菠菜性别相关的ISSR标记

菠菜为雌雄异株植物,用CTAB法提取其雌、雄株成株幼嫩叶片DNA,分别构建雌、雄株DNA池,以之为模板,用已优化的ISSR体系扩增,在74条ISSR引物中,I62扩增出一条约1 200 bp雌性连锁标记,回收纯化该特异扩增片段,将其连接于pUCm-T载体,转化进大肠杆菌JM109菌株,并检测及测序。回收克隆和测序后发现该片段全长1 176 bp,富含AT,AT占57.0%。根据测序结果设计1对25 bp的特异引物将这个雌性连锁的ISSR标记转化为稳定性和特异性更好的SCAR标记。该特异引物对随机选取的雌雄菠菜单株进行PCR扩增,在雌株中均有1 176 bp的特异条带,而雄株中均无。此特异条带的获得为菠菜性别相关基因的克隆奠定基础。     

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。     

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.     

热带小奥德蘑的序列特异性扩增标记开发

[目的]为了快速、准确地对热带小奥德蘑JZB2115055进行鉴定和保护,该研究开发了该菌的序列特异性扩增(SCAR)标记。[方法]采用26个ISSR引物对19个小奥德蘑属菌株进行PCR扩增,以引物P826扩增时,JZB2115055在700 bp～1 000 bp之间出现了一条特异条带,获得此条带的DNA序列并设计特异性引物对P826-1-2XF/R。[结果]以19个小奥德蘑DNA为模板,P826-1-2XF/R为引物在JZB2115055中能够特异性地扩增出2条条带,长度分别为431 bp、537 bp;该引物在2～19号菌株中扩增不出目的条带或者扩增条带在2 000～5 000 bp之间。[结论]开发了热带小奥德蘑JZB2115055的SCAR标记,能够在该菌中特异性地扩增出431 bp和537 bp大小的条带,而其他18株菌株不能扩增出特异条带,此标记能够快速、准确地进行该菌的鉴定和保护。     

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。     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)技术获得与大麻性别连锁的分子标记.将10株雄性大麻或10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNApool),以提供具有相同遗传背景的雌、雄性DNA样品.每个随机引物分别用三个不同的循环程序进行PCR扩增.在30个随机引物中,用引物401扩增得到一条约2.5kb雄性多态性片段.对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)分子标记.     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA（random amplified polymorphic DNA,RAPD)技术获得与大麻性别连锁的分子标记,将10株雄性大麻或10株雌性麻的单个DNA样品等量混合分别组成雄性或雌性DNA池（DNApool),以提供具有相同遗传背景的雄,雄性DNA样品。每个随机引物分别用三个不同的循环程序进行PCR扩增,在30个随机引物中,用引物S401扩增得到一条约2．5kb雄性多态性片段,对该片段进行了克隆和序列分析 ,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR（Sequence characterized amplified regions)分子标记。     

绿僵菌M189菌株特异性SCAR标记的建立及田间监测应用

为了监测生防真菌绿僵菌菌株在田间释放后的回收,有必要建立菌株DNA分子标记,以此将应用的菌株与其他菌株或田间的土著分离株鉴别开来。作者采用16条随机引物扩增了51株绿僵菌菌株的基因组DNA,得到81个多态性位点。其中M189菌株多态性位点30个,分析得到1个特异性位点,并将该位点的DNA片段测序后转化成为特异性SCAR标记。检验确定了该标记的敏感性,可以从供试的51株菌株中准确鉴定出目的菌株M189。并用该标记检测了从田间回收的3个分离株,确定其中1个与应用菌株M189一致。     

SCAR标记技术鉴别榆黄蘑品种

为了建立一套基于DNA分子标记技术快速鉴定榆黄蘑菌株的有效方法,本研究通过对生产上常用的16个榆黄蘑菌株进行SRAP多态性分析,从榆黄蘑2号菌株中扩增获得了一个片段长为537bp的特异片段,将其克隆、测序并设计引物,成功转化为稳定的SCAR标记。试验结果表明,利用该特异SCAR标记,能在1d时间内准确鉴定出榆黄蘑2号菌株。由此可见,SCAR分子标记是一种快速、稳定、准确鉴定榆黄蘑菌株的新方法。     

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.     

