Assessment of the genetic diversity and genetic relationships of Lilium in China using ISSR markers

China is a center of natural distribution and diversity of genus Lilium around the world. In the study, the genetic diversity and genetic relationships of Lilium in China were analyzed by inter-simple sequence Repeat (ISSR) markers. The 6 highly polymorphic ISSR primers were selected to amplify the 20 Lilium species. The results showed that a total of 114 DNA bands were amplified, all of which were polymorphic loci (P = 100%), the effective number of alleles (Ne) was 1.2753, and the difference value between observed number of alleles (Na) and effective number of alleles (Ne) was 0.7247, and the Nei's genetic diversity (H) was 0.2048, and the Shannon's information index (I) was 0.3503. These results indicated that there is significant genetic difference among Lilium species in China. Taking the average genetic similarity coefficient (Gs) 0.5313 as the threshold, the 20 tested Lilium species were clustered into 5 groups, which was not entirely consistent with traditional clustering by morphological traits. The results obtained from this study can provide a reference for the molecular study of Lilium germplasm resources.     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

走马胎种质资源遗传多样性的ISSR分析

走马胎( Ardisia gigantifolia)是紫金牛科( Myrsinaceae)紫金牛属( Ardisia)多年生小灌木植物。走马胎作为我国传统中药材,已有多年的药用历史。目前,走马胎不再局限于临床药用,在食疗和保健方面的开发利用崭露头角,大大扩展了其应用范围。随着走马胎市场需求量的增大,野生走马胎植物被过度采挖,导致野生走马胎资源几乎枯竭。人工栽培走马胎逐渐成为供应药用市场的主力军,但是人工栽培走马胎种质、种子来源混杂,常会造成质量和疗效的不稳定,而利用分子标记技术可以从分子水平上对走马胎进行种质的区分和评价。该研究利用ISSR分子标记技术,对来自广西地区的36份走马胎种质资源进行了遗传多样性分析,采用POPGEN32软件进行数据分析,用UPGMA软件绘制聚类图。结果表明：14条ISSR引物共检测到136个清晰的扩增位点,多态性位点112个,多态位点百分率为82.35％;Nei’ s基因多样性指数( H)为0.2965,Shannon多样性指数(I)为0.4417,基因分化系数(Gst)为0.8558。个体间的遗传相似系数为0.6678~0.8382,平均为0.7391。基于聚类分析可知,所有的个体被划分为5类,其中绝大多数来自相同或者邻近地区的个体严格按照地理位置聚为相同的一类或者亚类,只有少数个体在归类上与地理位置相悖。研究证明ISSR分子标记技术在评价走马胎种质资源亲缘关系和遗传变异等方面有很好的适用。该研究结果为该药用植物的种质资源评估和引种栽培提供了科学依据。     

Genetic diversity in Vicia amoena (Fabaceae) germplasm resource in China using SRAP and ISSR markers

The genetic diversity in 17 germplasms of Vicia amoena L. from north China was analyzed using SRAP and ISSR markers. Three hundred and sixty-eight (94.11%) polymorphic bands (211 and 157 obtained from 20 pairs of SRAP and ISSR primers, respectively) were scored. Although SRAP was more effective than ISSR markers with higher PIC, RP and larger variation range of genetic distance, both the markers were useful for assessing V. amoena genetic diversity. Cluster analysis showed that the 17 germplasms were clustered into 5 groups. The results of principal coordinate analysis supported UPGMA clustering. The germplasms from source areas where the annual average temperature ranged from −1.0 to 5 °C exhibited the highest level of genetic diversity with the highest PPI, I and H. These results have important implications in genome mapping, breeding purposes, and germplasm conservation.     

