Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.     

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

应用RAPD分子标记技术探讨3种石斛属植物的种间关系

采用RAPD分子标记技术,分析了金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的种间关系。10个引物产生的113条DNA扩增片段中,106条（93.81%）具有多态性,利用113个RAPD标记,计算遗传距离,利用非加权组平均法建立聚类图。结果表明,RAPD标记技术较好地从分子水平揭示金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的遗传背景、亲缘关系,并为后期在DNA水平上对药用石斛的开发利用提供资料。     

RAPD analysis of genetic variation between a group of rose cultivars and selected wild rose species

The genetic variability based on random-amplified polymorphic DNA markers was analysed among 10 cultivated rose varieties and 9 wild species from three different series of the genus Rosa. Using 13 different RAPD primers, 104 polymorphic DNA fragments with a high potential to differentiate rose genotypes could be produced. A dendrogram displaying the relative genetic similarities among the genotypes shows the existence of large genetic diversity among the cultivated roses as compared to the wild species. Furthermore, the main clusters found here are in agreement with known pedigrees and the classical taxonomy. However, the relationships between cultivated roses as inferred by RAPD markers do not correlate with the classical rose classification system. From the present data it is concluded that cultivated roses display a high level of genetic variability despite the fact that single morphological and physiological characters may be less polymorphic within rose groups. This contrasts with the widely accepted opinion of a lack of genetic variability in roses. This is also in accordance with the reported history of rose breeding which makes it highly probable that rose genomes comprise mosaics of different species genomes. As a consequence, it may be possible to utilize the high genetic variability of all genetic traits not under actual selection by breeders for future breeding programmes.     

用RAPD分子标记探讨沙拐枣属的种间关系

利用随机扩增多态性DNA（RAPD）技术分析了14种沙拐枣属（Calligonum L.)植物,通过对16个Sangon公司十聚体随机引物进行PCR扩增,3个引物能产生多态性带。对3个引物扩增产生的45条扩增产物,计算单匹配系数,应用UPGMA方法构建亲缘关系树状图。分析结果表明：（1）物种间遗传差异明显,具有丰富的遗传多样性;（2）14种沙拐枣属植物明显聚为4类,与传统的形态学分类结果基本一致。     

Assessment of genetic diversity in Lepidium sativum (Chandrasur) a medicinal herb used in folklore remedies in India using RAPD

Lepidium sativum L. is a fast growing edible herb which belongs to family Brassicaceae. The seeds of L. sativum are aperient, diuretic, tonic, demulcent, carminative, galatogogue and emmenagogue. They have been used in the treatment of bacterial and fungal infections, as an aperient and also possess antibacterial and antifungal properties. The seeds of this plant possess rapid bone fracture healing ability. Despite of its diverse medicinal properties no molecular data for diversity analysis is available till date. During this study random amplified polymorphic DNA (RAPD) markers were used to detect genetic variations of L. sativum. Initially 50 decamer primers were screened, out of which only 32 primers showed reproducible fragments with easily recordable bands. A total of 414 reproducible and clear bands were distinguished across the selected primers and statistical analysis showed 361 polymorphic bands and 53 monomorphic bands. Cluster analysis of the genotypes based on UPGMA divided the 18 genotypes into two main clusters, with first cluster having only HCS-20 genotype of L. sativum and other having rest of all 17 genotypes. The dendrogram based on similarity matrix revealed 23–66% genetic relatedness among 18 genotypes. The results of the present study can be used for molecular breeding and improvement of L. sativum for various desired traits through hybridization in future.     

Analysis of genetic diversity among selected grasspea (Lathyrus sativus L.) genotypes using RAPD markers

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variability among five selected genotypes of grasspea. Out of 30 random decamer primers tested for the present investigation 20 showed reproducible DNA amplification. A total of 257 loci were amplified of which 159 were polymorphic including 57 genotype-specific unique bands. Amplicons had molecular weights ranging from 3.0 kb to 0.1 kb. Majority amplicons were shared by most of the genotypes which indicated a very narrow genetic gap between them. The dendrogram constructed on the basis of RAPD data showed two clusters. The local genotype collected from Nayagarh was grouped along with IC-120451 and IC-120453, sharing a common node at an 82% similarity level. The other genotypes, IC-120478 and IC-120487, were located in the second clade having a common node at 84% similarity level. The investigation showed that though all the genotypes of grasspea were of apparently similar morphology there exists polymorphism at the molecular level, which can be exploited in breeding programmes aimed at crop improvement.     

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.     

Genomic relations among 31 species of Mammillaria haworth (Cactaceae) using random amplified polymorphic DNA

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.     

RAPD polymorphisms in spring wheat cultivars and lines with different level of Fusarium resistance

Random amplified polymorphic DNA (RAPD) markers have been used to characterize the genetic diversity among 35 spring wheat cultivars and lines with different levels of Fusarium resistance. The objectives of this study were to determine RAPD-based genetic similarity between accessions and to derive associations between Fusarium head blight (FHB) and RAPD markers. Two bulked DNA from either highly resistant lines or susceptible lines were used to screen polymorphic primers. Out of 160 screened primers, 17 primers generated reproducible and polymorphic fragments. Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm, which corresponded well with the results of principal component analysis and separated the 35 genotypes into two groups. Association analysis between RAPD markers and the FHB index detected three RAPD markers, H19(1000), F2(500) and B1(2400), significantly associated with FHB-resistant genotypes. These results suggest that a collection of unrelated genotypes can be used to identify markers linked to an agronomically important quantitative trait like FHB. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.     

