双孢蘑菇SCAR标记的建立及在菌株群鉴定中的应用

根据15条具有菌株群特异性的SRAP、ISSR、RAPD扩增产物DNA条带测序结果,设计64对SCAR引物,其中6对引物能成功扩增出目的条带,即有6条特异片段(A、C、E、G、H、J)被成功转化为SCAR标记.通过这6个SCAR标记的不同组合,将40个双孢蘑菇菌株分为9个类群和6个类群,与基于SRAP、ISSR、RAPD资料的聚类分析结果的组间距离值取13和16时的分类结果完全吻合.对A片段的SCAR-PCR检测结果是:可从40个双孢蘑菇菌株中鉴别出双孢蘑菇333号菌株(编号01).     

双孢蘑菇RAPD标记的遗传多样性分析

随机抽取108个随机引物对40个双孢蘑菇栽培菌株的DNA进行RAPD-PCR扩增实验,发现有71个随机引物能够扩增出清晰的、稳定的、具有多态性的RAPD条带。结果表明,71个随机引物共扩增出567条DNA带,平均每个引物扩增出7.99条DNA带,其中434条DNA带具有多态性,占总数的76.5%。基于RAPD-PCR扩增资料,采用欧氏距离平方系数和组间连接法,应用SPSS 11.0软件进行聚类分析,探讨40个品种间的亲缘关系与类群划分。当取欧氏距离平方系数阈值为20.5时,所有品种分为三大类;当取欧氏距离平方系数阈值取8.5时,40种双孢蘑菇菌株可被分为6个类群,此结果与双孢蘑菇菌株群的同工酶分类研究结果相当吻合。     

云南省烟草镰刀菌根腐病菌遗传多样性分析

【背景和目的】尖孢镰刀菌（Fusarium oxysporum）引起的根腐病是烟草主要的土传病害,在云南烟区普遍发生。为分析云南省不同地理来源尖孢镰刀菌的遗传差异性和亲缘关系。【方法】采用ISSR(inter-simple sequence repeat)和SRAP(sequencerelated amplified polymorphism)分子标记技术分析40株菌株遗传多样性。【结果】（1）采用ISSR技术,供试的7条引物共扩增出53条条带,其中多态性条带44条,平均多态性条带83.02%。菌株间的遗传相似系数为0.49～0.96,存在遗传背景差异。遗传相似系数为0.68时,40株菌株可分为3个类群。（2）采用SRAP技术,供试的7对引物共扩增出44条条带,其中多态性条带32条,平均多态性条带72.73%,菌株间的遗传相似系数为0.55～0.96。遗传相似系数为0.70时,40株菌株可分为3个类群,结果与ISSR一致。【结论】云南省烟草尖孢镰刀菌菌株间遗传差异大,ISSR和SRAP分子标记技术可用于烟草尖孢镰刀菌的遗传多样性分析。     

草菇有性世代间rDNA序列变异分析及RAPD分析

草菇的遗传与有性生殖过程存在很多的特殊性和不确定性,本研究以草菇V23菌株的成熟子实体弹射孢子,单孢分离后得13个单株,分别摇瓶培养、收集菌丝,提取总DNA,用通用引物ITS4、ITS5进行PCR扩增后连接、转化并测序,将单孢间和单孢与亲本间的ITS序列进行比对,分析草菇ITS序列多样性及变异性;同时以相应单孢菌株DNA为模板,用20条随机引物进行RAPD扩增。结果表明:草菇单孢间和单孢与亲本间的ITS序列相似度均在99%以上,相较于亲本的ITS序列,单孢序列中发现了有5个位点出现碱基转换,但没有两个以上的单孢发生同一转换;20条随机引物分别与14个样本进行RAPD扩增,其中3个引物出现多态性条带,带型结果发现单孢菌株间的带型差异呈现一定规律。草菇ITS序列变异较少,不适于单孢间的变异分析,而RAPD方法对摸索草菇有性世代间的遗传分离规律有重要价值。     

不同来源酿酒酵母菌株的随机扩增多态DNA分析

研究中试用了20个随机引物对16株不同来源的酿酒酵母菌株全基因组进行了随机扩增多态DNA分析,其中OPG06,OPG11和OPG20三条适宜引物具有鉴别作用,每一引物均可扩增1～10条DNA片段,大多数片段分子量大小在100～2000bp之间,共扩增出34条RAPD谱带,多态性为85.3%,获得了稳定清晰的菌株RAPD指纹图谱。RAPD分析结果表明,不同来源的酿酒酵母菌株之间的遗传相似系数在37.5%～94.1%之间,反映出较高的遗传差异性,并可通过聚类分析将16株不同来源的酿酒酵母菌株按亲缘关系的远近分为6个类群。结果表明,利用RAPD标记技术在基因水平上对酿酒酵母菌株进行分子鉴定和分型是可行的。     

