中国黑木耳主要栽培菌株ISSR指纹分析及SCAR标记

采用ISSR标记技术对来自全国的黑木耳34个主要栽培菌株进行DNA指纹分析,初步构建其标准化DNA指纹图谱;在ISSR指纹分析基础上进行聚类分析,并将黑木耳两个栽培菌株173和186中扩增获得的ISSR特异性DNA带转化为可以直接用于菌株快速鉴定的SCAR标记。研究表明,我国黑木耳栽培菌株遗传背景差异不大,存在同物异名现象,而采用ISSR指纹及其SCAR标记鉴定黑木耳栽培菌株具有重要意义。     

中国黑木耳主要栽培菌株ISSR指纹分析及SCAR标记

采用ISSR标记技术对来自全国的黑木耳34个主要栽培菌株进行DNA指纹分析,初步构建其标准化DNA 指纹图谱;在ISSR指纹分析基础上进行聚类分析,并将黑木耳两个栽培菌株173和186中扩增获得的ISSR特异性DNA带转化为可以直接用于菌株快速鉴定的SCAR标记。研究表明,我国黑木耳栽培菌株遗传背景差异不大,存在同物异名现象,而采用ISSR指纹及其SCAR标记鉴定黑木耳栽培菌株具有重要意义。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

利用SRAP和ISSR建立快速鉴定灵芝属菌株的SCAR标记

根据ERIC聚类分析的结果,把152株灵芝属菌株(包括128株来自中国的栽培菌株及24株国外菌株)建成48个DNA池。用SRAP和ISSR引物对48个DNA池进行扩增,筛选获得4个特异性标记,回收特异性条带,经克隆测序后设计了4对SCAR引物,并通过SCAR-PCR扩增验证,从而将SRAP标记和ISSR标记均成功地转化为特异性和稳定性更好的SCAR标记;将得到的4个SCAR标记在构成DNA池的152个菌株上验证,并建立多重PCR体系,最终证实了SCAR特异标记在菌株快速检测鉴定中的可行性和可靠性。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

黑木耳Au185菌株一个SCAR标记的建立

在对60个供试黑木耳菌株RAPD指纹图谱进行分析的基础上获得了黑木耳菌株Au185的RAPD特异性标记,将其克隆、测序,应用Primer5.0软件设计相应的特异引物对SC38-1,将RAPD特异性标记转化为黑木耳菌株Au185的稳定方便的SCAR标记,可快速、准确地鉴定出黑木耳菌株Au185。     

SCAR标记在黑木耳栽培菌株分类鉴定中的应用

应用序列特异性扩增区域（SCAR）标记技术分析50株黑木耳栽培菌株的遗传多样性。在SRAP标记分析过程中发现1条1 200 bp的特异性条带,经回收克隆测序转为SCAR标记。根据序列设计出1对特异性引物,经PCR可以扩增出1 000 bp大小的片段,说明成功构建出了＂黑931＂的指纹图谱。     

武夷山及周边地区黄精植物ISSR分子标记鉴定及HPLC指纹图谱研究

采用ISSR分子标记鉴定方法及HPLC色谱方法对武夷山及周边地区5份黄精植物样本进行DNA分子标记鉴定及指纹图谱分析,并通过聚类分析软件分别探讨其遗传相关聚类图谱。ISSR分子标记法能较好地对5份黄精植物样本进行区分,HPLC指纹图谱分析同样表明5份黄精样本化学成分存在一定的差别。DNA分子标记及HPLC指纹图谱分析的遗传相关聚类分析结果相似,表明武夷山及周边地区的野生黄精种质资源存在明显的差别。     

SCAR分子标记技术在香菇菌株鉴定上的应用研究

为了建立一套基于DNA分子标记技术快速鉴定香菇菌株的有效方法,本研究首先通过对生产上常用的14个香菇菌株进行RAPD多态性分析,从香菇菌株162中扩增获得了一个片段长为1166bp的特异RAPD标记XG1166,随之利用分子克隆技术将该特异RAPD标记成功转化为稳定的SCAR标记。用同样的方法,本研究又从另一香菇菌株申香10号中获得了一段长度为347bp的特异SCAR标记SX347。试验结果表明,利用本研究获得的香菇菌株162和申香10号的特异SCAR标记,能在一天时间内准确鉴定出香菇菌株162或申香10号菌株的真伪。由此可见,SCAR分子标记是一种快速、稳定、准确鉴定香菇菌株的新方法, 可应用于食用菌种质资源保护利用、品种分类与鉴定和假种辨别。     

Use of inter-simple sequence repeat markers to develop strain-specific SCAR markers forFlammulina velutipes

In this paper, we report for the first time on authentication of Flammulina velutipes cultivars by using strain-specific sequence-characterized amplified region (SCAR) markers developed from inter-simple sequence repeat (ISSR) markers. The genomic DNA polymorphism was analyzed by the ISSR technique in 7 strains of F. velutipes presently cultivated in China on a commercial scale. Eight primers selected from 20 ISSR primers amplified 104 clear and stable bands, of which 81 bands were polymorphic. Among the selected primers, primer ISSR9 can distinguish strain No. 12 from the other 6 strains by amplifying a unique and reproducible band of approximately 750 bp. According to the sequence of the strain-specific fragment, a pair of SCAR primers was designed to diagnose strain No. 12 on the molecular level. The validity of the SCAR marker was confirmed by using DNA samples from another 12 strains of F. velutipes obtained from different parts of China. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for F. Velutipes.     

Use of intersimple sequence repeats markers to develop strain-specific SCAR markers for Lentinula edodes

Although Lentinula edodes is the second most important cultivated mushroom worldwide, most industrially cultivated strains have been identified only through traditional phenotypic analysis. Here, we report for the first time the use of sequence characterized amplified region (SCAR) markers for strain differentiation. SCAR markers were created by first generating and sequencing single intersimple sequence repeats fragments, and then designing primers based on these sequences to amplify strain-specific fragments of a certain size. One SCAR primer pair, ISL450F/R7 (amplifying a band of c. 450 bp), was designed to identify one strain of L. edodes (strain No. 7). The SCAR primer pair was then used to correctly amplify the single unique fragment from DNA samples taken from a total of 85 strains representing three separate species. Our data provide the foundation for a precise and rapid PCR-based strain-diagnostic system for L. edodes.     

Strain-typing of Lentinula edodes in China with inter simple sequence repeat markers

To validate strain typing by inter simple sequence repeat (ISSR) analysis in Lentinula edodes cultivars, 17 Chinese L. edodes strains including 15 cultivated strains cultivated on a large scale and two wild strains were analyzed with the ISSR technique. With the use of two ISSR primers, a total of 32 DNA products were detected, of which, 31 DNA products (96.9% of the detected products) were polymorphic between two or more strains. The profiles of those two primers could be employed to differentiate all of the tested strains. A cluster analysis based on ISSR data revealed that the 17 strains could be classified into two distinct groups. One group consisted of eight strains in which the cultivated strains were H (high-temperature)-type or B (broad-temperature)-type, and the other group comprised cultivated strains that were of the L (low-temperature)-type or M (medium-temperature)-type. In contrast to the two wild strains, the genetic diversity of 15 cultivated strains was very rich based on a similarity coefficient analysis.     

Development of strain-specific SCAR markers for authentication of Ganoderma lucidum

In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated as GL₆₁₂F and GL₆₁₂R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum.     

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.