Examining genetic relationships of Chinese Pleurotus ostreatus cultivars by combined RAPD and SRAP markers

Combined randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) were used to assess the genetic diversity of Pleurotus ostreatus strains cultivated in China. For the RAPD and SRAP analyses, 479 and 282 polymorphic bands were obtained from 20 P. ostreatus strains using 20 and 13 selected primers or primer pairs, respectively. A combined RAPD/SRAP dendrogram grouped the 20 strains into five clades with a coefficient of 0.690. The comparison of RAPD and SRAP was evaluated in the present study. The combined RAPD/SRAP markers provided reliable information regarding the relationships among the P. ostreatus strains.     

Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD,ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5 %) RAPD, 211 (74.6 %) ISSR, and 98 (74.8 %) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.     

Comparison of ISSR and RAPD markers to characterize three Chilean Nothofagus species

The present study is the first report of fingerprinting on three Chilean Nothofagus species using ISSR and RAPD markers; 61 Nothofagus nervosa, 32 Nothofagus obliqua and 32 Nothafagus dombeyi individual trees, sampled from collection and natural sites, were analyzed. Among 45 primers tested, the 6 ISSR and 6 RAPD primers selected for the analysis generated a total of 63 ISSR and 42 RAPD fragments. A high proportion of polymorphic bands, ranging from 97% and 80%, was found using both markers. A similar number of private and marker bands was generated by both markers in all the species examined and one discriminant ISSR fragment was obtained for N. dombeyi. Jaccard and Dice similarity indices were used to evaluate pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate the intra- and inter-specific genetic diversity, and PCA analysis was employed to evaluate the resolving power of the markers to differentiate between the species. These analyses, carried out for both markers, allowed us to identify three main groups corresponding to the three Nothofagus species. The results of the present study can be seen as a starting point for future researches on the population and evolutionary genetics of these species.     

Comparison of RAPD,ISSR, and AFLP Molecular Markers to Reveal and Classify Orchardgrass (Dactylis glomerata L.) Germplasm Variations

Three different DNA-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) markers, were used for fingerprinting Dactylis glomerata genotypes and for detecting genetic variation between the three different subspecies. In this study, RAPD assays produced 97 bands, of which 40 were polymorphic (41.2%). The ISSR primers amplified 91 bands, and 54 showed polymorphism (59.3%). Finally, the AFLP showed 100 bands, of which 92 were polymorphic (92%). The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Three cluster analyses were performed to express–in the form of dendrograms–the relationships among the genotypes and the genetic variability detected. All DNA-based techniques used were able to amplify all of the genotypes. There were highly significant correlation coefficients between cophenetic matrices based on the genetic distance for the RAPD, ISSR, AFLP, and combined RAPD-ISSR-AFLP data (0.68, 0.78, 0.70, and 0.70, respectively). Two hypotheses were formulated to explain these results; both of them are in agreement with the results obtained using these three types of molecular markers. We conclude that when we study genotypes close related, the analysis of variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for RAPD, ISSR and AFLP markers. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationship among genotypes of Dactylis glomerata.     

杏鲍菇菌种遗传多态性的ISSR分析

采用ISSR(Inter-simple Sequence Repeat)分子标记技术并应用NTSYSpc 2.10e生物软件对20株杏鲍菇菌种进行遗传聚类分析。结果表明,从22条引物中筛选出11条扩增条带清晰、多态性好的ISSR引物,共获得74个ISSR标记位点,51个多态性位点,多态比率为68.92%,产物片段大小在100~5 000 bp之间。聚类分析发现,遗传相似系数在0.828时,20株杏鲍菇菌种分为4个类群,当遗传相似系数达到0.896时,分为12个类群,杏528寿与其他菌种的亲缘系数最远,具有独立的遗传体系。     

