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.     

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.     

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.     

Estimation of genetic distance based on RAPDs between 11 cotton accessions varying in heat tolerance

The genetic distance of 11 cotton genotypes varying in heat tolerance was studied using RAPD markers. Fifty-three random decamer primers were used for the estimation of genetic distance. Among the 53 RAPD primers, which were custom synthesized by GeneLink Inc., UK, 32 were polymorphic and 21 were monomorphic. The 32 polymorphic primers produced 273 fragments, with a mean of 8.3 fragments per primer. The number of polymorphic bands produced in the 11 cotton accessions ranged from 1 to 31. Primer GLC-20 produced 31 polymorphic bands, while two primers, GLB-5 and GLC-12, produced one polymorphic band each. A range of 88.89 to 42.48% genetic similarity was observed among the 11 cotton accessions. The highest genetic similarity was observed between FH-945 and BH-160 (88.89%), whereas the lowest value was found between NIAB-801/2 and FH-945 (42.48%). Unique amplification profiles were produced by most of the cultivars; the differences were sufficient to distinguish them from other genotypes. This confirms the efficacy of RAPD markers for the identification of plant genotypes. An accumulative analysis of amplified products generated by RAPDs was sufficient to assess the genetic diversity among the genotypes. This information should be helpful for formulating breeding and genome mapping programs.     

Genetic distance detected with RAPD markers among selected Australian commercial varieties and boron-tolerant exotic germplasm of pea (Pisum sativum L.)

The optimisation of polymerase chain reaction (PCR) for random amplified polymorphic DNA (RAPD) analysis in pea was investigated and the results were applied to an analysis of five representative Australian varieties and five selected boron-tolerant accessions derived from different geographical regions. Genotypes were compared using 34 random primers (Operon Technologies, Alameda, CA) which generated 180 polymorphic bands. Genetic similarity among genotypes was estimated on the basis of the percentage of common bands between genotypes and a dendrogram was constructed by the unweighted pair grouping method. A pattern of RAPD reaction corresponding to two main groups was discerned. The genetic divergence between Australian varieties and the boron-tolerant accessions suggests an intensive back-crossing programme would be required to transfer boron tolerance to a locally adapted genetic background. Our results show RAPD to be useful for clarifying phylogenic relationships within a species and also to provide useful genetic markers for varietal identification in pea.     

Development and study of SCAR markers in pea (Pisum sativum L.)

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F1 and F2. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism type.     

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。     

Microsatellite DNA polymorphism divergence in Chinese wheat (Triticum aestivum L.) landraces highly resistant to Fusarium head blight

Genetic differences between 20 Chinese wheat (Triticum aestivum L.) landraces highly resistant to Fusarium head blight (FHB) and 4 wheat lines highly susceptible to FHB were evaluated by means of microsatellite markers, in order to select suitable parents for gene mapping studies. Thirty-nine out of 40 microsatellite markers (97.5%) were polymorphic among the 24 wheat genotypes. A total of 276 alleles were detected at the 40 microsatellite loci. The number of alleles per locus ranged from 1 to 16, with an average of 6.9 alleles. Among these microsatellite loci, the largest polymorphism information content (PIC) value was 0.914 (GWM484), while the lowest PIC value was 0 (GWM24). The mean genetic similarity index among the 24 genotypes was 0.419, ranging from 0.103 to 0.673. Clustering analysis indicated that the highly susceptible synthetic wheat line RSP was less genetically related to and more divergent from the Chinese highly resistant landraces. These results were useful in the identification of suitable parents for the development of mapping populations for tagging the FHB resistance genes among these Chinese wheat landraces.     

RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B.S.P.

Summary The usefulness of random amplified polymorphic DNA (RAPD) in assessing the genetic stability of somatic embryogenesis-derived populations of black spruce [Picea mariana (Mill.) B.S.P.] was evaluated. Three arbitrary 11-mer primers were successfully used to amplify DNA from both in-vivo and in-vitro material. Twenty-five embryogenic cell lines, additional zygotic embryos and megagametophytes from three controlled crosses involving four selected genotypes of black spruce were used for the segregation analysis of RAPD variants. Ten markers were genetically characterized and used to evaluate the genetic stability of somatic embryos derived from three embryogenic cell lines (one cell line per cross, 30 somatic embryos per cell line). No variation was detected within clones. The utilization of RAPD markers both for the assessment of genetic stability of clonal materials and to certify genetic stability throughout the process of somatic embryogenesis is discussed.     

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.     

Development and Study of SCAR Markers in Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F₁ and F₂. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism origin.     

