李艳萍,冯瑞光,2015,花药培养快速培育聚合抗3种水稻病害基因的新种质研究,华北农学报,30(2):116-123)
Yuan L.,Song D.D.,Gao G.J.,and He Y.Q.,2011,Improvement of resistance to rice blast in PGMS line Y58S by molecular marker-assisted selection and anther culture,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(5):620-625(袁丽,宋丁丁,高冠军,何予卿,2011,利用分子标记辅助选择和花药培养改良光温敏不育系Y58S的稻瘟病抗性,基因组学与应用生物学,30(5):620-625)
Chen B.,Zhao H.,Geng S.S.,Zhang B.X.,Zhang Y.Y.,and Liu F.,2007,Studies on ploidy composing in regenerated plants from anther culture of pepper(Capsicum annuum L.),Huabei Nongxuebao(Acta Agriculture Boreali-Sinica),22(1):123-128(陈斌,赵泓,耿三省,张宝玺,张月云,刘凡,2007,辣椒花药培养再生株群体染色体倍性构成的多样性,华北农学报,22(1):123-128)
Chen B.,Geng S.S.,Zhao H.,Zhang X.F.,Zhang B.X.,and Liu F.,2010,Studies on the main agronomic traits and virus resistance of three DH population derived from anther culture of Capsicum annuum L.,Huabei Nongxuebao(Acta Agriculture Boreali-Sinica),25(1):231-238(陈斌,耿三省,赵泓,张晓芬,张宝玺,刘凡,2010,3个辣椒花药培养再生株群体主要农艺性状及苗期抗病性研究,华北农学报,25(1):231-238)
Guo Z.C.,Wang Y.Y.,Qian N.F.,Gu S.R.,Gong M.L.,and Xu H.J.,1973,Investigations on the anther culture in vitro of Nicotiana tabacum L.and Capsicum annuum L.,Zhiwu Xuebao(Acta Botanica Sinica),15(1):37-52(郭仲琛,王玉英,钱南芬,顾淑荣,龚明良,徐惠君,1973,菸草(Nicotiana tabacum)和辣椒(Capsicum annuum)花药离体培养的研究,植物学报,15(1):37-52)
Kim B.S.,and Hur J.M.,1991,Inheritance of resistance to bacterial spot and phytophthora blight in peppers,Journal of Korean Society for Horticultural Science,31:350-357
Li Y.F.,Zhang S.C.,Jiang X.Y.,Huang R.Z.,Lei K.R.,Huang Q.Z.,and Lin Q.,2017,The creation of processing type pepper cytoplasmic male sterility restoring line by anther culture,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2317-2321(李怡斐,张世才,蒋晓英,黄任中,雷开荣,黄启中,林清,2017,利用花药培养技术创制加工型辣椒细胞质雄性不育恢复系,分子植物育种,15(6):2317-2321)
Quirin E.A.,Ogundiwin E.A.,Prince J.P.,Mazourek M.O.,Briggs M.,Chlanda T.S.,Kim K.T.,Falise M.,and Kang B.C.,2005,Development of sequence characterized amplified region(SCAR)primers for the detection of Phyto.5.2,a major QTL for resistance to Phytophthora capsici Leon.in pepper,Theor.Appl.Genet.,110(4):605-612
Ristaino J.B.,and Johnston S.A.,1999,Ecologically based approaches to management of phytophthora blight on bell pepper,Plant Disease,83(12):1080-1089
Song D.D.,Yuan L.,Gao G.J.,and He Y.Q.,2011,Improving blast resistance and brown planthopper resistance of 29Bthrough MAS and anther culture,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),9(4):418-424(宋丁丁,袁丽,高冠军,何予卿,2011,利用花药培养和分子标记选择相结合改良水稻稻瘟病和褐飞虱抗性,分子植物育种,9(4):418-424)
Sun H.B.,Zou M.Z.,Ren H.Y.,Wang J.Y.,Yan S.Y.,Li Y.P.,and Feng R.G.,2015,Study of quick cultivation of new seeds with three diseases resistance by anther culture method,Huabei Nongxuebao(Acta Agriculture Boreali-Sinica),30(2):116-123(孙海波,邹美智,任洪岩,王景余,闫双勇,李艳萍,冯瑞光,2015,花药培养快速培育聚合抗3种水稻病害基因的新种质研究,华北农学报,30(2):116-123)
Yuan L.,Song D.D.,Gao G.J.,and He Y.Q.,2011,Improvement of resistance to rice blast in PGMS line Y58S by molecular marker-assisted selection and anther culture,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(5):620-625(袁丽,宋丁丁,高冠军,何予卿,2011,利用分子标记辅助选择和花药培养改良光温敏不育系Y58S的稻瘟病抗性,基因组学与应用生物学,30(5):620-625)