47份水稻品种资源的ISSR遗传多样性分析

为研究广东省惠州市种植的常规水稻品种的遗传多样性,本实验利用ISSR标记对47份水稻品种资源进行遗传多样性检测.从49条引物中筛选出5条重复性好,条带清晰的引物进行PCR扩增,共扩增出53条带,每个引物可以扩增出9～13条带,平均为10.6条,其中47条具有多态性,比率为88.7%.不同水稻品种间遗传相似系数变幅为0.319～0.936,平均达0.691,说明ISSR标记能够揭示材料间较高的遗传多样性.通过聚类,从分子水平对水稻品种资源的遗传关系进行分析,并对47份水稻品种资源进行分类,ISSR标记能将47份水稻品种完全区分开,为水稻品种资源的研究利用提供参考.     

一种新的DNA分子标记技术——起始密码子-微卫星扩增多态性

综合SCoT和ISSR分子标记技术开发了一种既能将标记位点与表达序列紧密联系,又具有相对较高的多态性的新的分子标记技术——起始密码子一微卫星扩增多态性（start codon-simple sequence repeat, SC-SSR）。SC—SSR标记是基于PCR的目的基因标记系统．上游引物用SCoT标记引物,瞄准基因区域,下游引物用ISSR标记引物,上下游引物间可自由组配。引物设计原则同SCoT标记和ISSR标记。使用50℃的退火温度,保证了扩增结果的稳定性。PCR结果采用琼脂糖凝胶电冰和聚丙烯酰胺凝胶电泳检测。SC—SSR分子标记结合了ISSR标记和SCoT标记的优点,具有操作简单、成本低廉、多态性丰富、重复性好、引物设计简单且通用性良好、同时与表达序列紧密连锁等诸多优点,可用于种质资源的鉴定评价、遗传图谱的构建、重要性状基因标记、gDNA与cDNA指纹分析乃至图位克隆等方面。     

利用RAPD分子标记对番茄杂交种纯度的鉴定研究

应用RAPD(RandomlyamplifiedpolymorphicDNA)分子标记对番茄京丹1号和毛粉802的F1代杂交种纯度进行鉴定的实验研究。该项研究使用了10个碱基的单随机引物和10个碱基的双随机引物进行扩增。在60个单引物扩增反应中获得7个京丹1号父本特有的核酸标记片段。但在14个双随机引物对京丹1号和毛粉802杂交组合的扩增反应中获得了7个京丹1号F1代杂交种特有的核酸标记片段和5个毛粉802父本特有的标记带。实验结果显示,双引物的扩增反应对鉴定双亲亲缘关系极近的杂交种纯度较单引物扩增反应更有效。其中,京丹1号的14个标记片段在北京蔬菜研究中心,种子纯度检测室又进行了重复扩增实验。实验结果为87%的RAPD标记可以在使用不同的PCR仪和不同来源的Taq酶的实验条件下得到。RAPD分子标记技术对鉴定双亲亲缘关系极近的杂交种纯度是真实可靠的。     

利用DNA ISSR分子标记技术对芍药属植物栽培品种的分类鉴定方法

以利用DNA ISSR分子标记逐级区分品种为主要研究目标,通过ISSR引物的设计与实验筛选,取得了进展。该方法类似于经典的植物形态检索表式的分类策略,不同之处在于利用基因组DNA分子性状作为品种分类的依据,即"ISSR引物结合位点"与"DNA ISSR片段长度差异"的组合信息。该方法不仅可以有效鉴定品种,而且可以很容易地生成富含遗传变异信息的0、1矩阵,通过运算揭示品种间的遗传关系与演化规律。该方法成本低廉、操作便捷,对于建立客观、科学的牡丹及芍药品种分类体系,对于芍药属品种种质资源的保存、评价与合理利用具有重要价值。应该加大支持力度推进其实际应用。     

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。     

乌塌菜种质资源遗传多样性的ISSR分析

利用ISSR技术对48份乌塌菜种质资源进行遗传多样性分析。从60条随机引物中筛选出稳定性强、条带清晰且多态性丰富的9条引物进行PCR扩增,共扩增出103条谱带,平均每个引物扩增出11.4条带,其中多态性带85条,多态性位点百分率为82.68%。不同乌塌菜种质间遗传相似系数变幅为0.59~0.97,说明ISSR标记能够揭示材料间较高的遗传多样性。利用UPGMA聚类分析,ISSR标记能将48份乌塌菜品种完全区分开,48份乌塌菜种质被划分为4个类群,聚类结果与叶片颜色相关,为乌塌菜品种资源的研究利用提供参考。     

分子标记对草原野生食用菌蒙古口蘑的鉴别分析

对采自内蒙古锡林郭勒草原的野生食用菌蒙古口蘑(Tricholoma mongolicum Imai.)等10个菌种进行了ITS和ISSR分子标记的鉴定,其中蒙古口蘑共计4个不同来源菌种,其它6个菌种为子实体与蒙古口蘑形态相近的常见草原菇类或市售食用菌,鉴定结果用RAPD进行辅助分析验证。从12条ISSR引物中筛选出2条可用引物,扩增总条带数为246条,多态性标记为215个,多态性频率为87.4%。经过NT-SYS软件聚类分析,发现在ISSR分析中结合ITS方法可以有效地将蒙古口蘑与其它菌种区分开,这与RAPD法的分析结果相一致,获得了可靠的野生食用菌分子标记方法。研究表明ISSR分子标记是蒙古口蘑等野生食用菌鉴定的理想手段之一。     

A new strategy employed for identification of sweet orange cultivars with RAPD markers

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.     