Genetic variation in Drechslera teres populations as indicated by RAPD markers

Random amplified polymorphic DNA (RAPD) was used to assess genetic variation among 48 isolates of Drechslera teres originating from different sites in Finland. RAPD profiles were generated with five arbitrary 10-mer primers and revealed polymorphisms suitable for screening differentiation in this fungal population. Using UPGMA clustering analysis, a similarity coefficient of approximately 63% was observed between all D. teres isolates studied. The variation was, however, distributed on a small scale as different genotypes were found from the same plant. The isolates could not be grouped according to geographic origin, aggressiveness, growth rate or morphological features, indicating that the primers used in this study were neutral markers for these characters. The primers were, however, able to differentiate between isolates of Helminthosporium species (D. teres, Drechslera graminea and Bipolaris sorokiniana).     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

Analysis of Genetic Diversity in Napier Grass (Pennisetum purpureum Schum) as Detected by RAPD and ISSR Markers

Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers were used to detect the DNA polymorphism among thirty Napier grass collections of wide geographical distribution. A total of 20 RAPD and 10 ISSR primers were used in this study. RAPD analysis produced 222 fragments of which, 195 were polymorphic with an average of 9.75 polymorphic fragments per primer. The ten ISSR primers produced a total of 98 fragments out of which 88 were polymorphic accounting for 89.8%. The Mantel test between two similarity matrices of the markers revealed a low correlation (r = 0.33) indicating low correspondence between polymorphism brought out by the two marker systems. The UPGMA clustering of genotypes eventhough was not similar when RAPD and ISSR derived dendrograms were compared, but showed a greater correspondence with geographical identity in both the marker systems employed. This correspondence was also evident when data from both the RAPD and ISSR markers were combined. The implications on collection and breeding of this important forage grass had been discussed.     

Detection of genetic diversity using RAPD-PCR and sugar analysis in watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasm

RAPD (random amplified polymorphic DNA) markers generated by 15 arbitrary decamers were used to determine the frequency of DNA polymorphism in 39 watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasms. Of the 15 primers tested, all except 1 (primer 275) directed the amplification of polymorphic products. A total of 162 amplification products were generated across all 39 genotypes. Among the 162 fragments, 35 (21%) appeared to be reliable polymorphic markers. The mean value by marker difference in this comparison was 0.24, and the highest, 0.69. Eight RAPD markers could be utilized in the unique variety discrimination 8 watermelon genotypes. From the phenograms constructed by UPGMA based on the comparison of RAPD markers, four clusters were resolved. Each group was also characterized and identified with morphological and genetic characteristics for each genotype. The free sugars of the edible parts of watermelons were analyzed by HPLC (high-performance liquid chromatography). Results from the phylogenetic analysis of band sharing data were consistent with sweetness as measured by HPLC. In conclusion, RAPD assays can be used for providing alternative markers for identifying genotypes and quantitative characteristics in watermelon.     

Identification and genetic mapping of random amplified polymorphic DNA (RAPD) markers to the sheep genome

The random amplified polymorphic DNA (RAPD) assay utilizes the polymerase chain reaction (PCR) and short primers of arbitrary nucleotide sequence to amplify DNA. In this study, the RAPD assay was used to identify and map polymorphic markers in the AgResearch International Mapping Flock (IMF) sheep pedigrees. Sires and dams of eight of the full-sib IMF pedigrees were screened with 131 different 10-mer oligonucleotide primers. An average of 85 RAPD polymorphisms was identified between each parental pair, and 53 markers were contributed to the AgResearch IMF collaboration. Forty-five of the RAPD markers were mapped in the AgResearch IMF genetic linkage map, and at least one marker was located on 17 of the 26 autosomes and both sex chromosomes. Three lines of evidence were used to check for the homology of scored polymorphisms in different pedigrees, pedigree evaluation, segregation analysis, and Southern blot analysis. These results demonstrate that the RAPD assay is a powerful approach for identifying polymorphisms that can be used as markers for constructing a sheep genetic linkage map. Received: 5 October 1995 / Accepted: 16 April 1996     

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.     

Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.     

Assessment of RAPD markers for barley doubled haploid lines resistant and susceptible to Fusarium culmorum at seedling and adult plant growth stages

Thirty doubled haploid (DH) lines of barley derived from F(1) of a cross between the six-rowed cultivar Pomo and two-rowed cultivar Maresi were examined for susceptibility to Fusarium seedling blight (SB) and head blight (FHB), measured by mycotoxin (nivalenol) content of kernels. RAPD (random amplified polymorphic DNA) polymorphism was analysed by using 53 decamer primers. Amplification products (APs) were 200 bp up to 2000 bp in size on average 5.7 per primer and the total number of APs was 284, 51.06% of which were polymorphic. Only 32 APs differentiated the examined DH lines - 19 APs for nivalenol content of kernels and 13 for seedling resistance. DH lines segregated with continuous distribution of resistance to FHB and SB. At the seedling stage all DH lines exhibited lower susceptibility than parental cultivars, but in the adult stage only two lines (MP 2 and MP 7) appeared to be more resistant to FHB, i.e. accumulated in kernels a lower amount of mycotoxin than cultivars Maresi and Pomo.