Assessing the genetic diversity of tobacco germplasm using intersimple sequence repeat and inter-retrotransposon amplification polymorphism markers

The genetic diversity of 118 tobacco accessions, including flue‐cured tobacco, sun‐/air‐cured tobacco, burley tobacco, oriental tobacco and wild tobacco, was characterised using intersimple sequence repeat (ISSR) and inter‐retrotransposon amplification polymorphism (IRAP) markers. ISSR and IRAP banding patterns and genetic distance (GD) values showed the low level of genetic diversity within and among cultivated tobacco types. There was higher GD and average heterozygosity among wild tobacco types than those among cultivated tobacco. Genetic diversity of tobacco germplasm was low, with a high level of genetic identity (>0.77) between the different types. However, neighbour‐joining cluster analysis of marker‐based GDs showed that the accessions from the same tobacco type, as classified by manufacturing quality traits, were nearly clustered into the same group. These results will help in the formulation of appropriate strategies for variety improvement in tobacco, and ISSR and IRAP markers of the genetic diversity will contribute to further study and improvement of tobacco.     

鸢尾属植物遗传多样性的 RAPD和ISSR分析

应用RAPD和ISSR标记技术,对来自不同产地的鸢尾属(IrisL.)4个野生种的遗传多样性进行了分析。结果表明,12个RAPD和ISSR引物分别扩增出225和196条带,多态性条带分别为215和196条,多态性条带百分率分别为95.56%和100.00%;种间总基因多样度分别为0.368 9和0.357 5、种内基因多样度分别为0.107 7和0.138 0,表明鸢尾属种间遗传多样性较高,且种间变异大于种内变异。4个野生种中,蝴蝶花(I.japonicaThunb.)的遗传组成较为丰富。此外,种内遗传关系与地理分布和环境差异有一定的相关性。     

基于ISSR遗传多样性分析的云南蓝果树保护措施探索

保存种群的遗传多样性和进化潜力是成功拯救保护濒危物种的关键。云南蓝果树(Nyssa yunnanensis W.C.Yin)是国家I级重点保护的极度濒危野生植物,也是国家实施极小种群野生植物保护工程的目的物种。为了制定针对性的保护措施,采用ISSR分子标记方法对64株极度濒危物种云南蓝果树子代个体进行遗传多样性分析。从100条引物中筛选出12条ISSR引物,共扩增出77个稳定、清晰的条带。其中58个为多态性条带,物种水平上的多态性条带百分率(PPL)变动范围为50.00%~87.50%,平均值为(74.65±0.01)%;等位基因观察值(Na)为1.7532;有效等位基因观察值(Ne)为1.5804;Nei's基因多样性指数(He)为0.3206,多态性信息含量指数(PIC)变动范围为0.15~0.44,平均值为0.27±0.01;Shannon's遗传多样性信息指数(I)随着样本数的增加而升高,但当样本数达到24及以上时,Shannon's指数的数值基本不再变化。基于遗传多样性分析结果,从地质历史原因和人为干扰因素等方面对云南蓝果树的濒危机制进行了探析,并提出了有针对性的云南蓝果树保护措施。     

Assessment of genetic diversity in Colletotrichum falcatum Went accessions based on RAPD and ISSR markers

Sugarcane is susceptible to red rot disease caused by phytopathogenic fungus Colletotrichum falcatum Went which ultimately affect the economy of farmers as well as sugar based industry. One of the various ways to control this devastating disease is to develop disease resistance sugarcane cultivar and this requires the complete understanding of genetic makeup of pathogen. Although South Gujarat is well known sugarcane cultivating area, less published data can be found about PCR-based genetic diversity in prevalent C. falcatum accessions. So, present investigation aims at finding molecular variation among the ten accessions of C. falcatum using RAPD and ISSR molecular markers. A total of 35 RAPD and 39 ISSR primers were screened across 10 C. falcatum accessions, of which 15 RAPD and 21 ISSR primers have showed consistent amplification. Statistics related to genetic variation were estimated using NTSYS-PC by means of Dice’s coefficient. The results revealed 80.6% and 68.07% polymorphism and similarity coefficient ranged from 0.43 to 0.91 and 0.73 to 0.93 in RPAD and ISSR analysis respectively. The dendrogram generated using RAPD, ISSR and combined RAPD-ISSR grouped accessions into different clusters which reveal considerable level molecular variation among the C. falcatum accessions. It is also evident from PCA plots that accessions are rather dispersed with tested marker systems indicating good genetic base. So, in nut shell, we found considerable genetic variation and relatedness within C. falcatum accessions collected from different areas of south Gujarat, India using RAPD and ISSR markers.     

