利用分子标记鉴定白菜细胞质雄性不育类型的研究
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摘要
通过对9份白菜细胞质雄性不育材料的不育胞质类型进行分子标记鉴定,以期为今后更好的利用分子标记来进行辅助育种奠定基础。通过CTAB法提取白菜的基因组DNA,根据Gen Bank中orf138基因保守序列设计2对特异引物,对9份白菜基因组DNA进行PCR扩增,将扩增出的条带进行测序,在NCBI中进行同源性比对,最终确定其不育胞质类型。结果表明:2对特异引物PI/PII和PIII/PIV只在4份白菜雄性不育基因中均扩增出条带,在另外5份材料中未扩增出条带,与田间育性鉴定相符,获得4份白菜Ogura胞质雄性不育的分子标记;在Gen Bank中用BLAST进行同源性比对分析,发现L1-CI、L3-CI、L3-F1 3个不育材料的特异片段与已报道的青花菜Ogu CMS所具有的Ogu orf138基因(Genbank登录号:HQ149728)同源度高达100%,L1-F1的同源度达99%,出现了一个变异位点,另外5份材料的胞质不育类型有待进一步研究。通过该方法鉴定出9份材料中有4份材料是萝卜胞质雄性不育的材料,得到了4条分子标记。
Different sterile cytoplasm types of nine cabbage cytoplasmic male sterile materials were identifiedby molecular marker in the study in order to better use molecular marker to conduct the assisted breeding inthe future.Genomic DNA was isolated from Chinese cabbage by CTAB method.The design of two pairs ofspecific primers was performed on conserved flanking region of orf138 gene in the Gen Bank.PCR wereperformed with genomic DNA of nine Chinese cabbage materials.The bands were sequenced.The homologouscomparison was conducted in NCBI,and finally,the type of sterile cytoplasm was determined.The resultsshowed that the bands were amplified only in four Chinese cabbage male sterile materials with two pairs ofspecific primers PI/PII and PIII/PIV,while the other five materials did not obtain the relative bands.The resultwas consistent with the field sterility identification.And then four molecular markers of Chinese cabbageOgura cytoplasmic male sterility(CMS) were obtained.After conducting a homologous comparative analysis with BLAST in Gen Bank,it was found that the homologous degree was 100% in specific segments of the threesterility materials(L1-CI,L3-CI and L3-F1) and Ogu orf138 gene(Gen Bank accession No.:HQ149728) ofthe reported broccoli Ogu CMS.The homologous degree of L1-F1 was 99% with a variation point.The type ofcytoplasmic male sterility of the other five materials needed further research.Four materials of the nine wereidentified as the radish cytoplasmic male sterility materials and four molecular markers were obtained.
Different sterile cytoplasm types of nine cabbage cytoplasmic male sterile materials were identifiedby molecular marker in the study in order to better use molecular marker to conduct the assisted breeding inthe future.Genomic DNA was isolated from Chinese cabbage by CTAB method.The design of two pairs ofspecific primers was performed on conserved flanking region of orf138 gene in the Gen Bank.PCR wereperformed with genomic DNA of nine Chinese cabbage materials.The bands were sequenced.The homologouscomparison was conducted in NCBI,and finally,the type of sterile cytoplasm was determined.The resultsshowed that the bands were amplified only in four Chinese cabbage male sterile materials with two pairs ofspecific primers PI/PII and PIII/PIV,while the other five materials did not obtain the relative bands.The resultwas consistent with the field sterility identification.And then four molecular markers of Chinese cabbageOgura cytoplasmic male sterility(CMS) were obtained.After conducting a homologous comparative analysis with BLAST in Gen Bank,it was found that the homologous degree was 100% in specific segments of the threesterility materials(L1-CI,L3-CI and L3-F1) and Ogu orf138 gene(Gen Bank accession No.:HQ149728) ofthe reported broccoli Ogu CMS.The homologous degree of L1-F1 was 99% with a variation point.The type ofcytoplasmic male sterility of the other five materials needed further research.Four materials of the nine wereidentified as the radish cytoplasmic male sterility materials and four molecular markers were obtained.
引文
[1]卢玉华,蔬菜栽培学各论[M].北京:中国农业出版社,2000:175.
[2]张德双,徐家炳,曹鸣庆,等.大白菜转育新型甘蓝型油菜细胞质雄性不育系的研究[J].华北农学报,2002,17(1):60-63.
[3]刘玉梅,方智远,孙培田,等.十字花科作物雄性不育性获得的主要途径及其利用[J].中国蔬菜,2002(2):52-55.
[4]施展,万正杰,徐跃进,等.大白菜hau胞质雄性不育系的鉴定及不育相关基因结构分析[J].园艺学报,2012,39(3):469-476.
[5]Zhang L,Yasumoto K,Yamagishi H.Identification of cytoplasmic male sterility in Chinese radish following PCR analysis of mitochondrial DNA[J].Plant Mol Biol Rep,2012,30:817-825.
