瓠瓜类型砧木品种根系DNA提取方法比较及指纹图谱构建  

Comparison of Root DNA Extraction Methods and Construction of DNA Fingerprinting on Bottle Gourd(Lagenaria siceraria L.) Type of Rootstock Cultivars

作　　者：宋慧[1] 张香琴[1] 应泉盛[1] 严蕾艳[1] 王迎儿[1] 王毓洪[1] 

机构地区：[1]宁波市农业科学研究院蔬菜研究所,宁波市瓜菜育种重点实验室,宁波315040

出　　处：《分子植物育种》2015年第2期401-408,共8页Molecular Plant Breeding

基　　金：国家自然基金(31000907);公益性行业(农业)科研专项(201403032);国家西甜瓜产业技术体系(nycytx-36);宁波市瓜类砧木育种创新团队(2014B81002);宁波市瓜菜育种重点实验室(2014A22007)共同资助

摘　　要：为利用瓠瓜砧木根系提取DNA进行品种真实性分子检测,本研究比较了CTAB法(方法 1)、SDS简易法(方法 2)、SDS法(方法 3)、TPS法(方法 4)、快速制备法(方法 5)和简易快速制备法(方法 6)提取瓠瓜类型砧木根系基因组DNA的效果。研究结果表明,SDS简易法(方法 2)提取瓠瓜根系总DNA质量好,蛋白污染小,PCR扩增条带清晰稳定,满足瓠瓜根系DNA分子检测的要求。利用该法提取的‘甬砧1号’、‘甬砧3号’、‘甬砧4号’、‘甬砧5号’、‘神通力’和‘京欣砧1号’的根系DNA可重复上述6份瓠瓜材料幼叶DNA的指纹图谱结果(01001,10010,10101,01010,00110和00000)。新增加‘华欣948’、‘5756’、‘1715’和‘56YZ2’等4份瓠瓜砧木材料,通过根系DNA构建的指纹图谱分别是00110、01011、11000和01000。10份瓠瓜类型砧木品种根系DNA指纹图谱出现概率为1/13 500,图谱出现概率低,表明可以利用根系取代叶片提取DNA,用于构建指纹图谱进行品种鉴定与保护。In order to identify the authenticity of the bottle gourd（Lagenaria siceraria L.） cultivars by using root DNA,six methods were employed to extract the root DNA from bottle gourd type of rootstock cultivars,which were CTAB（Method 1）,Modified SDS（Method 2）,SDS（Method 3）,TPS（Method 4）,Rapid extraction（Method 5）,and Simple-rapid extraction（Method 6）.The quality and purity of DNA were compared by agarose gel electrophoresis,K5600 micro-spectrophotometer,and PCR amplification.The results showed that DNA extracted by modified SDS（Method 2） showed the highest purity and quality,less protein contamination,and the amplification bands were clear and stable,modified SDS（Method 2） was selected for molecular experiment.The fingerprinting result generated from root DNA of‘Yongzhen No.1＇,‘Yongzhen No.3＇,‘Yongzhen No.4＇,‘Yongzhen No.5＇,‘Shentongli＇and‘Jingxinzhen No.1＇was same to which from leave DNA（01001,10010,10101,01010,00110 and 00000）.The results of‘Huaxin 948＇,‘5756＇,‘1715＇and‘56YZ2＇were 00110,01011,11000 and 01000,respectively.The fingerprinting appearance probability of ten cultivars derived from root DNA was 1/13 500,which showed that the root of bottle gourd could be instead of the leave for DNA extraction used to construct the molecularfingerprinting for cultivar identification and protection.

关 键 词：瓠瓜 根系 DNA制备 指纹图谱 

分 类 号：S642.9[农业科学—蔬菜学]