苜蓿品种资源亲缘关系的RAPD分析
摘要
本试验以RAPD技术为研究方法，对吉林省农科院畜牧分院42个供试品种进行了品种鉴定和分类地位的研究。结果表明：
采用CTAB改良法快速提取到了较高质量苜蓿基因组总DNA用于RAPD扩增，并在RAPD反应中筛选出19个10碱基随机引物，它们的多态性百分率大多在62.7％以上，确定了RAPD-PCR苜蓿最适反应体系为：模板DNA浓度为135ng／μL，Mg~(2+)浓度为2.5mmol／L，Taq DNA聚合酶1U，dNTP的浓度为1.5mmol／L，10碱基引物浓度为15pmol，10×缓冲液浓度为2.5mmol／L，其余用重蒸馏水补充。扩增程序为：预变性94℃ 5min，变性94℃ 1min，退火40℃ 1min，延伸72℃ 1.5min，共40个循环；72℃最终延伸5min。
建立了42个品种的指纹图谱。找到了诺瓦苜蓿、甘农一号、Magna601 3个品种的特征标记。42个品种中的CW-321、WL-323接种、WL-232HQ、朝阳苜蓿、菲尔兹、CW-340、金字塔、大叶苜蓿、辛普劳、WL-323ML、Ladak+、Magnum-Wet、CW-306、农宝、多叶苜蓿、霍普兰德、苜蓿王、兼用型、胜利者、得龙苜蓿、金钥匙、肇东苜蓿、公农一号、草原二号、敖汉苜蓿、熊一号、紫花苜蓿27个品种可以用一个引物的两条带和其它品种区分开。
对供试苜蓿42个品种进行了RAPD扩增，根据遗传距离进行了聚类分析，将供试品种基本按照来源的不同分成了五类，第一类包括辛普劳和Magna601，两个都是美国品种；第二类包括WL-323ML、CW-200、CW-340、敖汉苜蓿、金字塔、菲尔兹、大叶苜蓿，主要是美国品种；第三类包括公农二号、草原一号、草原二号、苜蓿王、霍普兰德、公农一号、龙牧803、龙牧801、肇东苜蓿，主要是吉林、内蒙、黑龙江品种，其中除了霍普兰德和苜蓿王两个美国品种之外，其它品种叶色相同、大部分为叶片不柔软，9个品种中除了苜蓿王以外都是抗寒品种，说明大部分品种的生态型相同；第四类包括兼用型、农宝、CW301、胜利者、Ladak+、多叶苜蓿，主要是美国和加拿大品种；第五类WL-232HQ、wL-252HQ、CW272、诺瓦苜蓿、Magnum-Wet、CW-321、WL-525、金钥匙、得龙苜蓿、紫花苜蓿、阿尔冈金、熊一号、甘农一号、朝阳苜蓿、WL-323接种、MagnumV、WL324、CW-306，主要是美国、甘肃、宁夏品种。证明RAPD技术可作为苜蓿分类鉴定的依据。
Forty-two Medicago varieties from the offset farming academe of Jilin Agricultural Academe of Science were used to identify the varieties and research the sort status by RAPD in this experiment .The results showed that:
An improved CTAB method could extracted high-quality DNA from gene group of Medicago and filter nineteen 10 random primer in RAPD amplification procedures, their polymorphism rate were over 60. 5%. An optimal reaction system for RAPD-PCR was: template DNA 135ng/μL, Mg~(2+) 2.5mmol/L, Taq DNA polymeraselunit, dNTP 1. 5mmol /L, 10-code primer 15pmol, 10×buffer 2.5mmol/L, others complement with ddH_2O. The amplification procedures was: initial denaturation at 94 ℃ for 5 minutes; denaturation at 94℃ for 1 minute, annealing at 40 ℃ for 1 minute, elongation at 72 ℃ for 1.5minutes, 45 cycles; final elongation at 72℃ for 5 minutes.
Fingerprints of 42 varieties were established. The marker of Nuowamuxu, Gannong Nol and Magna601 were founded.Among 42 varieties, 27 varieties can be distinguished from other varieties by two bands in one primer. CW-321, WL-323Jiezhong, WL-232HQ, Zhaoyangmuxu, Feierzi, CW-340, Pyramid, Dayemuxu, Xinpulao, WL-323ML, Ladak+, Magnum-Wet, CW-306, Nongbao, Duoyemuxu, Huopulande, Muxuwang, Jianyongxing, Winner, Delongmuxu,Jinyaoshi, Zhaodongmuxu, Gongnong Nol, CaoyuanNo2, Aohanmuxu, Xiong Nol and Alfalfa were included.
By using RAPD amplification to conduct clstering analyse based on genetic distance, 42 varieties were classified into five groups by origin.