[6]励启腾,孟平红,吴跃勇.大白菜细胞质雄性不育系的选育[J].贵州农业科学,1995(3):1-4.
[7]柯桂兰,赵稚雅,宋胭脂,等.大白菜异源胞质雄性不育系CMS3411-7的选育及应用[J].园艺学报,1992,19(4):333-340.
[8]王学芳,张彦锋,李殿荣,等.不结球白菜异源胞质不育系及其优良组合的选育[J].安徽农业科学,2004,32(1):87-88.
[9]刘惠吉,王华.同源四倍体白菜异源胞质雄性不育系的选育[J].南京农业大学学报,2004,27(23):30-33.
[10]Ogura H,Studies on the new male-sterility in Japanese radish with special reference to the utilization of this sterility towards the practical raising of hybrid seeds[J].Memoirs of the Faculty of Agriculture,1968,6(2):39-78.
[11]侯喜林,曹寿椿,吴志行.十字花科作物几种主要细胞质雄性不育类型及其利用(上)[J].长江蔬菜,1999(5):1-3.
[12]李桂花,张衍荣,曹健,等.雄性不育在十字花科蔬菜中的研究进展[J].江西农业大学学报,2003,25(2):181-186.
[13]张明龙,章徐鸯,林宝刚,等.国内外油菜细胞质雄性不育(CMS)系统的研究进展[J].黑龙江农业科学,2004(1):36-38.
[14]Williams P H,Heyn F W.The origin and development of cytoplasmic male sterile Chinese cabbage[A].Chinese Cabbage,Proceeding of the first international symposium[C].Taiwan:Asian Vegetable Research and Development Center,1981:293-300.
[15]Robertson D,Earle J D P,Mutschler M A.Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B.campestris[J].Theoretical and Applied Genetics,1987,74(3):303-309.
[16]朱玉英,姚文岳,张素琴,等.Ogura细胞质甘蓝雄性不育系选育及其利用[J].上海农业学报,1988,14(2):19-24.
[17]傅廷栋,杨光圣,杨小牛,等.甘蓝型油菜波里马细胞质雄性不育的发现、研究与利用[J].自然科学进展:国家重点实验室通讯,1995,5(3):287-293.
[18]张德双,张凤兰,徐家炳.大白菜CMS96细胞质雄性不育系的特点分析[J].华北农学报,2005,20(1):59-62.
[19]王国芳,张鲁刚,李冬霞.白菜细胞质雄性不育的恢保关系与分子鉴定[J].西北农林科技大学学报:自然科学版,2014,42(7):183-190.
[20]吉立柱,孙德岭,孙立全.十字花科细胞质雄性不育的类型及机理[J].江苏农业科学,2007(3):106-110.
[21]Bonhomme S,Budar F,Lancelin D,et al.Sequence and transcript analysis of the Nco 2.5 Ogura-specific fragment correlation with cyto-plasmic male sterility in Brassica hybrids[J].Mol Gen Genet,1992,235:340-348.
[22]Liu Z Y,Peter S O,Long M H,et al.A PCR assay for rapid discrimination of sterile cytoplasm types in maize[J].Crop Science,2002,42:566-569.
[23]荆赞革,裴徐梨,唐征,等.青花菜Ogu不育胞质分子鉴定和序列分析[J].广西植物,2015(02):231-235-294.
[24]许小勇,孙希禄,方智远,等.大白菜CMS雄性不育发生相关基因的克隆与分析[J].西北农林科技大学学报:自然科学版,2014(12):119-125.
[25]Krishnasamy S,Makaroff C A.Characterization of the radish mitochondrial orf B locus possible relationship with male sterility in Ogura radish[J].Curr Genet,1993,24(1-2):156-163.
[26]Yamagishi H,Terachi T.Intra-and inter-specific variations in the mitochondrial gene orf138 of Ogura-type male-sterile cyto-plasm from Raphanus sativus and R.raphanistrum[J].Theor Appl Genet,2001,103(5):725-732.
[27]Grelon M,Budar F,Bonhomme S,et al.Ogura cytoplasmic malesterility(CMS)-associated orf138 is translated into a mito-chondrial membrane polypeptide in male-sterile Brassica cybrids[J].Mol Gen Genet,1994,243(5):540-547.
[28]Hiesel R,Haeseler A V,Brennicke A.Plant mitochondrial nucleic acid sequences as a tool for phylogenetic analysis[J].Proc Natl Acad Sci,1994,91(2):634-638.
[2]张德双,徐家炳,曹鸣庆,等.大白菜转育新型甘蓝型油菜细胞质雄性不育系的研究[J].华北农学报,2002,17(1):60-63.
[3]刘玉梅,方智远,孙培田,等.十字花科作物雄性不育性获得的主要途径及其利用[J].中国蔬菜,2002(2):52-55.
