Molecular-genetic polymorphisms of cultivars of common hops (Humulus lupulus L.) using ISSR-PCR analysis

Genetic diversity among 26 Russian and European cultivars of the common hop (Humulus lupulus L.) 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 certain cultivars were revealed. Based on the coefficient of dissimilarity values, 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 selective studies aimed at the classification and identification of common hop cultivars are discussed.     

40个黄皮品种的ISSR分析

采用ISSR-PCR分子标记技术对40个黄皮品种的遗传多样性进行分析。从96条ISSR引物中筛选出15条引物用于PCR扩增,共扩增出165条带,其中多态性条带100条,多态性比率为60.6%。应用SPSS软件计算各品种间的Jaccard相似系数介于0.714~1.000,UPGMA法将40个品种分成5组。     

A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars

 Commercial scale fingerprinting of potato cultivars is made difficult by the need for speed, reliability and the ability to distinguish between large numbers of genotypes. There are also problems in extrapolating the results of small experimental studies to predict the performance of techniques or primers for larger applications. The potential of ISSR-PCR for fingerprinting purposes was evaluated using four primers on 34 potato cultivars. The complex band profiles generated were reproducible between repeat PCRs, DNA extractions, electrophoreses and gel scorings. Two primers were each able to distinguish all cultivars. The combined use of any two of the four primers also allowed complete diagnosis. It is concluded that ISSR-PCR provides a quick, reliable and highly informative system for DNA fingerprinting that is amenable for routine applications. Two possible correlates of the ability of primers to distinguish between genotypes were then examined. Marker Index failed to correlate significantly with genotype diagnosis, but a strong and seemingly linear relationship was observed between Resolving Power of a primer and its ability to distinguish genotypes (r²=0.98). Resolving Power of one or a pair of primers was found to provide a moderately accurate estimate of the number of genotypes identified. Possible implications for future studies on DNA fingerprinting are discussed. Received: 7 May 1998 / Accepted: 15 July 1998     

牡丹品种鉴定用ISSR引物的筛选与开发

用于牡丹品种鉴定的DNAISSR-PCR反应体系已经建立。利用DNAISSR分子标记分析少量牡丹品种时,容易获得各品种的特有ISSR标记。然而,中国牡丹品种约有1500个,在小批量品种范围内找到的品种特有ISSR标记有可能出现在其它品种中。因此,利用DNAISSR分子标记对数量庞大的中国牡丹品种进行区分和鉴定时,寻找品种特有标记成为突出的技术难题。标记是由引物通过PCR扩增产生的。因此,关键在于找到理想的ISSR引物。对已知的ISSR引物的筛选未获得良好的PCR扩增结果。报道牡丹鉴定用ISSR引物的设计与开发新途径。     

不同品种牡丹ISSR遗传多样性分析

旨在为牡丹的合理利用及其资源管理奠定基础。对洛阳地区35个品种的牡丹进行遗传多样性分析。利用改良的CTAB法和ISSR-PCR来提取DNA以及进行DNA扩增,从UBC公布的220条ISSR引物中最后筛选出了12条扩增条带清晰的引物。筛选后总共获得了130条清晰的扩增条带,多态性条带的占比为96.1%。统计条带之后,使用popgene32分析得出35种牡丹的平均Shannon信息指数H=0.465 9,平均Nei’s基因多样性指数He=0.294 5,说明35个牡丹品种遗传多样性丰富。利用NTSYSpc-2.10e分析软件进行聚类分析后,将洛阳的35个牡丹品种分为5大类。实验中的35种牡丹在进行分类时并不一定按照花色进行分类,只有在亲缘关系较近时才会聚在一起。     

蚬壳花椒ISSR-PCR反应体系的建立及优化

通过单因素试验及正交设计方法对影响蚬壳花椒ISSR-PCR扩增的主要因素(模板DNA、Mg2+的浓度、引物、dNTPs,TaqDNA聚合酶的用量以及退火温度)进行优化,以建立蚬壳花椒ISSR-PCR反应的最佳体系。结果表明:最佳反应体系(20μL)为模板DNA 60ng、MgCl2 2.5mmol/L、dNTPs 0.15mmol/L、引物0.6μmol/L、TaqDNA聚合酶2.4U。在此基础上,从100条引物中筛选出18条扩增稳定、多态性好的ISSR引物并经过10份蚬壳花椒种质检验,证明该体系具有扩增条带清晰、稳定、重复性好等优点。该反应体系的建立为蚬壳花椒种质资源分类、遗传多样性分析提供了更客观可靠的方法。     