IGS2-RFLP、SCoT和ISSR在白灵侧耳遗传多样性分析中的比较研究

应用IGS2-RFLP、SCoT和ISSR标记分析新疆5个采集地点的28个白灵侧耳野生样本的遗传多样性,比较这3种分子标记在白灵侧耳种质资源遗传多样性研究中的效用。结果显示,IGS2-RFLP的3个内切酶、5个SCoT引物、5个ISSR引物分别检测到42、59和77条多态性条带,多态性比率分别为91.3%、92.4%和92.8%。3种标记检测出的有效等位基因数(ne)和位点平均的预期杂合度(Hep)没有显著性差异。表明3种标记都适宜作为遗传学标记对白灵侧耳野生种质进行多样性分析。多态性检测效率最高的标记为ISSR,E=15.4,Ai=23.4,其次为IGS2-RFLP,E=14.0,Ai=22.4,SCoT则为最低,E=11.8,Ai=18.2。3种标记中,SCoT和ISSR标记的聚类结果极其相似,且均能较为准确地反映样本的地理来源,虽然二者的聚类图不完全相同。IGS2-RFLP的标记效率较高,准确性和可重复性最好,可用于菌株标准图谱的制作,更适用于品种权保护中的菌株鉴定鉴别;SCoT和ISSR标记的多态性高,信息量大,评价范围广,则更适用于白灵侧耳种质资源的遗传多样性研究。     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region

The genetic variability and relationships among 20 Mangifera indica genotypes representing 15 endangered and 5 cultivars, obtained from Indian Gir forest region, were analyzed using 10 random amplified polymorphic DNA (RAPD) and 21 inter simple sequence repeat (ISSR) markers. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection. Also, the average numbers of polymorphic loci per primer, average polymorphic information content (PIC) and primer index (PI) values were more for RAPD than for ISSR. But, total number of genotype specific marker loci, Nei’s genetic diversity (h), Shannon’s information index (I), total heterozygosity (Ht), average heterozygosity (Hs) and mean coefficient of gene differentiation (Gst) were more for ISSR as compared to RAPD markers. The regression test between the two Nei’s genetic diversity indexes showed low regression between RAPD and ISSR based similarities but maximum for RAPD and RAPD + ISSR based similarities. The pattern of clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared. Thus, both the markers were equally important for genetic diversity analysis in M. indica.     

Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers

Genetic variation within and between five populations of Oryza granulata from two regions of China was investigated using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pairwise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. Our results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Received: 29 March 2000 / Accepted: 15 May 2000     

Genetic variation,population structure and identification of yellow catfish, <Emphasis Type="Italic">Mystus nemurus</Emphasis> (C&;V) in Thailand using RAPD,ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.     

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。     

苦瓜种质遗传多样性的RAPD和ISSR分析

采用RAPD和ISSR分子标记技术对38份苦瓜种质进行遗传多样性分析。结果表明:10个RAPD和10个ISSR引物分别扩增出93条和81条带,多态性比率分别为50.54%和61.29%;RAPD和ISSR标记检测供试材料的遗传相似性系数(GS)范围,分别为0.287~1和0.221~1,ISSR(平均GS值0.672)检测多态性效果高于RAPD(平均GS值0.694)。RAPD标记聚类分析将供试种质分为3个类群6组,分类结果与苦瓜瓜瘤的表型分类比较相似;ISSR标记聚类分析将供试种质分为3个类群7组,ISSR标记划分类群与形态上以颜色分类比较接近。RAPD和ISSR标记的遗传相似性系数呈显著相关(r=0.550)。两个标记整合后聚类分析可检测到更大的遗传变异,结果与苦瓜的农艺性状分类和地理分布有一定的相关性。     

Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers

The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.     