烟草品种的DNA指纹图谱和品种鉴定

随机扩增多态性DNA(RAPD)是目前常用的DNA指纹图谱技术.采用改进的"ROSE法"提取DNA,并建立了重复性较好的RAPD标准分析条件.在此标准条件下,检测了烟草10个品种材料基因组的多态性.经10个引物扩增,在得到的53条扩增带中,有10个片段为10个材料所共有,多态性达81.1%.其中引物P7的扩增图谱,各品种间可以互相区别,差异明显,可用作烟草10个品种鉴定的DNA指纹图谱.     

克罗诺杆菌的随机多态性扩增DNA(RAPD)基因分型研究

研究了克罗诺杆菌标准菌株和样品中分离菌株的分子特征,为克罗诺杆菌的种间鉴别和分子溯源提供一种有效手段。采用随机引物扩增多态性DNA分析对38株菌株进行基因分型。筛选出1条随机引物,每一菌株可扩增出1～7条DNA片段,片段在400-3500bp,可将8株标准菌株的种间区分开来。以相似性50%为标准,38株克罗诺杆菌按照其遗传差异可以分为8个基因型类群。所建立的RAPD技术可用于克罗诺杆菌的种间鉴别和分子溯源研究。     

尖孢镰刀菌胡麻专化型（Fusarium oxysporum f.sp.lini）ISSR标记聚类分析

结合形态学特征和分子生物学方法对我国尖孢镰刀菌胡麻专化型菌株进行鉴定、聚类分析和遗传变异分析。结合传统分类方法和ITS序列分析,确定6省区96株菌株为尖孢镰刀菌。采用ISSR（简单重复序列区间）分子标记技术对这96株菌株进行分析,12条引物共扩增出800个条带,多态率条带数为797条,多态率为99.62%;在相似性系数为0.88处,96株供试菌株被分为5个类群。聚类结果表明,胡麻枯萎病菌种内存在明显的多态性,且ISSR类群与地理来源存在相关性。     

云南品牌卷烟主体烟叶原料的ISSR多态性分析及标记

为考察云南品牌卷烟主体烟叶原料的遗传多样性,从27个ISSR引物中筛选出7个引物,分别在17份烟草材料中进行PCR扩增。结果显示,能够产生40条稳定的条带,其中多态性带28条。通过UPGMA聚类分析,将17个烤烟品种分为2类。品种间遗传相似指数范围为0.50~1.00。同时,利用1个多态性强的ISSR引物、通过聚丙烯酰胺凝胶可以将这17份烤烟材料区分开,每个品种均有各自独特的指纹图谱,表明ISSR标记适于烟草品种鉴定和遗传多样性分析。     

烤烟品种的RAPD分析

利用RAPD标记研究了29个烤烟品种的分子指纹和遗传关系。100个随机引物经10个品种筛选,选出18个随机引物对29个品种基因组DNA进行扩增,得到了29个烤烟品种的分子指纹图谱,参照这些图谱,可对不同烤烟品种进行真实性鉴定。以扩增到的79条多态性DNA片段为基础计算29个品种间遗传相似系数,利用UPGMA法作聚类图,可将参试品种分为3个类群。云南省主栽品种K326与近年选育的品种V2、云烟85、云烟317等被归为一类,遗传基础过于狭窄,建议选用一些遗传关系较远的品种进行亲本选配、品种布局;北方烟区主栽品种NC89、地方品种净叶黄及G140等则归为另一类。      

Molecular characterization of mulberry (Morus spp.) genotypes via RAPD and ISSR

BACKGROUND: In recent years, DNA‐based markers have been used quite extensively because of their many advantages over the traditional morphological and biochemical markers. Many studies have shown that molecular markers are useful in delineating the genetic relationships among closely related mulberry genotypes and cultivars. Thus, in the present study, polymer chain reaction based DNA fingerprinting techniques were used to investigate the genetic relationships among mulberry genotypes growing in different agro‐climatic regions of Turkey. RESULTS: 20 RAPD primers generated a total of 173 bands, of which 157 (90.75%) were polymorphic. As for 11 ISSR primers, 124 bands (96.55%) were polymorphic in a total of 128. The similarity index for RAPD technique ranged between 0.24–0.98; 25?s203 with 25?s112 were found to be the closest genotypes, while 24Ke10 and 25?s123 were the most distant ones. According to the ISSR result, the genetic similarity index changed between 0.21–095; 25?s203 with 25?s112 genotypes were the closest, while 25?s08 and 01KaD2 were the most distant ones. CONCLUSION: The RAPD and ISSR markers were found to be promising for assessing genetic diversity in mulberry genotypes. Copyright © 2011 Society of Chemical Industry     

