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.     

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.     

温州盘菜地方品种鉴定与指纹图谱的分子标记分析

利用基因组DNA的RAPD、ISSR与SRAP等3种分子标记技术,以日本芜菁品种作为外类群,对来自于温州不同地区具有代表性的10个盘菜品种进行品种鉴定与遗传多样性分析。10个RAPD引物共产生多态性条带70条,多态率为71．7％;12个ISSR引物共产生142条清晰带,其中多态性条带70条,多态率为49．3％;8个SRAP引物组合共产生105条谱带,其中多态性谱带78条,多态性比率为74．3％,表明品种间存在较高的多态性。用单个引物NAURP299、NAUISR43以及SRAP引物组合mel／em2,都可以将11个品种完全区分开来。基于3种标记的聚类分析结果表明,11个材料可以分为3大类,一定程度上能够揭示品种之间园艺学性状的相似性及亲缘关系远近。     

菊花芽变和相似品种的RAPD分析

本实验在形态学分析的基础上,采用5对中国传统菊花的芽变品种和2组形态相似品种,运用随机扩增DNA多态性（random amplified polymorphic DNA,RAPD）分子标记方法对其进行了品种鉴定。通过反复试验,建立了稳定的RAPD反应体系;从22个20碱基随机引物中筛选出4个高效引物,扩增出13条带,扩增的多态性片段多在501～1000bp之间,其中有2个引物（ch1．21和ch1．25）能将试验中的7组材料全部清楚地区分开。研究结果表明：4个高效引物的RAPD分析能够有效地将选定的样本区分开。因此作者提出RAPD分析方法可以应用到菊花芽变及相似品种鉴定中。这就为进一步开发更多RAPD随机引物,开展菊花的品种鉴定和保护工作提供了参考资料。     

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.     

Radiomutants of chrysanthemum (Dendranthema grandiflora Tzvelev) of the Lady group: RAPD analysis of the genetic diversity

Random amplified polymorphic DNA (RAPD) markers were used to study the molecular characterization of 10 new radiomutants of chrysanthemum. The original cultivar ‘Richmond’ differed in genetic distance from its Lady group mutants. The analysis of genetic similarity indices revealed low diversity within the radiomutants. The dendrogram obtained after cluster analysis separated the new cultivars as a group that differed from the original cultivar ‘Richmond’. The Lady group cultivars, derived from one original cultivar by radiomutation, could be distinguished from each other by using RAPD markers of only a single primer or sets of two or three primers. Polymerase chain reaction analysis proved the efficiency of the RAPD method for DNA fingerprinting of the original cultivar ‘Richmond’ and its new radiomutants.     

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.     

Analysis of molecular variance and population structure in southern Indian finger millet genotypes using three different molecular markers

The genetic relationship among 42 genotypes of finger millet collected from different geographical regions of southern India was investigated using random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and simple sequence repeats (SSR) markers. Ten RAPD primers produced 111 polymorphic bands. Five ISSR primers produced a total of 61 bands. Of these, 23 bands were polymorphic. The RAPD and ISSR fingerprints revealed 71.3 and 37.4% polymorphic banding patterns, respectively. Thirty-six SSR primers yielded 83 scorable alleles in which 62 were found to be polymorphic. Out of 36 SSR primers used, 14 primers (46.6%) produced polymorphic bands. The SSR primer UGEP7 produced a maximum number of six alleles. Mean polymorphic information content (PIC) of RAPD, ISSR and SSR were 0.44, 0.28, and 0.14, respectively. Molecular variances among the population were 2, 11, and 1% for RAPD, ISSR, and SSR markers, respectively. SSR produced 99% molecular variance within individuals. RAPD and ISSR markers produced a low level of molecular variance within individuals. The STRUCTURE (model-based program) analysis revealed that the 42 finger millet genotypes could be divided into a maximum of four subpopulations. Based on the Bayesian statistics, each RAPD and SSR marker produced three subpopulations (K=3), while ISSR marker showed four subpopulations (K=4). This study revealed that RAPD and SSR markers could narrow down the analysis of population structure and it may form the basis for finger millet breeding and improvement programs in the future.     

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.     

Molecular characterisation of cultivars of apple (Malus × domestica Borkh.) using microsatellite (SSR and ISSR) markers

In this study, two microsatellite-based methodologies (SSR and ISSR) were evaluated for potential use in fingerprinting and determination of the similarity degree between 41 commercial cultivars of apple previously characterised using RAPD and AFLP markers. A total of 13 SSR primer sets was used and 84 polymorphic alleles were amplified. Seven ISSR primers yielded a total of 252 bands, of which 176 (89.1%) were polymorphic. Except for cultivars obtained from somatic mutations, all cultivars were easily distinguishable employing both methods. The similarity coefficient between cultivars ranged from 0.20 to 0.87 for SSR analysis and from 0.71 to 0.92 using the ISSR methodology. Dendrograms constructed using UPGMA cluster analysis revealed a phenetic classification that emphasises the existence of a narrow genetic base among the cultivars used, with the Portuguese cultivars revealing higher diversity. This study indicates that the results obtained based on the RAPD, AFLP, SSR and ISSR techniques are significantly correlated. The marker index, based on the effective multiplex ratio and expected heterozygosity, was calculated for both analyses (MI = 1.7 for SSR and MI = 8.4 for ISSR assays) and the results obtained were directly compared with previous RAPD and AFLP data from the same material. The SSR and ISSR markers were found to be useful for cultivar identification and assessment of phenetic relationships, revealing advantages, due to higher reproducibility, over other commonly employed PCR-based methods, namely RAPD and AFLP. This revised version was published online in August 2006 with corrections to the Cover Date.     

