华山新麦草3Ns染色体特异SCAR标记的开发
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摘要
为快速准确地建立小麦-华山新麦草杂交后代的分子标记鉴定方法,采用150条10bp的随机引物R1-R150对华山新麦草、普通小麦7182、中国春及华山新麦草1Ns-7Ns全套二体附加系共10个材料进行了RAPD分析,从中获得了华山新麦草3Ns染色体上2个特异RAPD分子标记片段FR-113和FR-125。克隆这两个片段并测序,发现其全长分别为543bp和515bp;分析序列后对其分别设计SCAR引物S-113和S-125,重新对10个材料进行SCAR分析,结果只在华山新麦草和小麦-华山新麦草3Ns二体附加系上扩增出了特异条带,由此证明已将华山新麦草3Ns染色体上获得的两个RAPD标记成功转化为稳定可靠的SCAR标记。这两个SCAR标记可用于检测小麦背景下的华山新麦草3Ns染色体,同时为小麦-华山新麦草杂交后代提供了分子鉴定依据。
To establish an effective way for rapid identification of wheat-Psathyrostachys huashanica Keng hybrids by molecular markers,150 random primers R1-R150(10bp)were used to perform RAPD analysis on Psathyrostachys huashanica Keng,common wheat 7182,Chinese Spring and 1Ns-7Ns addition lines of Psathyrostachys huashanica Keng.Among the 150 primers,R-113 and R-125 were confirmed to amplify a specific DNA fragment from Psathyrostachys huashanica Keng and its3 Ns addition line,respectively.The amplified fragments by R-113 and R-125 were recovered,cloned and sequenced.Based on the sequences,two pairs of SCAR primers,S-113 and S-125,were designed to perform SCAR analysis on the 10 tested materials.The result showed that the same bands could be amplified only from Psathyrostachys huashanica Keng and its 3Ns addition line,indicating that the RAPD markers R-113 and R-125 had been successfully converted into the reliable SCAR markers S-113 and S-125.These two SCAR markers can be used for detecting the 3Ns chromosome of Psathyrostachys huashanica Keng under common wheat background,which provided a scientific basis for molecular identification of wheat-Psathyrostachys huashanica Keng hybrids.
To establish an effective way for rapid identification of wheat-Psathyrostachys huashanica Keng hybrids by molecular markers,150 random primers R1-R150(10bp)were used to perform RAPD analysis on Psathyrostachys huashanica Keng,common wheat 7182,Chinese Spring and 1Ns-7Ns addition lines of Psathyrostachys huashanica Keng.Among the 150 primers,R-113 and R-125 were confirmed to amplify a specific DNA fragment from Psathyrostachys huashanica Keng and its3 Ns addition line,respectively.The amplified fragments by R-113 and R-125 were recovered,cloned and sequenced.Based on the sequences,two pairs of SCAR primers,S-113 and S-125,were designed to perform SCAR analysis on the 10 tested materials.The result showed that the same bands could be amplified only from Psathyrostachys huashanica Keng and its 3Ns addition line,indicating that the RAPD markers R-113 and R-125 had been successfully converted into the reliable SCAR markers S-113 and S-125.These two SCAR markers can be used for detecting the 3Ns chromosome of Psathyrostachys huashanica Keng under common wheat background,which provided a scientific basis for molecular identification of wheat-Psathyrostachys huashanica Keng hybrids.
引文
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[5]Du Y N(杜雅楠),Chen X H(陈新宏),Zhao J X(赵继新),et al.Analysis of drought resistance of Triiticum-Psathyrostachys deritives at germination and seedling stages[J].Journal of Triticeae Crops(麦类作物学报),2010,30(4):670-675(in Chinese with English abstract).
[6]Chen S Y(陈漱阳),Hou W S(侯文胜),Zhang A J(张安静),et al.Breeding and cytogenetic study of Triticum aestivumPsathyrostachys huashanica alien addition lines[J].Acta Genetica Sinica(遗传学报),1996,23(6):447-452(in Chinese with English abstract).
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[10]Su J N(苏佳妮),Wu J(武军),Zhao J X(赵继新),et al.Identification of a translocation line 24-6from offsprings between wheat-Psathyrostachys huashanica[J].Journal of Triticeae Crops(麦类作物学报),2014,35(5):615-620(in Chinese with English abstract).
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[12]Ding G H(丁国华),Qin Z W(秦智伟),Zhou X Y(周秀艳),et al.A novel RAPD and SCAR marker of the resistant gene for downy mildew(dm)in cucumber[J].Acta Botanica Boreali-Occident Sinica(西北植物学报),2007,27(9):1747-1751(in Chinese with English abstract).
[13]Jia J Q(贾举庆),Lei M P(雷孟平),Liu C(刘成),et al.Exploitation and application of a new SCAR marker linked to strip rust resistance gene Yr17in wheat[J].Journal of Triticeae Crops(麦类作物学报),2010,30(1):11-16(in Chinese with English abstract).