采用RAPD和ISSR标记研究古尔班通古特沙漠东南缘梭梭种群的遗传结构

梭梭(Haloxylon ammodendron(CA Mey.)Bunge)是一种沙漠旱生优势树种,具有重要的生态和经济价值,然而,我们对梭梭种群的遗传多样性和遗传结构所知甚少.本文采用RAPD和ISSR标记对来自古尔班通古特沙漠东南缘的4个天然梭梭种群的遗传多样性和遗传结构进行了检测.5个RAPD引物和8个ISSR引物分别扩增出61和195条带,多态性位点比率分别为83.6%和89.7%,Shannon信息指数分别为0.333和0.367,RAPD和ISSR分析均表明梭梭种群的遗传多样性水平较高.利用分子方差分析(AMOVA)研究梭梭种群的遗传结构,结果表明,大部分遗传变异存在于种群内,通过RAPD分析发现138.2%的遗传变异发生在种群内;通过ISSR分析发现89.4%的遗传变异发生在种群内;而种群间的遗传分化很小.通过RAPD标记没有检测到种群间的遗传分化,ISSR分析表明10.6%的遗传变异发生在种群内.我们推测梭梭种群较高的遗传多样性水平可能源于对异质、高胁迫环境的长期适应,但还需要进一步的研究加以证实.种群间遗传分异低的主要原因是种群间存在强大的基因流.     

Molecular Marker Based Coefficient of Parentage Analysis for Establishing Distinctness in Indian Rice (Oryza sativa L) Varieties

Molecular characterization of 32 Indian rice varieties of different agro-climatic zones resulted in mean heterozygosity value of 0.622, 0.819 and 0.890 over polymorphic loci and marker index value of 1.00, 6.75 and 4.16, respectively for RAPD, ISSR and STMS primers. The three marker systems resulted in 201 polymorphic bands (94.36%) out of a total of 213 bands. The probability of a chance identical match between two varieties was very low (2.08x10¹⁰) in combined molecular marker analysis as compared to individual marker system (RAPD, 7.5 x 10^-4; ISSR, 1.5 x 10^-3 and STMS, 3.9 x 10^-6). The combined average genetic similarity for molecular markers and coefficient of parentage (COP) across all 496 pairwise combinations revealed a non-significant relationship (r = 0.215).     

RAPD and ISSR markers in the evaluation of genetic divergence among accessions of elephant grass

Considering the expected genetic variability of elephant grass (Pennisetum purpureum), due to its cultivation in different continents, we characterized and estimated the genetic divergences between 46 accessions of elephant grass with different edaphoclimatic adaptations, using RAPD and ISSR markers. We evaluated, comparatively, the consistency of the information achieved with these markers. Twenty-six RAPD and 25 ISSR primers were employed. The RAPD markers produced 185 bands, 72% of which were polymorphic, with a mean of 5.11 polymorphic bands per primer. The 25 ISSR starters produced 216 bands; 76% were polymorphic, with a mean of 6.56 polymorphic bands per primer. The correlation between the genetic distances achieved by the RAPD and ISSR markers was 0.76, which is highly significant by the Mantel test. Based on UPGMA grouping, considering the point of sudden change, five and six groups were formed for the data from the RAPD and ISSR markers, respectively. These markers provided partially concordant groups, indicating that these techniques can provide consistent information and consequently could be used in studies of genetic diversity among accessions.     

利用RAPD和ISSR分子标记分析怀地黄种质遗传多样性

用RAPD与ISSR技术对怀地黄的8个品种和2个脱毒品系进行了种质遗传多样性分析。分别从80条RAPD引物和44条ISSR引物中筛选出适合怀地黄种质分析的17条RAPD引物和10条ISSR引物,用于RAPD和ISSR分析。17条RAPD引物共扩增出177条带, 多态性位点数为109; 多态性位点比率为61.58%;平均多样性指数(I)为0.3135;每个位点的有效等位基因数（Ne）是1.3641; 10条ISSR引物共扩增出110条带. 多态性位点数为79; 多态性位点比率为71.58%;平均多样性指数(I)为0.3577;每个位点的有效等位基因数（Ne）是1.4037。 基于扩增条带数据库建立了各自的Jaccard遗传相关系数矩阵,构建了相似的分子树状图,将10个供试材料分为2类:一类群含组培85.5、大田85.5、组培9302、大田9302、金状元和金白6个材料;另一类群含北京1号、大红袍、地黄9104和野生地黄4个材料。两种分子标记的分析结果呈极显著正相关(r＝0.649)。结果表明,RAPD与ISSR标记适合于怀地黄种质遗传多样性分析,ISSR标记技术是一种多态性和重复性优于RAPD技术的实用技术。     

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis>)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.     

