Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

21个兰花品种的延长RAPD(ERAPD)和PCR-RFLP分析

【研究目的】明确中国兰花品种间在DNA水平上的遗传多样性和亲缘关系。【方法】应用ERAPD（延长随机引物扩增）、叶绿体和线粒体基因组PCR-RFLP对21个中国兰花品种进行分子鉴定。【结果】RAPD分析中共有8个(10%)引物和88条(83.81%)多态性带纹;ERAPD分析中能获得一条2.5KbRAPD特异性片段;叶绿体基因组（cpDNA）的PCR-RFLP分析中有4个（66.67%）引物和12个(25%)引物-酶组合;线粒体基因组中（mtDNA）的PCR-RFLP分析中有2个（33.33%）引物和1个（4%）引物-酶组合能揭示材料间遗传差异。【结论】3种分子标记均能揭示品种间在DNA水平上存在遗传差异,而且遗传学分类与形态学分类基本吻合。     

Random amplified polymorphic DNA variation among German and Dutch asparagus cultivars

DNA polymorphism among nine cultivars of Asparagus officinalis L. was measured using random amplified polymorphic DNA (RAPD). Of 69 reproducible amplification products from 12 arbitrary decamer primers, 49 RAPD markers were polymorphic and could be used to distinguish six German and three Dutch asparagus cultivars. Even with very small sample sizes, genetic similarity measurements based on the RAPD data allowed accurate grouping of the nine cultivars into distinct clusters, with the exception of two individuals which clustered to closely related varieties. Two German cultivars showed high genetic similarity and were distinct from the remaining German varieties. The German and Dutch cultivars were clearly separated by a relatively large genetic distance.     

Genetic diversity of hexaploid wheat cultivars estimated by RAPD markers, morphological traits and coefficients of parentage

Estimates of genetic diversity can be based on different types of data. The aim of this research were to study genetic diversity among Croatian wheat cultivars by random amplified polymorphic DNA (RAPD) markers, morphological traits and pedigree records; to analyse differences between wheat cultivars from two breeding centres; and to evaluate usability of RAPD markers for estimation of genetic diversity among wheat cultivars in comparison with morphological traits and pedigree record data. Studies were conducted on 14 wheat cultivars and breeding lines from two breeding centres in Croatia. For the RAPD analysis 36 primers were screened and the 14 most polymorphic ones yielded 341 polymorphic bands. Twelve morphological traits were used for morphological analysis. Pedigrees were composed of seven generations of ancestors. RAPD markers showed a high level of polymorphism among the cultivars examined and the breeding lines. No significant correlations were observed among the methods tested.     

Study of genetic diversity in safflower genotypes using agro-morphological traits and RAPD markers

This research was conducted to study the genetic diversity in safflower (Carthamus tinctorius L.) using agro-morphological traits and RAPD markers. Sixteen selected lines derived from landraces growing in various agro-climatic regions of Iran along with four exotic genotypes were evaluated in a randomized complete block design with three replications under field conditions. Days to emergence, days to initial flowering, days to flowering, days to maturity, plant height, branches per plant, capitula per plant, seeds per capitulum, 1,000-seed weight, seed yield per plant, seed yield, and reaction to powdery mildew (Leveillula taurica Arnaud) were evaluated in this study. Genetic diversity of the genotypes was assessed by RAPD markers. The results indicated significant differences among genotypes for the agro-morphological traits and clustering based on these traits classified the genotypes into five groups. Analysis of the RAPD markers revealed 15 polymorphic primers out of 50 used primers. Based on RAPD data, the highest genetic similarity was observed between the cultivars of “AC Sunset,” “AC Sterling” from Canada and the lowest relatedness observed between a local breeding line “E₂₄₂₈” and genotype “GE₆₂₉₂₃” from Germany. Cluster analysis based on RAPD markers and 54% coefficient of similarity divided the genotypes into five distinct groups. Comparing the clusters based on agro-morphological traits with those from molecular markers showed slight similarities. The finding of high genetic variation for agro-morphological traits and polymorphism at DNA level reveal that agronomic traits can be improved by selection programs.     