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.     

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.     

Comparative Analysis of RAPD and ISSR Markers for Characterization of Sesame (Sesamum indicum L) Genotypes

The determination of genetic differences among crop genotypes has become the primary need to grant patent and the protection of Plant Breeder Rights (PBR). In the present study RAPD and ISSR markers were employed for the characterization of 16 sesame genotypes. Twenty six RAPD and 17 ISSR primers that generated clear and reproducible banding patterns amplified 194 and 163 bands, respectively among 16 sesame genotypes. Both RAPD and ISSR primers showed maximum discrimination power, and produced putative variety specific bands, which could be used for the identification of all the sesame genotypes, individually. However, only AG and CA based ISSR primers were found effective in the discrimination of genotypes. A poor correlation was observed between the matrices produced by RAPD and ISSR primers, which might be due to the array of different sites of the genome. Though, there was greater similarity among sesame genotypes (0.78 for RAPD and 0.71 for ISSR), the observed genetic diversity (0.22 for RAPD and 0.29 for ISSR), was found effective for the characterization of sesame genotypes. It is suggested that putative variety specific RAPD and ISSR markers could be converted to Codominant sequence characterized amplified region/sequence tagged site (SCAR /STS) markers to develop robust variety specific markers.     

Relationships among <Emphasis Type="Italic">Leymus</Emphasis> species assessed by RAPD markers

The DNA genetic diversity of 40 accessions of genus Leymus was analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 352 products were amplified by 34 10-mer arbitrary primers, among which 337 products (95.74 %) were found to be polymorphic. 5–14 polymorphic bands were amplified by each polymorphic primer, with an average of 9.91 bands. The data of 352 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Great genetic diversity in genus Leymus was observed, the genetic diversity among the different species more abundant than that of the different accessions, and the different accessions in a species or the species from the same areas were clustered together.     

Detection of genetic variability in various isolates of cattle tick, <Emphasis Type="Italic">Boophilus microplus</Emphasis> from Tamil Nadu,India using PCR-RAPD analysis

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.     

Determination of variability in monosporidial lines of Tilletia indica by RAPD analysis

Genetic variability in 23 monosporidial lines developed from five isolates of Tilletia indica causing Karnal bunt of wheat isolated from four wheat growing states of India was determined by using 19 rapid amplified polymorphic DNA (RAPD) markers. Amplification profile generated with all the 19 primers produced 3–16 numbers of bands of 1.5–5 kb size. High level of polymorphism (95.2%) suggested wide range of variability. Maximum Jaccard's similarity coefficient (80%) was observed between KB2MsB and KB2MsC followed by KB5MsC and KB5MsE with 75% similarity, whereas it was minimum between KB3MsA and Kb4MsB (47%). The dendrogram derived from the fingerprint analysis with 19 RAPD primers by using UPGMA showed different levels of genetic similarity among monosporidial lines. At 35% genetic similarity, the monosporidial lines were grouped in two clusters. Some primers, viz., OPN-1, OPN-6, OPN-9, OPN-12, OPN-13, OPN-18, OPM-2, OPM-8, OPM-10, OPB-8, OPB-17 and OPB-20 showed 100% polymorphism. The RAPD fingerprint generated by OPN-1 and OPM-3 were analysed and showed high range of variation in genetic make-up of monosporidial lines.     

分子标记鉴定常山胡柚优良基因型的初步研究

本研究利用RAPD和ISSR分子标记对常山胡柚的优良基因型进行鉴定,并探讨常山胡柚的起源。从100个RAPD引物中筛选出12个多态性引物用于正式扩增,共得到117条DNA带,其中多态性DNA带64条,占扩增片段的54．7%;从105个ISSR引物中筛选出11个多态性引物用于正式扩增,共得到94条DNA带,其中多态性DNA带58条,占扩增片段的61．7%。RAPD和ISSR分析揭示了常山胡柚及其近缘种的一些特异性条带。ISSR共产生了15条特异条带,RAPD共产生12特异性条带。实验数据用AMOVA软件计算遗传距离,用NTSYS-pc软件构建UPGMA聚类树状图。结果显示,所有的基因型及不同种之间均能够彼此区分,分析得到的指纹图谱对常山胡柚种和基因型的鉴定具有潜在的应用价值,可用于优良基因型的鉴定。聚类分析结果显示常山胡柚和甜柚聚为一枝,确定了甜柚是杂交亲本之一,但是常山胡柚和柚的遗传距离较远,说明常山胡柚可能是甜橙、柚和柑桔属其他种的多重自然杂交的结果。     

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.     

国产甘草属植物的RAPD分析及其分类学研究

应用RAPD技术,探讨甘草属(G ly cy rrh iza L.)13种1变种30个植物类群的遗传差异和几个争议种的分类地位。从60个随机引物中筛选出14个多态性好的引物进行RAPD实验,DNA片段的二态数据用U PGM A聚类法构建系统发育树。共扩增出250条带,多态性带204条,约占总数的81.7%。聚类结果显示RAPD分子标记构建的系统发育树与经典分类系统一致。甘草属植物具有丰富的遗传多样性,同种内不同居群间的遗传分化较大。黄甘草、胀果甘草、乌拉尔甘草三者亲缘关系较近,平卧甘草与粗毛甘草存在很大的遗传差异,作为独立种较合理。RAPD标记可为甘草属植物的系统分类研究提供分子生物学依据。