The first group include Xinpulao and Magna601,they are both American varieties;the second .group include WL-323ML, CW-200, CW-340, Aohanmuxu, Pyramid, Feierzi and Dayemuxu,most of them are
采用CTAB改良法快速提取到了较高质量苜蓿基因组总DNA用于RAPD扩增，并在RAPD反应中筛选出19个10碱基随机引物，它们的多态性百分率大多在62.7％以上，确定了RAPD-PCR苜蓿最适反应体系为：模板DNA浓度为135ng／μL，Mg~(2+)浓度为2.5mmol／L，Taq DNA聚合酶1U，dNTP的浓度为1.5mmol／L，10碱基引物浓度为15pmol，10×缓冲液浓度为2.5mmol／L，其余用重蒸馏水补充。扩增程序为：预变性94℃ 5min，变性94℃ 1min，退火40℃ 1min，延伸72℃ 1.5min，共40个循环；72℃最终延伸5min。
建立了42个品种的指纹图谱。找到了诺瓦苜蓿、甘农一号、Magna601 3个品种的特征标记。42个品种中的CW-321、WL-323接种、WL-232HQ、朝阳苜蓿、菲尔兹、CW-340、金字塔、大叶苜蓿、辛普劳、WL-323ML、Ladak+、Magnum-Wet、CW-306、农宝、多叶苜蓿、霍普兰德、苜蓿王、兼用型、胜利者、得龙苜蓿、金钥匙、肇东苜蓿、公农一号、草原二号、敖汉苜蓿、熊一号、紫花苜蓿27个品种可以用一个引物的两条带和其它品种区分开。
对供试苜蓿42个品种进行了RAPD扩增，根据遗传距离进行了聚类分析，将供试品种基本按照来源的不同分成了五类，第一类包括辛普劳和Magna601，两个都是美国品种；第二类包括WL-323ML、CW-200、CW-340、敖汉苜蓿、金字塔、菲尔兹、大叶苜蓿，主要是美国品种；第三类包括公农二号、草原一号、草原二号、苜蓿王、霍普兰德、公农一号、龙牧803、龙牧801、肇东苜蓿，主要是吉林、内蒙、黑龙江品种，其中除了霍普兰德和苜蓿王两个美国品种之外，其它品种叶色相同、大部分为叶片不柔软，9个品种中除了苜蓿王以外都是抗寒品种，说明大部分品种的生态型相同；第四类包括兼用型、农宝、CW301、胜利者、Ladak+、多叶苜蓿，主要是美国和加拿大品种；第五类WL-232HQ、wL-252HQ、CW272、诺瓦苜蓿、Magnum-Wet、CW-321、WL-525、金钥匙、得龙苜蓿、紫花苜蓿、阿尔冈金、熊一号、甘农一号、朝阳苜蓿、WL-323接种、MagnumV、WL324、CW-306，主要是美国、甘肃、宁夏品种。证明RAPD技术可作为苜蓿分类鉴定的依据。
Forty-two Medicago varieties from the offset farming academe of Jilin Agricultural Academe of Science were used to identify the varieties and research the sort status by RAPD in this experiment .The results showed that:
An improved CTAB method could extracted high-quality DNA from gene group of Medicago and filter nineteen 10 random primer in RAPD amplification procedures, their polymorphism rate were over 60. 5%. An optimal reaction system for RAPD-PCR was: template DNA 135ng/μL, Mg~(2+) 2.5mmol/L, Taq DNA polymeraselunit, dNTP 1. 5mmol /L, 10-code primer 15pmol, 10×buffer 2.5mmol/L, others complement with ddH_2O. The amplification procedures was: initial denaturation at 94 ℃ for 5 minutes; denaturation at 94℃ for 1 minute, annealing at 40 ℃ for 1 minute, elongation at 72 ℃ for 1.5minutes, 45 cycles; final elongation at 72℃ for 5 minutes.
Fingerprints of 42 varieties were established. The marker of Nuowamuxu, Gannong Nol and Magna601 were founded.Among 42 varieties, 27 varieties can be distinguished from other varieties by two bands in one primer. CW-321, WL-323Jiezhong, WL-232HQ, Zhaoyangmuxu, Feierzi, CW-340, Pyramid, Dayemuxu, Xinpulao, WL-323ML, Ladak+, Magnum-Wet, CW-306, Nongbao, Duoyemuxu, Huopulande, Muxuwang, Jianyongxing, Winner, Delongmuxu,Jinyaoshi, Zhaodongmuxu, Gongnong Nol, CaoyuanNo2, Aohanmuxu, Xiong Nol and Alfalfa were included.
By using RAPD amplification to conduct clstering analyse based on genetic distance, 42 varieties were classified into five groups by origin.
The first group include Xinpulao and Magna601,they are both American varieties;the second .group include WL-323ML, CW-200, CW-340, Aohanmuxu, Pyramid, Feierzi and Dayemuxu,most of them are
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[2] 白静仁．我国苜蓿品种资源的发展及利用．中国草地，1990，4：57-60．
[3] 苜蓿的科学与技术．中国农科院苜蓿所饲料室．农牧渔业部畜牧局草原处，1986．
[4] 卢欣石．中国苜蓿遗传多样性及基因生态类型研究．甘肃农业大学博士学位论文，1997．
[5] 洪绂曾．积极稳步发展中国的苜蓿产业．首届中国苜蓿大会论文集，中国草原学会，北京市农村工作委员会．2001
[6] 田明英，许淑桂．果园种牧草，改土有捷径．林果特产，28
[7] 鲁会玲．坡地果草间作，提高水土保持综合效益．国土与自然资源研究，2005 (2)：61-62
[8] 徐柱．面向21世纪的中国草地资源．中国草地，1998，(5)：1-8
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