[4]施展,万正杰,徐跃进,等.大白菜hau胞质雄性不育系的鉴定及不育相关基因结构分析[J].园艺学报,2012,39(3):469-476.
[5]Zhang L,Yasumoto K,Yamagishi H.Identification of cytoplasmic male sterility in Chinese radish following PCR analysis of mitochondrial DNA[J].Plant Mol Biol Rep,2012,30:817-825.
[6]励启腾,孟平红,吴跃勇.大白菜细胞质雄性不育系的选育[J].贵州农业科学,1995(3):1-4.
[7]柯桂兰,赵稚雅,宋胭脂,等.大白菜异源胞质雄性不育系CMS3411-7的选育及应用[J].园艺学报,1992,19(4):333-340.
[8]王学芳,张彦锋,李殿荣,等.不结球白菜异源胞质不育系及其优良组合的选育[J].安徽农业科学,2004,32(1):87-88.
[9]刘惠吉,王华.同源四倍体白菜异源胞质雄性不育系的选育[J].南京农业大学学报,2004,27(23):30-33.
[10]Ogura H,Studies on the new male-sterility in Japanese radish with special reference to the utilization of this sterility towards the practical raising of hybrid seeds[J].Memoirs of the Faculty of Agriculture,1968,6(2):39-78.
[11]侯喜林,曹寿椿,吴志行.十字花科作物几种主要细胞质雄性不育类型及其利用(上)[J].长江蔬菜,1999(5):1-3.
[12]李桂花,张衍荣,曹健,等.雄性不育在十字花科蔬菜中的研究进展[J].江西农业大学学报,2003,25(2):181-186.
[13]张明龙,章徐鸯,林宝刚,等.国内外油菜细胞质雄性不育(CMS)系统的研究进展[J].黑龙江农业科学,2004(1):36-38.
[14]Williams P H,Heyn F W.The origin and development of cytoplasmic male sterile Chinese cabbage[A].Chinese Cabbage,Proceeding of the first international symposium[C].Taiwan:Asian Vegetable Research and Development Center,1981:293-300.
[15]Robertson D,Earle J D P,Mutschler M A.Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B.campestris[J].Theoretical and Applied Genetics,1987,74(3):303-309.
[16]朱玉英,姚文岳,张素琴,等.Ogura细胞质甘蓝雄性不育系选育及其利用[J].上海农业学报,1988,14(2):19-24.
[17]傅廷栋,杨光圣,杨小牛,等.甘蓝型油菜波里马细胞质雄性不育的发现、研究与利用[J].自然科学进展:国家重点实验室通讯,1995,5(3):287-293.
[18]张德双,张凤兰,徐家炳.大白菜CMS96细胞质雄性不育系的特点分析[J].华北农学报,2005,20(1):59-62.
[19]王国芳,张鲁刚,李冬霞.白菜细胞质雄性不育的恢保关系与分子鉴定[J].西北农林科技大学学报:自然科学版,2014,42(7):183-190.
[20]吉立柱,孙德岭,孙立全.十字花科细胞质雄性不育的类型及机理[J].江苏农业科学,2007(3):106-110.
[21]Bonhomme S,Budar F,Lancelin D,et al.Sequence and transcript analysis of the Nco 2.5 Ogura-specific fragment correlation with cyto-plasmic male sterility in Brassica hybrids[J].Mol Gen Genet,1992,235:340-348.
[22]Liu Z Y,Peter S O,Long M H,et al.A PCR assay for rapid discrimination of sterile cytoplasm types in maize[J].Crop Science,2002,42:566-569.
[23]荆赞革,裴徐梨,唐征,等.青花菜Ogu不育胞质分子鉴定和序列分析[J].广西植物,2015(02):231-235-294.
[24]许小勇,孙希禄,方智远,等.大白菜CMS雄性不育发生相关基因的克隆与分析[J].西北农林科技大学学报:自然科学版,2014(12):119-125.
[25]Krishnasamy S,Makaroff C A.Characterization of the radish mitochondrial orf B locus possible relationship with male sterility in Ogura radish[J].Curr Genet,1993,24(1-2):156-163.
[26]Yamagishi H,Terachi T.Intra-and inter-specific variations in the mitochondrial gene orf138 of Ogura-type male-sterile cyto-plasm from Raphanus sativus and R.raphanistrum[J].Theor Appl Genet,2001,103(5):725-732.
[27]Grelon M,Budar F,Bonhomme S,et al.Ogura cytoplasmic malesterility(CMS)-associated orf138 is translated into a mito-chondrial membrane polypeptide in male-sterile Brassica cybrids[J].Mol Gen Genet,1994,243(5):540-547.
[28]Hiesel R,Haeseler A V,Brennicke A.Plant mitochondrial nucleic acid sequences as a tool for phylogenetic analysis[J].Proc Natl Acad Sci,1994,91(2):634-638.
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