一串红品种（系）遗传多样性RADP分析

(农业应用新技术北京市重点实验室/北京农学院园林系,北京102206)     

DNA fingerprinting of strawberry (Fragaria × ananassa) cultivars using randomly amplified polymorphic DNA (RAPD) markers

Forty-one of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the United States and Canada were examined for RAPD (randomly amplified polymorphic DNA) marker polymorphisms using 10mer primers (>50% GC content). A set of 10 primers produced 15 polymorphic fragments ranging in size between 450 and 1200 bp, which were more than sufficient to distinguish among all tested cultivars. Ten of the markers derived from seven primers were absolutely required for distinguishing the cultivars. A DNA fingerprinting table was constructed based on these results. In addition, similarity coefficients were calculated based on RAPD marker data and a dendogram was constructed using the unweighted pair group method of arithmetic averages (UPGMA). These results were compared with known pedigree data for the cultivars. Our results demonstrate that RAPD markers can be used effectively for strawberry cultivar identification. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic diversity in cabbage cultivars using RAPD and SSR markers

Genetic relationship and diversity among seven cabbage cultivars were analyzed using RAPD and SSR markers. These cultivars are of great commercial value in India and are confirmed for their reaction to black rot caused by Xanthomonas campestris pv. campestris. However, so far the extent of genetic diversity and relatedness has not been studied in these cultivars. A total of 17 selected RAPD primers generated 90 bands, 76 of which were polymorphic (84.44%). In addition, 27 selected SSR primers generated 67 amplified bands with 59 of which were polymorphic (87.6%). Though both the marker techniques were able to discriminate the cultivars effectively, analysis of combined data of markers (RAPD and SSR) resulted in better distinction of cultivars. By combining both the markers, a total of 157 bands were detected of which 135 bands (85.98%) were polymorphic, i.e. an average of 5.95 bands per primer. High level of polymorphism (> 85%) recorded with two different marker systems indicated a high level of genetic variation existing among the cultivars. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pairs of cultivars varied from 0.21 to 0.77 in RAPD, 0.42 to 0.82 in SSR, and 0.43 to 0.89 with combined markers. A high correspondence had been recorded between the values of genetic variations generated by UPGMA, clustering, and scatter plot diagrams. The cultivars ‘January King Sel. Improved’ and ‘Golden Acre’ are highly divergent cultivars as demonstrated by both the marker systems.     

Analysis of loquat germplasm (Eriobotrya japonica Lindl) by RAPD molecular markers

The loquat’s adaptation to Spain has proved very successful. In the Valencia area, the crop has met with very good environmental conditions for its development. Many new cultivars have been selected by growers and a European loquat germplasm collection has been established in Valencia at IVIA. An efficient sampling as well as implementation of germplasm resources requires the accurate identification of plant material. Molecular markers offer an effective tool for cultivar fingerprinting, estimation of genetic similarity and relationships. In this study, as a tool for germplasm management, RAPD markers were tested. Thirty-six primers were used to screen 33 cultivars. Twenty-three primers proved polymorphic. These primers generated 29 polymorphic amplification fragments that were selected as markers. Twenty-two cultivars out of 33 were identified by unique combinations of RAPD markers. Four different combinations were shared by two or more cultivars each. Cluster analysis based on the similarity matrix obtained from Nei’s coefficient among cultivars showed groupings that agreed according to geographical and genetic origin. RAPD technology was useful in distinguishing those cultivars obtained through hybridization but could not be used to distinguish those obtained by selection of mutations. This revised version was published online in August 2006 with corrections to the Cover Date.     

RAPD genetic diversity of Albanian olive germplasm and its relationships with other Mediterranean countries

RAPD markers were used for the study of 19Albanian olive cultivars and two wild olives (oleasters). A total of 76polymorphic bands (4.8 polymorphic markers per primer) out of 107 reproducible were obtained using 16 primers. The number of bands per primer ranged from 4 to 10,whereas the number of polymorphic bands ranged from 1 to 9, corresponding to 71%of the total amplification products. All the accessions could be identified by the combination of four primers: OPA-19;OPA-02; OPK-16 and OPP-19. The dendrogram,based on Jaccard's index, included three major groups according to their origin: 1)most of the cultivars from the area of Berat (South of Albania) 2) cultivars from the Centre and Centre-North of Albania and3) cultivars from the Centre and North-West of Albania along with the oleaster from Elbasan. In order to evaluate the origin of Albanian cultivars they were compared to those diffused in other countries like Greece, Italy and Turkey, due to geographical and historical affinity among these countries, by using a one way AMOVA. Although most of the genetic diversity was attributable to differences among cultivars within each country (91.47%) significantφ-values among countries(φ_st = 0.085; p < 0.001)suggested the existence of RAPD phenotypic differentiation. Significant φ-values in all pairs formed by Albania with the other countries were observed. These results are consistent with the autochthonous origin of Albanian cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variation within and between two cultivars of red clover (Trifolium pratense L.): Comparisons of morphological,isozyme, and RAPD markers