The use of ISSR and RAPD markers for detecting DNA polymorphism,genotype identification and genetic diversity among barley cultivars with known origin

The potential of bulk analyses of RAPD and ISSR-PCR markers for fingerprinting purposes was evaluated using ten RAPD and ten ISSR primers. The phylogenetic relationships of 16 barley cultivars from different countries, and all having a known pedigree, were analysed using 353 PCR markers (125 RAPDs and 228 ISSRs). The band profiles generated were reproducible in spite of the different DNA extractions, PCR techniques, electrophoretic methods and gel scorings used. The RAPD primer S10 and four ISSR primers (811, 820, 835 and 881) were both able to distinguish all cultivars. A strong and quite linear relationship was observed between Resolving Power (Rp) of a primer and its ability to distinguish genotypes. The dendrograms obtained using these two molecular markers are in agreement with their known origin, showing clusters that separate very well the spring/winter and six-rows/two-rows cultivars. Thus, bulk analyses of RAPD and ISSR PCR markers provides a quick, reliable and highly informative system for DNA fingerprinting and also permit to establish genetic relationships which agree with, by other means, known origin of the cultivars.     

ISSR-PCR technique: a useful method for characterizing new allotetraploid somatic hybrids of mandarin

Somatic hybrids between Redblush and Duncan grapefruit (Citrus paradisi Macfadyen) with Avana, Tardivo di Ciaculli mandarin (C. deliciosa Tenore) and Fortune mandarin (C. reticulata Blanco) were obtained by symmetric protoplast fusion. Isozyme banding pattern analysis and flow cytometry were used for early screening. DNA of the hybrids was then extracted and subjected to inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) using different primers anchored at the 5' and 3' ends. PCR products were resolved on 1.5% agarose gels and stained with ethidium bromide. All the bands from the parental cultivars were present in the somatic hybrids. The usefulness and efficiency of the ISSR-PCR method as a screening technique for Citrus somatic hybrids are discussed.     

拟茎点霉ISSR-PCR反应体系的优化

旨在探讨TaqDNA聚合酶、dNTP、Mg2+、引物和模板DNA等因素对ISSR-PCR扩增的影响,采用单因子试验和正交设计方法相结合建立并优化拟茎点霉反应体系。在此基础上进行引物筛选,同时通过梯度PCR试验,确定引物的最佳退火温度。通过对19个拟茎点霉菌株的检验,结果表明已确立的体系稳定可靠,对不同模板均有较好的适用性和通用性,为利用ISSR技术对拟茎点霉进行遗传分析奠定基础。     

ISSR-PCR in differentiation of gene pools of cattle breeds

The genetic structure of five cattle breeds from Ukraine was investigated using ISSR-PCR method. Breeding- and sex-specific spectra of amplification products were revealed with two threenucleotide microsatellite primers. The possibility of using ISSR-PCR markers for characterization of genetic relations between breeds of cattle were evaluated.     

Allelic state of the molecular marker for golden nematode (Globodera rostochiensis) resistance gene H1 among Ukrainian and world potato (Solanum tuberosum ssp. tuberosum) cultivars

The purpose of this study was to determine the allelic state of the resistance gene H1 against the Ro1 and Ro4 pathotypes of the golden potato cyst nematode (Globodera rostochiensis) among Ukrainian and world potato (Solanum tuberosum ssp. tuberosum) cultivars. The allelic state of the TG689 marker was determined by PCR with DNA samples isolated from potato tubers and primers, one pair of which flanked the allele-specific region and the other one was used to control the DNA quality. Among 77 potato cultivars analyzed, the allele of the marker associated with the H1-type resistance was found in 74% of Ukrainian and 90% of foreign cultivars, although some of them proved to be susceptible to the potato cyst nematode in the field. The obtained data confirm the presence of H1 resistance against golden nematode pathotypes Ro1 and Ro4 among Ukrainian potato cultivars and the efficiency of the used marker within the accuracy that has been declared by its authors.     