我国江淮产区主要芝麻品种的SRAP指纹图谱分析

利用SRAP分子标记对江淮主产区8个芝麻品种进行指纹图谱分析。32对有效扩增的引物中有27对扩增出多态性,占84．37％;27对多态性引物共扩增465个位点,多态性位点158个,占33．98％;平均每对引物可检测位点17．22个,其中多态性位点平均5．85个;8个品种相互间的相似系数在0．7861—0．9711之间,平均为0．9079;用一对引物Em07／Me09便可将8份芝麻品种区分开来,用其构建的指纹图谱的置信概率达到99．6948％。     

烟草黑胫病菌全基因组DNA遗传多态性RAPD标记

应用ＲＡＰＤ分子标记技术,从８０个随机引物中筛选出１０个分别为ＯＰＡ０２、ＯＰＡ０１３、ＯＰＡ０１４、ＯＰＡ０１６、ＯＰＡ０１８、ＯＰＢ０４、ＯＰＢ０５、ＯＰＤ０２、ＯＰＤ０３、ＯＰＤ０１１,对来自云南省６大主产烟区的２４个烟草黑胫病菌株全基因组ＤＮＡ进行随机扩增。结果表明：１０个引物共标记出８９条ＲＡＰＤ图带,其中多态性图带６３条,多态率７０．７％。引物ＯＰＤ０１１、ＯＰＤ０２、ＯＰＡ０１６、ＯＰＢ０４对每个受试样品均可标记出分子量为０．８７Ｋｂ和１．１Ｋｂ的ＤＮＡ片段,此４个引物对受试样品全基因组ＤＮＡ具有特征性分子标记的特性,利用核酸分子杂交技术对田间寄主体内的黑胫病菌的有无进行分子检测。被标记出的分子量为０．８７Ｋｂ和１．１Ｋｂ的特征性ＤＮＡ片段,可用来制备特异性探针,对田间烟草黑胫病菌发生动态进行检测和预报。     

蓖麻DNA的提取及SRAP反应体系的建立

为建立蓖麻快速有效的DNA提取方法和SRAP标记的技术体系,本研究采用SDS法和CTAB法对蓖麻不同叶片（新鲜嫩叶、老叶和陈旧的嫩叶、老叶）的基因组DNA进行提取,并以DNA模板用量、Mg^2＋浓度、引物浓度和Taq酶用量4个因素的不同水平配制了10个反应体系对蓖麻SRAP反应体系进行优化。结果表明,SDS法和CTAB法均可获得较高质量的DNA,多数样品的A260／A280值介于1．7～1．9之间,但以CTAB法更为省工省时,不同叶片DNA的提取效果,以嫩叶效果最佳;设计的10个反应体系中,以反应体系A（总体积为15μL,40ng DNA模板,1．5mmol／L Mg^2＋浓度,0．2mmoL／L dNTPs,0．3μmol／L引物浓度和1Unit Taq酶）最为经济适用,将该体系用于蓖麻的SRAP扩增能获得稳定、清晰的多态性条带。本实验为蓖麻的遗传多样性研究及目标性状的分析定位提供了技术支撑。     

Effects of Primers and Taq Polymerase on Randomly Amplified Polymorphic DNA Analysis for Typing Listeria monocytogenes From the Environment of a Shrimp Processing Plant

Ninety-nine randomly selected isolates of Listeria monocytogenes from several processing environment locations, in a shrimp processing plant, obtained during a 5-month sampling period were subjected to randomly amplified polymorphic DNA (RAPD) analysis with the use of four primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. Eighteen composite RAPD types were discerned with the use of the four primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates, and type 9 comprised 49 indistinguishable isolates. These results indicate that the shrimp processing plant was dominated by these 2 RAPD types that comprised 63.6% of the 99 randomly selected isolates.     

Effects of Primers and Taq Polymerase on Randomly Amplified Polymorphic DNA Analysis for Typing Listeria monocytogenes From the Environment of a Shrimp Processing Plant

Ninety-nine randomly selected isolates of Listeria monocytogenes from several processing environment locations, in a shrimp processing plant, obtained during a 5-month sampling period were subjected to randomly amplified polymorphic DNA (RAPD) analysis with the use of four primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. Eighteen composite RAPD types were discerned with the use of the four primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates, and type 9 comprised 49 indistinguishable isolates. These results indicate that the shrimp processing plant was dominated by these 2 RAPD types that comprised 63.6% of the 99 randomly selected isolates.