Summary Morphological, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation within and between cultivars of red clover (Trifolium pratense L.), an important temperate forage legume. Two cultivars of red clover, Essi from Europe and Ottawa from Canada, were evaluated. Six monogenic morphological characters were observed for 80 plants from each of these two cultivars. All six morphological loci were polymorphic in the cultivar Essi whereas only four loci were polymorphic in the cultivar Ottawa. Forty plants from each cultivar were assayed for isozyme markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. Thirteen and nine of these loci were polymorphic in Essi and Ottawa, respectively. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random 10-mer primers were screened for RAPD markers. Nine primers which gave clear and consistent amplified products were used to assay 20 individuals from each cultivar. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One hundred and eight of 116 putative loci were polymorphic in Essi and 90 of 98 loci were polymorphic in Ottawa. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. This high polymorphism makes these markers useful for germplasm characterization and genetic studies in red clover.     

Cultivar identification and genetic relationship among selected breeding lines and cultivars in chickpea (Cicer arietinum L.)

Twenty two RAPD and 22 ISSR markers were evaluated for their potential use in determination of genetic relationships in chickpea (Cicer arietinum L.) cultivars and breeding lines. We were able to identify six chickpea cultivars/breeding lines by cultivar-specific markers. All of the cultivars tested displayed a different phenotype generated either by the RAPD or ISSR primers. Though ISSR primers generated less markers than RAPD primers, the ISSR primers produced higher levels of polymorphism (% of polymorphic markers per primer) than RAPD primers. A high level of within cultivar homogeneity was observed in chickpea. Cultivars/breeding lines originating from a common genetic background showed closer genetic relationship. Chickpea lines with similar seed type(kabuli or desi) had a tendency to cluster together. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity in soybean genotypes from north‐eastern China and identification of candidate markers associated with maturity rating

Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to estimate the genetic relationships among 101 soybean cultivars developed in north‐eastern China. Fifty‐three fragments of the 100 RAPD markers and 35 SSR markers tested were polymorphic across the 101 soybean cultivars. Similarity values among these soybean cultivars ranged from 45.2% to 100% for RAPD data, and ranged from 36.1% to 100% for SSR data. The similarity matrices for SSR data and RAPD data were moderately correlated (r = 0.31, P < 0.05). Cluster analyses indicated that the cultivars released from the same seed company were mostly grouped together. A principal component analysis, based on the combined RAPD and SSR data, yielded a good separation of soybean varieties with different maturity ratings [represented by soybean Heat Unit (HU)]. The varieties with HU < 2200 were well separated from those with HU > 2200. Four RAPD markers and eight SSR markers were significantly associated with the maturity ratings of soybean.     

Variation in Capsicum annuum revealed by RAPD and AFLP markers

Genetic relationships were examined among thirty-four pepper (Capsicum annuum) cultivars of different types. Two types of PCR-based markers were used, RAPD and AFLP, and their relative effectiveness was compared. A dendrogram based on RAPD markers separated the large-fruited sweet cultivars from the small-fruited pungent peppers, and the former group showed less divergence than the latter. The percentage of polymorphic markers was lower for AFLP than for RAPD markers (13 and 22% respectively). However, AFLP primers amplified on average six times more products than RAPD markers. The average numbers of polymorphic products per primer were 1.6 and 6.5 for RAPD and AFLP primers, respectively, i.e., AFLP primers were four times more efficient than RAPD primers in their ability to detect polymorphism in pepper. While four blocky type cultivars were indistinguishable by RAPD, two AFLP primer pairs were sufficient to distinguish the four cultivars from each other. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

Summary The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.     

Polymorphism and DNA markers for asparagus cultivars identified by random amplified polymorphic DNA

Summary DNA polymorphism among five Asparagus officinalis L. cultivars-Imperial, Snow, Steline, UC-157 and Larac, as detected by random amplified polymorphic DNA (RAPD), is reported. Thirty one decamer primers were tested. and twenty six of them yielded amplification products. Fourteen primers gave products with at least one polymorphic DNA fragment. Among a total of 119 amplified fragments 33 were polymorphic. These RAPD markers enabled the identification of asparagus cultivars. Unique markers for cultivars were: Snow-bands 475 bp, 772 bp, 412 bp, 935 bp and 820 bp amplified by primers D5, OPA-07, OPA-09, OPA-10 and OPA-18, respectively. Steline-bands 645 bp, 680 bp and 997 bp amplified by primers A32, OPA-03 and OPA-09, respectively. A band 903 bp, amplitied by primer OPA-12, is a marker for Imperial, and a band 420 bp, amplified by primer D52, is a marker for Larac. Cultivar UC-157 could be identified by a combination of shared polymorphic bands. The pairwise marker difference between cultivars ranged from 0.08 to 0.17. A phenogram of the genetic relationship based on RAPD fits with the known origin of the cultivars.     