白粉寄生孢ISSR-PCR体系的建立及遗传多样性的初步分析

通过正交设计和单因素水平优化的方法对白粉寄生孢ISSR-PCR程序中的一些反应参数和ISSR引物进行优化和筛选,并利用建立的ISSR-PCR的反应体系来分析白粉寄生孢的遗传多样性。5个ISSR引物对73个菌株的扩增条带表明,ISSR标记在中国白粉寄生孢中存在较高的多态性,ISSR标记揭示白粉寄生孢的遗传多样性和寄主植物(真菌)多样性存在一定的相关性。     

Identification of a DNA marker linked to sex determination in <Emphasis Type="Italic">Calamus tenuis</Emphasis> Roxb., an economically important rattan species in northeast India

Calamus tenuis (Roxb.), a versatile, dioecious rattan species predominant in northeast India, has emerged as an economical material for light furniture and cottage industries. For the genetic improvement of the species, it is essential to be able to recognize male and female plants at the seedling stage. Screening of genomic DNA with inter-simple sequence repeat (ISSR) primers was used to discover sex-specific polymerase chain reaction (PCR) amplification products. Thirty ISSR primers were screened on female and male C. tenuis plants from five different provinces of Assam, India. A putative female-specific marker was identified. The applicability of ISSR-PCR analysis for development of sex-linked molecular markers in Calamus is discussed.     

獐ISSR-PCR反应体系的建立与优化及引物筛选

利用正交试验L₁₆(4⁵)对影响獐(Hydropotes inermis)ISSR-PCR反应的Taq DNA聚合酶浓度、dNTP浓度、引物浓度、Mg²⁺浓度及模板DNA浓度5个因素在4个水平上进行优化,同时对退火温度进行梯度PCR反应,以建立适合于獐ISSR-PCR反应的最佳体系.最终确定獐25μL ISSR-PCR反应体系为:Taq酶1.25 U·25μL^-1、Mg²⁺浓度2.5 mmol·L^-1、引物浓度0.3μmol·L^-1、DNA模板量350 ng·25μL^-1、dNTP浓度0.15 mmol·L^-1.在此基础上,利用优化的反应体系成功筛选出10条用于獐相关研究的ISSR引物并确定了各自的最佳退火温度,为今后利用ISSR技术进行獐的物种鉴定与分类、亲缘关系、系统发育和生理病理学研究奠定了技术基础,也为开展其它大型资源动物如黑麂的保护遗传学研究提供理论基础.     

基于ISSR分子标记的叶子花亲缘关系分析和指纹图谱构建

为分析品种遗传多样性和遗传距离并构建品种聚类图和指纹图谱,该研究从DNA模板浓度、引物浓度、退火温度和循环次数等方面优化了叶子花ISSR-PCR反应体系和反应程序,利用11个ISSR引物对131个叶子花品种进行PCR扩增,扩增产物经琼脂糖凝胶电泳检测.结果表明:优化的ISSR-PCR反应体系中DNA模板浓度为0.5 n...     

西藏嵩草ISSR-PCR反应体系优化研究

西藏嵩草( Kobresia tibetica )为莎草科嵩草属多年生草本,根状茎短,秆密集丛生。生于海拔2550~4950 m的河滩地、湿润草地、高山灌丛草甸,分布于甘肃、青海、四川西部、西藏东部,其根系发达,喜湿,耐寒,生活力强。西藏嵩草繁殖以营养繁殖为主、有性繁殖为辅,其茎叶茂盛,有较高的营养价值,产草量高,是青藏高原夏、秋两季的主要放牧饲草。该研究以青藏高原高寒沼泽化草甸的建群种和优势种———西藏嵩草为材料,对影响其ISSR-PCR反应的因素( Mg2＋、Taq DNA聚合酶、dNTP、引物和模板DNA)进行5因素4水平的正交试验,以确定西藏嵩草ISSR分析的最佳反应体系,并筛选适宜的ISSR引物及各引物的最佳退火温度。结果表明：建立了适宜于西藏嵩草ISSR-PCR反应的最佳体系为20μL反应液中包括10× PCR buffer 2μL、1.5 mmol?L-1 Mg2＋、1.0 U Taq DNA聚合酶、0.100 mmol?L-1 dNTP、0.3μmol?L-1引物和30~40 ng DNA模板;同时从100条ISSR引物中筛选出了扩增结果清晰、稳定的12条引物,各引物的最佳退火温度为48.0~53.2℃(引物不同,其最佳退火温度也不同);西藏嵩草ISSR-PCR适宜的扩增程序为首先预变性94℃5 min,然后变性94℃20 s、复性48.0~53.2℃1 min、延伸72℃80 s、38个循环,最后72℃延伸6 min。体系稳定性验证结果表明,该体系在西藏嵩草其他样品中所得条带清晰且多态性丰富,为后续西藏嵩草的遗传多样性分析和优良牧草种质资源筛选研究奠定了基础。该研究结果对以西藏嵩草为优势种的高寒沼泽化草甸研究及湿地生态系统的修复和保护具有重要意义。     