[14]Meng F J(孟凡娟),Xu X Y(许向阳),Li J F(李景富),et al.RAPD and SCAR markers linked to Fulvia fulva resistant gene Cf-6in tomato[J].Scientia Agricultura Sinica(中国农业科学),2008,41(7):2191-2196(in Chinese with English abstract).
[15]Gan Y(干滟),Zeng F Y(曾凡亚),Zhao Y(赵云),et al.Development of SCAR markers to authenticate the hybrid seed lots of Shuza No.6(Brassica napus L.)[J].Acta Agronomica Sinica(作物学报),2001,27(6):721-728(in Chinese with English abstract).
[16]You M S(尤明山),Li B Y(李保云),Tang Z H(唐朝晖),et al.Establishment of E-genome specific RAPD and SCAR markers for Thinopyrumspp.[J].Journal of China Agricultural University(中国农业大学学报),2002,7(5):1-6(in Chinese with English abstract).
[17]Dai J(戴剑),Hong D L(洪德林),Zhang D D(张大栋),et al.Study on a rapid,high efficient DNA extraction method[J].Journal of Triticeae Crops(麦类作物学报),2011,31(3):437-442(in Chinese with English abstract).
[18]Ding C B(丁春邦),Zhou Y H(周永红).Fertilization and embryo development in hybridization between wheat and Psathyrostachys huashanica[J].Journal of Sichuan Agricultural University(四川农业大学学报),1997,15(1):18-20(in Chinese with English abstract).
[19]Liu C(刘成),Yang Z J(杨足君),Liu C(刘畅).Analysis of St-chromosome-containing triticeae polyploids using specific molecular markers[J].Hereditas(遗传),2007,29(10):1271-1279(in Chinese with English abstract).
[20]Yan H F(闫红飞),Liu C Y(刘春燕),Gao S G(高士刚),et al.A SCAR marker for leaf rust resistance gene Lr45[J].Scientia Agricultura Sinica(中国农业科学),2009,42(1):124-129(in Chinese with English abstract).
[21]Liu C(刘成),Li G R(李光蓉),Yang Z J(杨足君),et al.Specific DNA band isolation and SCAR marker construction of rye genome[J].Acta Botanica Boreali-Occident Sinica(西北植物学报),2006,26(12):2434-2438(in Chinese with English abstract).
[22]Meng Y X(孟亚雄),Zhong J(仲军),Ma X L(马小乐),et al.RAPD analysis of Puccinia striiformisf.sp tritici in Gansu and SCAR marker establishment for Su11-14[J].Acta Pratac Sinica(草业学报)2010,19(6):180-186(in Chinese with English abstract).
[23]Qi X L(亓晓莉),Peng D L(彭德良),Peng H(彭焕),et al.Rapid molecular diagnosis based on SCAR marker system for cereal cyst nematode[J].Scientia Agricultura Sinica(中国农业科学),2012,45(21):4388-4395(in Chinese with English abstract).
[24]Zhao J Y(赵静媛),Chen S M(陈素梅),Chen F D(陈发棣).Conversion of RAPD marker linked to creeping plant type in ground-cover chrysanthemum to SCAR marker[J].Scientia Silvae Sinicae(林业科学),2009,45(9):147-150(in Chinese with English abstract).
[2]Wan Y F(万永芳),Yan J(颜济),Yang J L(杨俊良),et al.Study on wild relatives of wheat for resistance to head scab[J].Acta Phytopathologica Sinica(植物病理学报),1997,27(2):107-111(in Chinese with English abstract).
[3]Jing J X(井金学),Fu J(傅杰),Yuan H X(袁红旭),et al.A preliminary study on heredity of the resistance to stripe rust in three wild relatives of wheat[J].Acta Phytopathologica Sinica(植物病理学报),1999,29(2):147-150(in Chinese with English abstract).
[4]Wang M N(王美南),Shang H S(商鸿生).Evaluation of resistance in Psathyrostachys huashaica to wheat take-all fungus[J].Journal of Northwest A&F University(西北农业大学学报),2000,28(6):69-71(in Chinese with English abstract).
[5]Du Y N(杜雅楠),Chen X H(陈新宏),Zhao J X(赵继新),et al.Analysis of drought resistance of Triiticum-Psathyrostachys deritives at germination and seedling stages[J].Journal of Triticeae Crops(麦类作物学报),2010,30(4):670-675(in Chinese with English abstract).
[6]Chen S Y(陈漱阳),Hou W S(侯文胜),Zhang A J(张安静),et al.Breeding and cytogenetic study of Triticum aestivumPsathyrostachys huashanica alien addition lines[J].Acta Genetica Sinica(遗传学报),1996,23(6):447-452(in Chinese with English abstract).
[7]Hou W S(侯文胜),Zhang A J(张安静),Yang Q H(杨群慧),et al.Breeding and cytogenetic study of Triticum aestivumand Psathyrostachys huashanica alien substitution lines[J].Acta Botanica Boreali-Occident Sinica(西北植物学报),1997,17(3):368-373(in Chinese with English abstract).