Efficiency of ISSR and RAPD markers in genetic divergence analysis and conservation management of Justicia adhatoda L., a medicinal plant

Genetic variation within and among population is the basis for survival of the population both in short and long term. Thus, studying the plant genetic diversity is essential for any conservation program. Indigenous medicinal plants like Justicia adhatoda L. which are facing high rate of depletion from the wild population need immediate attention. DNA-based dominant molecular marker techniques, random amplification of polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) were used to unravel the genetic variability and relationships across thirty-two wild accessions of J. adhatoda L., a valuable medicinal shrub widespread throughout the tropical regions of Southeast Asia. Amplification of genomic DNA using 38 primers (18 RAPD and 20 ISSR) yielded 434 products, of which 404 products were polymorphic revealing 93.11 % polymorphism. The average polymorphic information content value obtained with RAPD and ISSR markers was 0.25 and 0.24, respectively. Marker index (RAPD = 3.94; ISSR = 3.53) and resolving power (RAPD = 4.24; ISSR = 3.94) indicate that the RAPD markers were relatively more efficient than the ISSR assay revealing the genetic diversity of J. adhatoda. The Shannon diversity index obtained with RAPD and ISSR markers was 0.40 and 0.38, respectively. The similarity coefficient ranged from 0.26 to 0.89, 0.33 to 0.93 and 0.31 to 0.90 with RAPD, ISSR and combined UPGMA dendrogram, respectively. PCA derived on the basis of pooled data of both the markers illustrated that the first three principal coordinate components accounted 79.27 % of the genetic similarity variance. The mantel test between two Jaccard’s similarity matrices gave r = 0.901, showing the fit correlation between ISSR- and RAPD-based similarities. Based on the results, ex-situ methods may be the most suitable and efficient measure for long-term conservation.     

Comparison of RAPD and ISSR markers for assessment of genetic diversity among endangered rare Dalbergia oliveri (Fabaceae) genotypes in Vietnam

Dalbergia oliveri is a leguminous tree of the Fabaceae family. This species is popular and valuable in Vietnam and is currently listed on the Vietnam Red List and on the IUCN Red List as endangered. Two PCR techniques using RAPD and inter-simple sequence repeat (ISSR) markers were used to make a comparative analysis of genetic diversity in this species. Fifty-six polymorphic primers (29 RAPD and 27 ISSR) were used. The RAPD primers produced 63 bands across 35 genotypes, of which 24 were polymorphic. The number of amplified bands varied from one to four, with a size range from 250 to 1400 bp. The percentage polymorphism ranged from 0 to 75. Amplification of genomic DNA of the 35 genotypes, using ISSR analysis, yielded 104 fragments, of which 63 were polymorphic. The number of amplified fragments using ISSR primers ranged from one to nine and varied in size from 250 to 1500 bp. The percentage polymorphism ranged from 0 to 100. ISSR markers were relatively more efficient than RAPDs. The mental test between two Jaccard's similarity matrices gave r ≥0.802, showing good fit correlation between ISSRs and RAPDs. Clustering of isolates remained more or less the same for RAPDs compared to combined RAPD and ISSR data. The similarity coefficient ranged from 0.785 to 1.000, 0.698 to 0.956 and 0.752 to 0.964 with RAPD, ISSR, and the combined RAPD-ISSR dendrogram, respectively.