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessions including Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions. Received: 5 April 2001 / Accepted: 13 July 2001     

Assessment of genetic diversity in indigenous turmeric (Curcuma longa) germplasm from India using molecular markers

Curcuma longa L., commonly known as turmeric, is one of the economically and medicinally important plant species. It is predominantly cultivated in the tropical and sub tropical countries. India is the largest producer, and exporter of turmeric in the world, followed by China, Indonesia, Bangladesh and Thailand. In the present study, Directed Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR), methods were used to estimate the genetic variability in indigenous turmeric germplasm. Cumulative data analysis for DAMD (15) and ISSR (13) markers resulted into 478 fragments, out of which 392 fragments were polymorphic, revealing 82 % polymorphism across the turmeric genotypes. Wide range of pairwise genetic distances (0.03–0.59) across the genotypes revealed that these genotypes are genetically quite diverse. The UPGMA dendrogram generated using cumulative data showed significant relationships amongst the genotypes. All 29 genotypes studied grouped into two clusters irrespective of their geographical affiliations with 100 % bootstrap value except few genotypes, suggesting considerable diversity amongst the genotypes. These results suggested that the current collection of turmeric genotypes preserve the vast majority of natural variations. The results further demonstrate the efficiency and reliability of DAMD and ISSR markers in determining the genetic diversity and relationships among the indigenous turmeric germplasm. DAMD and ISSR profiling have identified diverse turmeric genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable turmeric genotypes.     

Genetic diversity analysis of Perinereis aibuhitensis based on ISSR and SRAP markers of Chinese coast populations

The objective of this study was to obtain an overview of the genetic relationships within Perinereis aibuhitensis using Inter-Simple Sequence Repeat (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) markers that were derived from related populations residing in the Chinese coasts. The percentage of polymorphic bands, Nei's gene diversity and Shannon's information index revealed a high level of genetic diversity at the species level. The analysis of molecular variance revealed that 81.22% (ISSR) and 76.29% (SRAP) of variability were partitioned among individuals within populations, which indicated the coherent trend by Nei's genetic differentiation (Gst) (0.2568/0.2876). The gene flow number (Nm) was 1.4470/1.2385, which indicated that there was limited gene exchange between populations. The phylogenetic tree of the ten P. aibuhitensis populations was separated into four major clusters using the neighbor-joining (NJ) method. These results provide a simple and useful basis for P. aibuhitensis germplasm research and aquaculture breeding.     

65份野生油茶种质遗传多样性的ISSR和RAPD标记分析

利用ISSR和RAPD标记,对名邛台地野生油茶种质进行遗传多样性分析。从60条简单重复序列引物中筛选出16条引物,在65份样品中共扩增出213条带,其中多态位点为203个,多态位点百分率为95.31%;从30条寡居核苷酸引物中筛选出8条引物,共扩增出105条带,其中多态性位点94个,多态位点百分率为89.52%。结果表明:名邛台地野生油茶种质具有较丰富的遗传多样性,ISSR和RAPD标记可以应用于油茶种质遗传多样性分析。     

ISSR analysis of genetic diversity in sacred lotus cultivars

In this study, inter-simple sequence repeats (ISSR) markers were applied to assess genetic diversity and genetic relationships of 92 cultivars of sacred lotus (Nelumbo nucifera Gaertn.), one of the most famous flowers in China. Our results showed that sacred lotus exhibited a low level of genetic diversity (percentage of polymorphic bands, PPB = 55.8%), which may result from its asexual mode of reproduction and long-term artificial selection. Clustering analyses indicated that these cultivars could be divided into two clades. Most cultivars of Chinese lotus species origin were included in one clade, and one cultivar of American lotus species origin was nested in the other clade. The hybrid cultivars from hybridization between the two subspecies were interspersed in these two clades. Seven cultivars native to Thailand formed a distinct subclade among the cultivars of Chinese lotus species origin. Genetic differentiation between two subspecies, and between cultivars from Thailand and other cultivars could be attributed to geographic isolation. The monophyly of three cultivars of Sanshui Winter Lotus and their closest relationships to Chinese lotus species origin suggests that they might have a common origin and may consist completely or mainly of genetic material from N. nucifera subsp. nucifera.     