[8]Zhao J X(赵继新),Chen X H(陈新宏),Wang X L(王小利),et al.C-banding identification of alien substitution lines and alien additional lines in Triticum-Psathyrostachys[J].Journal of Northwest A&F University:Natural Science Edition(西北农林科技大学学报:自然科学版),2003,31(6):1-4(in Chinese with English abstract).
[9]Wu J(武军),Zhao J X(赵继新),Chen X H(陈新宏),et al.Cytology characteristic and GISH analysis on the progenies derived from common wheat(T.aestivum L.)×Psathyrostachys huashanica[J].Journal of Triticeae Crops(麦类作物学报),2007,27(5):772-775(in Chinese with English abstract).
[10]Su J N(苏佳妮),Wu J(武军),Zhao J X(赵继新),et al.Identification of a translocation line 24-6from offsprings between wheat-Psathyrostachys huashanica[J].Journal of Triticeae Crops(麦类作物学报),2014,35(5):615-620(in Chinese with English abstract).
[11]Williams J G,Kubekik A R,Livak K J,et al.DNA polymorphisms amplified by arbitrary primers are useful as genetic markers[J].Nucleic Acids Research,1990,18(22):6531-6535.
[12]Ding G H(丁国华),Qin Z W(秦智伟),Zhou X Y(周秀艳),et al.A novel RAPD and SCAR marker of the resistant gene for downy mildew(dm)in cucumber[J].Acta Botanica Boreali-Occident Sinica(西北植物学报),2007,27(9):1747-1751(in Chinese with English abstract).
[13]Jia J Q(贾举庆),Lei M P(雷孟平),Liu C(刘成),et al.Exploitation and application of a new SCAR marker linked to strip rust resistance gene Yr17in wheat[J].Journal of Triticeae Crops(麦类作物学报),2010,30(1):11-16(in Chinese with English abstract).
[14]Meng F J(孟凡娟),Xu X Y(许向阳),Li J F(李景富),et al.RAPD and SCAR markers linked to Fulvia fulva resistant gene Cf-6in tomato[J].Scientia Agricultura Sinica(中国农业科学),2008,41(7):2191-2196(in Chinese with English abstract).
[15]Gan Y(干滟),Zeng F Y(曾凡亚),Zhao Y(赵云),et al.Development of SCAR markers to authenticate the hybrid seed lots of Shuza No.6(Brassica napus L.)[J].Acta Agronomica Sinica(作物学报),2001,27(6):721-728(in Chinese with English abstract).
[16]You M S(尤明山),Li B Y(李保云),Tang Z H(唐朝晖),et al.Establishment of E-genome specific RAPD and SCAR markers for Thinopyrumspp.[J].Journal of China Agricultural University(中国农业大学学报),2002,7(5):1-6(in Chinese with English abstract).
[17]Dai J(戴剑),Hong D L(洪德林),Zhang D D(张大栋),et al.Study on a rapid,high efficient DNA extraction method[J].Journal of Triticeae Crops(麦类作物学报),2011,31(3):437-442(in Chinese with English abstract).
[18]Ding C B(丁春邦),Zhou Y H(周永红).Fertilization and embryo development in hybridization between wheat and Psathyrostachys huashanica[J].Journal of Sichuan Agricultural University(四川农业大学学报),1997,15(1):18-20(in Chinese with English abstract).
[19]Liu C(刘成),Yang Z J(杨足君),Liu C(刘畅).Analysis of St-chromosome-containing triticeae polyploids using specific molecular markers[J].Hereditas(遗传),2007,29(10):1271-1279(in Chinese with English abstract).
[20]Yan H F(闫红飞),Liu C Y(刘春燕),Gao S G(高士刚),et al.A SCAR marker for leaf rust resistance gene Lr45[J].Scientia Agricultura Sinica(中国农业科学),2009,42(1):124-129(in Chinese with English abstract).
[21]Liu C(刘成),Li G R(李光蓉),Yang Z J(杨足君),et al.Specific DNA band isolation and SCAR marker construction of rye genome[J].Acta Botanica Boreali-Occident Sinica(西北植物学报),2006,26(12):2434-2438(in Chinese with English abstract).
[22]Meng Y X(孟亚雄),Zhong J(仲军),Ma X L(马小乐),et al.RAPD analysis of Puccinia striiformisf.sp tritici in Gansu and SCAR marker establishment for Su11-14[J].Acta Pratac Sinica(草业学报)2010,19(6):180-186(in Chinese with English abstract).
[23]Qi X L(亓晓莉),Peng D L(彭德良),Peng H(彭焕),et al.Rapid molecular diagnosis based on SCAR marker system for cereal cyst nematode[J].Scientia Agricultura Sinica(中国农业科学),2012,45(21):4388-4395(in Chinese with English abstract).
[24]Zhao J Y(赵静媛),Chen S M(陈素梅),Chen F D(陈发棣).Conversion of RAPD marker linked to creeping plant type in ground-cover chrysanthemum to SCAR marker[J].Scientia Silvae Sinicae(林业科学),2009,45(9):147-150(in Chinese with English abstract).