ISSR鉴定亲缘关系非常近的芒果栽培品种

用ISSR技术鉴定7个吕宋芒品种(系)和柳州吕宋芒。从30个引物中筛选出6个多态性好的ISSR引物建立DNA指纹图谱用于区分吕宋芒品种(系)。分析DNA指纹图谱,发现这6个引物中每个引物都能区分吕宋系列品种(系),表明ISSR-PCR技术对芒果品种(系)的鉴定非常有效,能区分亲缘关系很近的品种(系)。基于69条多态性条带的聚类分析结果,发现吕宋芒和其它供试的7个品种(系)同源性低,而这7个品种(系):高州吕宋芒、湛江吕宋芒、田阳香芒、金钱芒、柳州吕宋芒、粤西一号、攀西红吕宋同源性较高,可归为一类。     

Genetic variation and cultivar identification in Cymbidium ensifolium

As a popular flowering species with many cultivars, Cymbidium ensifolium (L.) is commercially important in horticulture. However, so far little has been known about genetic diversity and conservation genetics of this species. Understanding of the genetic variation and relationships in cultivars of C.?ensifolium is a prerequisite for development of future germplasm conservation and cultivar improvement. Here we report assessment of genetic variations in C.?ensifolium cultivars using the DNA fingerprinting technique of inter-simple sequence repeats (ISSR). A total of 239 ISSR loci were identified and used for evaluation of genetic variation with a selection of 19 ISSR primers. Among these ISSR loci, 99.16% were polymorphic with wide genetic variation as shown by Nei??s gene diversity (H?=?0.2431) among 85 tested cultivars. ISSR fingerprinting profiles showed that each cultivar had its characteristic DNA pattern, indicating unequivocal cultivar identification at molecular level. Eighteen cultivar-specific ISSR markers were identified in seven cultivars. The cultivar Sijiwenhan was confirmed as hybrid by four ISSR primers. Several cultivars with same name but different geographical origins were distinguished based on their ISSR profiles. A dendrogram generated with ISSR markers could group 73 of 85 cultivars into four major clusters. Further analysis of ISSR variation revealed that about 69% of total genetic variation in this species is due to genetic divergence inside geographical groups. Our results suggest that both germplasm collection and in?situ conservation are important for future planning of C.?ensifolium species conservation.     

Molecular diversity and relationships among <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> cultivars based on inter-simple sequence repeat (ISSR) markers

Spring orchid (Cymbidium goeringii) is a popular flowering plant species. There have been few molecular studies of the genetic diversity and conservation genetics on this species. An assessment of the level of genetic diversity in cultivated spring orchid would facilitate development of the future germplasm conservation for cultivar improvement. In the present study, DNA markers of intersimple sequence repeats (ISSR) were identified and the ISSR fingerprinting technique was used to evaluate genetic diversity in C. goeringii cultivars. Twenty-five ISSR primers were selected to produce a total of 224 ISSR loci for evaluation of the genetic diversity. A wide genetic variation was found in the 50 tested cultivars with Nei’s gene diversity (H = 0.2241) and 93.75% of polymorphic loci. Fifty cultivars were unequivocally distinguished based on ISSR fingerprinting. Cultivar-specific ISSR markers were identified in seven of 50 tested cultivars. Unweighted pair-group mean analysis (UPGMA) and principal coordinates analysis (PCA) grouped them into two clusters: one composed the cultivars mainly from Japan, and the other contained three major subclusters mainly from China. Two Chinese subclusters were generally consistent with horticultural classification, and the third Chinese subcluster contained cultivars from various horticultural groups. Our results suggest that the ISSR technique provides a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in C. goeringii.     