Analysis of diversity of russian and Ukrainian bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars for high-molecular-weight glutenin subunits

The allelic diversity of high-moleculat-weght glutenin subunits (HMWGS) in Russian and Ukrainian bread wheat cultivars was analyzed. The diversity of spring wheat cultivars for alleles of the Glu-1 loci is characterized by medium values of the polymorphism polymorphism information content (PIC), and in winter wheats it varies from high at the Glu-A1 locus to low at the Glu-D1 locus. The spring and winter cultivars differ significantly in the frequencies of alleles of the glutenin loci. The combination of the Glu-A1b, Glu-B1c, and Glu-D1a alleles prevails among the spring cultivars, and the combination of the Glu-A1a, Glu-B1c, and Glu-D1d alleles prevails among the winter cultivars. The distribution of the Glu-1 alleles significantly depends on the moisture and heat supply in the region of origin of the cultivars. Drought resistance is associated with the Glu-D1a allele in the spring wheat and with the Glu-B1b allele in the winter wheat. The sources of the Glu-1 alleles were identified in the spring and wheat cultivars. The analysis of independence of the distribution of the spring and winter cultivars by the market classes and by the alleles of the HMWGS loci showed a highly significant association of the alleles of three Glu-1 loci with the market classes in foreign cultivars and independence or a weak association in the Russian and Ukrainian cultivars. This seems to be due to the absence of a statistically substantiated system of classification of the domestic cultivars on the basis of their quality.     

番茄叶霉病菌ISSR-PCR体系的建立

以番茄叶霉病菌(Passalora fulva)基因组DNA为模板,采用单因素试验和正交设计试验对该菌ISSR-PCR体系中的一些重要参数(Mg2+、dNTPs、引物、模板DNA、TaqDNA聚合酶、缓冲液、循环次数)和引物进行筛选和优化,并对退火温度进行了梯度优化,建立了番茄叶霉病菌ISSR-PCR的最佳反应体系(20μL):Mg2+1.5 mmol/L,dNTPs 0.4 mmol/L,引物1.5μmol/L,模板DNA45 ng,TaqDNA聚合酶1.0 U,1倍的PCR缓冲液,循环40次,退火温度50℃。     

正交设计优化紫萼藓科植物的ISSR-PCR反应体系

目的:建立紫萼藓科(Grimmiaceae)植物ISSR-PCR反应的最佳体系。方法:通过L16(45)正交试验,研究了dNTP浓度、镁离子浓度、模板DNA浓度、引物浓度、Taq DNA聚合酶浓度这5个因素在4个水平上对ISSR-PCR的影响。结果:建立了紫萼藓科ISSR-PCR反应的最佳反应体系,其中dNTPs浓度为0.8mmol/L、Mg2+浓度为3.0mmol/L、模板为15ng、引物浓度为1.4μmol/L、Taq酶量2U,总体积为25μl。反应程序为:扩增程序为:94℃预变性5min;94℃变性30s,48～52℃退火1min,72℃延伸2min,共40个循环;72℃延伸10min;4℃保存。采用引物UBC812等均能够得到有效扩增。结论:该优化体系的建立为下一步对紫萼藓进行ISSR分子标记奠定了基础。     

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.