Analysis of Specific RAPD and ISSR Fragments in Maize (Zea mays L.) Somaclones and Development of SCAR Markers on Their Basis

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

Analysis of specific RAPD- and ISSR-fragments in somaclonal maize (Zea mays L.) and development of SCAR markers based on them

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

DNA分子标记在木耳属真菌中的应用研究进展

介绍了目前已在木耳属真菌中广泛应用的几种DNA分子标记技术(RFLP、RAPD、AFLP、ISSR、SRAP、SCAR、ITS、IGS),以及这些标记在木耳属真菌系统发育、分类研究、种间和种内鉴定、遗传多样性分析等方面的应用,并展望了这些标记技术在木耳属真菌中的应用前景。     

Evaluation of the Use of SCAR Markers for Screening Genetic Diversity of Lentinula edodes Strains

In this study, three molecular marker systems including sequence related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and inter-simple sequence repeats (ISSR) were screened to select polymorphisms of 24 main commercial strains of Lentinula edodes cultivated widely in China. Twenty-nine sequence characterized amplified region (SCAR) markers were developed to set up a dendrogram using UPMGA based on nucleotide sequences of some SRAP, RAPD, and ISSR polymorphic fragments. The grouping showed that the 24 strains were apparently clustered into five groups at a level of 0.68 similarity coefficient, and those that have similar breeding background clustered preferentially into the same subgroup. Results also revealed that the 24 strains had a low level of genetic diversity, and the breeding source of L. edodes should be broadened by exploiting wild types and introducing exotic strains. In addition, the tested strains of L. edodes could be clearly distinguished and identified from others by using different combinations of SCAR primers. Thus, results of this work demonstrated that SCAR was an excellent genetic marker system to characterize and investigate genetic diversity of L. edodes. Furthermore, this provided an alternative method to identify the genetic relationship of different strains of other fungi.     

Analysis of Genetic Diversity Among Chinese Auricularia auricula Cultivars Using Combined ISSR and SRAP Markers

DNA polymorphism among 34 Chinese Auricularia auricula cultivars was analyzed using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Thirty ISSR primers amplified a total of 129 DNA fragments of which 125 (96.9%) were polymorphic, whereas 11 SRAP primer combinations amplified 154 fragments of which 148 (96.1%) were polymorphic. Both methods were highly effective in discriminating among the test strains. Phylogenetic trees constructed on the basis of ISSR, SRAP, and combined ISSR/SRAP analyses using the Unweighted Pair-group Method with Arithmetic Averages (UPGMA) method distributed the 34 strains into four or five major groups. Clustering analysis based on all the three data sets indicated a high level of genetic diversity among A. auricula, although the combined ISSR/SRAP data were more concordant with the main agronomic characters of strains and their geographical centers of cultivation. Our findings will facilitate future A. auricula breeding programs and the development of bioactive products from this commercially important medicinal mushroom.     

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.     

中国南瓜海南农家品种间的遗传特异性分析和DNA指纹图谱构建

在收集中国南瓜海南农家品种的基础上,本研究应用ISSR和SRAP标记技术对28份海南农家品种间的遗传特异性进行了分析,并构建指纹图谱,为中国南瓜海南农家品种鉴定、评价、保护和利用提供科学依据。结果表明,所供试的品种间存在显著的遗传特异性,具有特殊的遗传基础或背景,所筛选的6个ISSR引物和11对SRAP引物共产生了10个特异标记和11条唯一缺失带;应用ISSR引物组合UBC807/UBC814/UBC844/UBC868和UBC808/UBC814/UBC844/UBC868,以及SRAP引物组合Me1/Em2 Me1/Em10 Me2/Em3和Me1/Em1 Me1/Em10 Me8/Em3分别绘制了四张28份中国南瓜海南农家品种的DNA指纹图谱,所构建的DNA指纹图谱直观、简单。ISSR标记和SRAP标记技术可有效应用于中国南瓜海南农家品种DNA指纹图谱的构建和遗传特异性鉴定。