Identification of closely related citrus cultivars with inter-simple sequence repeat markers

 Inter-simple sequence repeat (ISSR) markers generated by 22 primers were tested for their ability to distinguish among samples from 94 trees of 68 citrus cultivars. Within each of the six cultivar groups studied, most of these cultivars are so closely related that they are difficult to distinguish by other molecular-marker techniques. ISSR markers involve PCR amplification of DNA using a single primer composed of a microsatellite sequence anchored at the 3′ or 5′ end by 2–4 arbitrary, often degenerate, nucleotides. The amplification products were separated on non-denaturing polyacrylamide gels and detected by silver staining. ISSR banding profiles were very repeatable on duplicate samples. Different citrus species had very different fingerprint patterns. Within Citrus sinensis (L.) Osbeck and C. paradisi Macf., in which all cultivars have originated by the selection of mutants, ISSR markers distinguished 14 of 33 sweet orange and 1 of 7 grapefruit cultivars. Five of six lemon cultivars were discriminated by ISSR markers. Many differences were found among mandarin cultivars; however, all five satsuma cultivars analyzed had identical ISSR fingerprints. Four of five citrange cultivars were distinguishable, but ‘Troyer’ and ‘Carrizo’ had identical ISSR fingerprints. ‘Kuharske Carrizo’ citrange, which has better citrus nematode resistance than other ‘Carrizo’ citrange accessions, had unique ISSR fingerprints. Three ISSR markers that differentiated certain sweet orange cultivars were hybridized to Southern blots of sweet orange DNA digested with different restriction endonucleases. The sweet orange cultivars tested could be distinguished by these ISSR-derived RFLP markers. Moreover, one ISSR marker unique to ‘Ruby’ blood orange was observed in its progeny trees. Received: 9 September 1996 / Accepted: 4 April 1997     

''''挽春''''(Paeonia rockii ''''Wan Chun'''')——中国西北紫斑牡丹品种群中的一个优良品种

紫斑牡丹品种群是中国的主要牡丹品种群之一。文中描述了一个优良紫斑牡丹品种‘挽春’(Paeoniarockii‘W an Chun’)的特征、习性和表现,该品种在紫斑牡丹品种群中具有代表性。紫斑牡丹品种可用于园艺观赏、经济林营造、荒山绿化以及荒漠治理。根据多年的调查和相关文献,按照紫斑牡丹品种的生长适应程度和引种栽培难易程度,将中国有可能引种栽植紫斑牡丹品种的地区划分为4类。介绍了各类地区在引种栽培方面需要注意的关键技术措施,分析了紫斑牡丹品种向全国各地推广过程中存在的问题,并提出了建议。     

Assessment of Genetic Polymorphism in Hop (Humulus lupulus L.) Cultivars by ISSR–PCR Analysis

Genetic diversity among 26 Russian and European cultivars of the common hop (Humulus lupulusL.) was studied using the ISSR–PCR technique. Twenty-one primers used provided amplification of 183 DNA fragments, 106 of which (57.9%) were found to be polymorphic. The ISSR markers, specific for some cultivars were revealed. Based on the genetic distances, cluster analysis was performed and a dendrogram was constructed, on which most of the hop cultivars formed two clusters according to their origin. Advantages of the ISSR–PCR analysis in breeding studies for classification and identification of common hop cultivars are discussed.     

‘挽春’(Paeonia rockii‘Wan Chun’)——中国西北紫斑牡丹品种群中的一个优良品种

紫斑牡丹品种群是中国的主要牡丹品种群之一。文中描述了一个优良紫斑牡丹品种‘挽春’(Paeonia rockii‘Wan Chun’)的特征、习性和表现,该品种在紫斑牡丹品种群中具有代表性。紫斑牡丹品种可用于园艺观赏、经济林营造、荒山绿化以及荒漠治理。根据多年的调查和相关文献,按照紫斑牡丹品种的生长适应程度和引种栽培难易程度,将中国有可能引种栽植紫斑牡丹品种的地区划分为4类。介绍了各类地区在引种栽培方面需要注意的关键技术措施,分析了紫斑牡丹品种向全国各地推广过程中存在的问题,并提出了建议。     

Development of Simple Sequence Repeat Markers from Functional Genes and Establishment of Molecular Identity for Tree Peony

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are generally superior to random markers because they are located in genes and therefore may affect gene expression or function. However, extremely limited genic SSRs are available for tree peony. We used the functional gene sequences available from Paeonia to develop genic SSRs. A total of 132 SSR loci were identified from 35 cDNA sequences, of which trinucleotide (58, 43.9%) and hexanucleotide repeat (37, 28.0%) were dominant. Moreover, 121 primer pairs were successfully designed and synthesized, of which 49 primer pairs (40.5%) provided efficient and reliable amplification. By screening 16 tree peony varieties, we developed eight polymorphic genic SSRs with 37 alleles, ranging from 2 to 11 for each marker. Transferability analysis indicated that 100% of the genic SSRs could be amplified in eight other Paeonia samples. Based on eight polymorphic genic SSRs and 12 polymorphic EST-SSRs developed by predecessors, the molecular identity of 190 tree peony cultivars was constructed by capillary electrophoresis. The results showed that 146 alleles and 338 genotypes were detected, with 2–13 alleles and 3–36 genotypes for each marker. All cultivars were completely identified and exhibited unique DNA identity. In addition, the identification efficiency of different primers combinations was analyzed, and 190 germplasms were identified using 6 core primers. This study provides valuable genic SSR resources for marker-assisted selection breeding of the genus Paeonia. The DNA identity of cultivars is of great significance for the protection, utilization and management of tree peony resources.