用ISSR标记技术分析山药品种遗传多样性

利用ISSR标记技术对28个山药品种的遗传多样性进行分析。结果表明,从44条ISSR引物中可筛选出7条能够扩增出清晰、稳定条带的引物;这7条ISSR引物对28个山药品种扩增条带间存在较大差异,多态性条带比率为83．01％,Shannon多样性指数为0．3191;构建的分子树状图将28个山药品种划分为4组：第一组含有日本白、花山药和日本园3个品种;第二组为小叶山药;第三组为嵩野1号;其余23个品种归入第四组。而且主成分分析结果支持上述的聚类分析结果。这为利用ISSR标记技术鉴定山药品种,为有效地利用山药种质资源提供了依据。     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

An analysis of genetic diversity in Marphysa sanguinea from different geographic populations using ISSR polymorphisms

The genetic diversity of five wild populations of marine polychaete Marphysa sanguinea found in China was investigated using Inter-Simple Sequence Repeat-PCR (ISSR-PCR) polymorphisms. The results of the ISSR-PCR showed that 108 (90.8%) of the 119 ISSR loci tested were polymorphic. The Shannon's information index value was 0.4981, Nei's gene diversity was 0.3418, and the coefficient of gene differentiation (Gst) was 0.3671, which indicated that the among-population component accounted for 36.7% of the total variation, while the within-population component accounted for 63.3%. A UPGMA tree showed that the five populations clustered into two branches. Populations from Dalian, Xingcheng, and Rushan clustered together, while the two Guangxi populations, A and B, clustered into a unique group. The results indicated that the genetic diversity among the five populations of M. sanguinea is high, which will provide useful information for the protection of biodiversity among marine polychaetes.     

The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers

Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identification of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson's diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets.     

云南黑籽南瓜种质遗传多样性的RAPD和ISSR分析

摘要: 采用RAPD和ISSR分子标记技术对来源于云南省6个地州13份的黑籽南瓜种质进行遗传多样性分析。结果表明：6个RAPD和6个ISSR引物分别扩增出43条和41条带,多态性比率分别为90.70%和51.21%;RAPD和ISSR标记检测供试材的遗传相似性系数(Gs)范围,分别为0.340-0.895和0.162-0.941,ISSR(平均GS值0.698)检测多态性效果高于RAPD(平均GS值0.481)。RAPD标记聚类分析将供试种质分为3个类群5组;ISSR标记聚类分析将供试种质分为4个类群6组,RAPD和ISSR标记的遗传相似性系数呈显著相关(r=0.536)。基于UPGMA聚类结果,可为黑籽南瓜的引种栽培或品种改良提供参考。     

Detecting DNA polymorphism and genetic diversity in Lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker

Genetic diversity and interrelationships among 31 lentil genotypes were evaluated using 10 Inter-Simple Sequence Repeat (ISSR) and 10 directed amplification of minisatellite DNA region (DAMD) primers. A total of 43 and 48 polymorphic bands were amplified by ISSR and DAMD markers, respectively. Average polymorphism information content (PIC) for ISSR and DAMD markers were 0.37 and 0.41, respectively. All 31 lentil genotypes could be distinguished by ISSR markers into three groups and by DAMD markers into two groups. Various molecular markers show a different efficiency for evaluating DNA polymorphism in lentil and indicate that the patterns of variation are clearly influenced by the genetic marker used. Comparatively, the genetic diversity of examined lentil genotypes by two different marker techniques (ISSR and DAMD) was high and indicated that ISSR and DAMD are effective and promising marker systems for fingerprinting in lentil and give useful information on its genetic relationships.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.