Comparing RAPD and AFLPTM analysis in discrimination and estimation of genetic similarities among apple (Malus domestica Borkh.) cultivars

Forty-one apple (Malus × domestica Borkh.) cultivars were screened for RAPD (Random Amplified Polymorphic DNA) and AFLP(Amplified Fragment Length Polymorphism) markers. RAPD analysis was performed with 35 arbitrary 10-mer primers, selected from 60 primers tested (kits A, C and E, Operon Technologies, Inc.). Of a total of 362bands observed, 208 (57.5%) were polymorphic. Three-hundred-and-eighty-one AFLP fragments were obtained with 8primer combinations, of which 218 (57.2%) were polymorphic. Cultivars differentiated through mutation were included in this study and showed identical patterns when analysed with both RAPD and AFLP analysis. The estimated genetic relationships were correlated (r = 73.7%) between the analysis with the two different markers. UPGMA analysis was performed and dendrograms were constructed using either the data apart from each(RAPD and AFLP) method or combined in a single joint matrix. The relationships among the forty-one studied cultivars were basically consistent with the known lineage and geographic origins of the cultivars. The four Portuguese cultivars included in this study clustered together and diverged from the other cultivars. Apparently they constitute an independent genetic pool, which could be of interest for apple plant breeders. This revised version was published online in July 2006 with corrections to the Cover Date.     

蛇龙珠葡萄品种亲缘关系的RAPD分析

利用随机扩增多态性DNA（RAPD）技术对4个酿酒葡萄品种和11个鲜食葡萄品种的亲缘关系进行了研究。从30个随机引物中筛选出16个扩多态性引物，用于15个样品的正式扩增。共扩增出134条DNA带，其中多态性扩增带99条，占73.9％。根据DNA扩增结果计算出品种的遗传距离，并构建聚类树状图。分析结果表明，红地球、瑞必尔与其它13个品种的遗传距离最远，巨峰系列的葡萄品种间遗传距离值较小，蛇龙珠品种与其它3个酿酒葡萄品种在遗传基因上存在差异，与品丽珠的亲缘关系最近。     

Genetic diversity in mustard (Brassica juncea L.) germplasm from Pakistan as determined by RAPDs

The genetic diversity and the relationships among a collection of mustard (B. juncea) germplasm, including 41 accessions collected from Pakistan, 6 oilseed cultivars/ lines and 5 Japanese vegetable cultivars, were evaluated using random amplified polymorphic DNA (RAPD) markers. A total of 198 polymorphic amplified products were obtained from 30 decamer primers. Of these, 14 were unique to the accession PAK-85835 and 37 were specific to PAK-85839. Based on pair-wise comparisons of RAPD amplification products, genetic similarity was estimated using similarity coefficients of Nei & Li (1979) and a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA). Cluster analysis based on these genetic similarities placed most of the collected germplasm and oilseed cultivars/lines close to each other, showing a low level of polymorphism between the oilseed accessions collected in Pakistan. However, the clusters formed by the oilseed collections and cultivars were distinct from those formed by the vegetable cultivars. A low level of genetic variability of oilseed mustard in Pakistan was attributed to the selection for similar traits and horticultural uses. The farmers' preference for more remunerative crops and perhaps the close parentage of these accessions further contributed towards their little diversity. The study demonstrated that the RAPD is a simple and fast technique to compare the genetic relationships and the patterns of variation among accessions of this crop. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variation among cultivars of red clover (Trifolium pratense L.) detected by RAPD markers amplified from bulk genomic DNA

Summary The use of random amplified polymorphic DNA (RAPD) markers obtained from bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. To determine the appropriate number of individuals to include in the bulked samples representing each cultivar, DNA samples from two, three, four, five, ten and twenty individuals were pooled. Twenty was found to be an appropriate number of red clover individuals per bulk in order to amplify only the DNA sequences shared among most individuals in each cultivar. Fourteen 10-mer primers were used to amplify genomic DNA from combined leaf samples of 15 red clover cultivars from European, Japanese and North American origins. A total of 79 amplified products, of which 55 were polymorphic, was obtained. Cultivar-specific bands were observed with 13 primers. The amplification patterns obtained from two primers could distinguish all 15 red clover cultivars. Rogers' genetic distances for all 105 pairwise comparisons were calculated to evaluate relationships among these cultivars. Cluster analysis based on these genetic distances separated these 15 cultivars into three groups, with two of the groups consisting of a single Japanese cultivar each, while the third group included cultivars from European, North American, and Japanese origins.