Morphological traits and high resolution RAPD markers for the identification of the main strawberry varieties cultivated in Argentina

Eight genotypes of the main Fragaria×ananassa cultivars grown in Argentina were analysed using the random amplified polymorphic DNA (RAPD) technique combined with electrophoresis in polyacrylamide gels. The high resolution of this procedure allowed the detection, with only 13 random primers, of 37 genotype‐specific bands that can be used as markers for verifying the identity of cultivars. By using this approach, three different accessions of the cultivar ‘Pájaro’ exhibited differences in amplification profiles, confirming the need for DNA analysis to prevent misidentification of cultivars. In addition, RAPD bands and morphological traits were used to assess genetic relatedness among cultivars. Comparison of both dendrograms revealed that there is no correlation between the clustering obtained with molecular and morphological characters.     

Genetic diversity and identification of cymbidium cultivars as measured by random amplified polymorphic DNA (RAPD) markers

DNA from thirty-six cymbidium cultivars was examined using polymerase chain reaction (PCR) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) markers in identifying cultivars and determining levels of genetic variability. A total of 132 RAPD markers, 78% of which were polymorphic, were produced from 15 10mer arbitrary primers. All the cultivars were distinguishable when a number of primers was considered. One cultivar, Blue Smoke ‘Green Meadow’ could be distinguished from all the rest based only on lack of the OPA5-370 fragment. Genetic distances among the cultivars were estimated based on the amount of band sharing and ranged from 0.08–0.50 with an average of 0.29. Cluster analysis of genetic distance estimates grouped siblings together with each other and parents with offsprings, thereby agreeing with known parentage information and corroborating isozyme data obtained from a separate study. The possible application of the observed polymorphism and variation to cymbidium breeding is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence of gene introgression in apple using RAPD markers

Summary A genomic remnant of Malus floribunda clone 821 introgressed into the cultivated apple M. x domestica Borkh. was identified using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). Using a set of 59 oligonucleotide decamer primers, polymorphic DNA markers were identified among three pooled DNA samples. Based on the presence or absence of bands among bulked apple scab-resistant selections and cultivars, bulked scab-susceptible cultivars, and a M. floribunda clone 821 sample, one primer, A 15, identified amplified fragments in the scab-resistant bulked sample that was also unique to the M. floribunda clone 821. The unique band from M. floribunda clone 821 was amplified in four out of 17 scab-resistant selections/cultivars. This RAPD, designated OA15₉₀₀, identifies an introgressed fragment that has as yet no known function.     

Genetic variation of sweet potatoes (Ipomoea batatas L.) cultivated in Chile determined by RAPDs

RAPD (randomly amplified polymorphic DNA) technology was applied to analyze the genetic variability of sweet potato germplasm existing in Chile and elsewhere. Analysis of 28 cultivars from all over the world showed polymorphic bands with all 18 primers tested. A total of 124 RAPD bands were scored with an average of 6.9 polymorphic bands per primer. These results confirm that sweet potato exhibits high genetic variation. Two groups were distinguished: one containing Peruvian cultivars, and another containing cultivars from the rest of the world. Analysis of 14 accessions from Central Chile and one from Northern Chile showed polymorphic bands with 24 of 26 primers tested, but almost all of the 140 polymorphic bands merely showed the distinctness of the Northern accession. The almost complete uniformity of the other 14 accessions shows that sweet potato germplasm collected in Central Chile has very little genetic variability and may be derived from a single cultivar. Based on these results and on historical records, some hypotheses are proposed to explain the origin of sweet potatoes cultivated in Chile. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

滩地13个杨树无性系遗传多样性的RAPD分析

利用RAPD分子标记手段,研究杨树无性系的遗传多样性。在探讨杨树叶片基因组DNA提纯方法和优化PCR扩增反应体系的基础上,采用18个引物对13个杨树无性系的基因组DNA进行扩增;每个引物扩增的DNA片段数为1~14个,共产生126条带,其中多态带53条,占42%,平均每个引物扩增出3条多态带。结果表明：通过对126条谱带的聚类,分析了供试杨树无性系的系统发育,并通过比较各品种间的特异带或特征带的差异进行无性系鉴别和测定,运用特殊谱带,建立了杨树无性系的分子检索表。     

基于RAPD标记的芥蓝种质资源遗传多样性分析

调查了44份芥蓝种质的植物学性状,并利用RAPD分子标记分析了其遗传多样性。结果从150条随机引物中筛选出20个引物,20个引物共扩增出177条谱带,其中多态谱带105条,平均每个引物扩增出8.9条谱带和5.3条多态性谱,多态性比率为59.32%。基于RAPD标记,利用NTSYS-pc2.11构建了聚类树状图谱,遗传距离为0.70时,44份芥蓝资源可聚成六大类群。芥蓝种质存在着一定的遗传多样性,但原产华南而且主要产区也在华南,遗传多样性要小于芸薹属其他蔬菜。     

黑龙江部分大豆品种分子ID的构建

以黑龙江13个育种单位6个积温带的83份大豆品种为材料, 选择分布在大豆基因组19个连锁群的43对SSR引物进行检测, 共检测出等位变异157个, 每个引物检测到的等位变异数变化范围为2~7个, 平均为3.65个。将聚丙烯酰胺凝胶电泳得到的谱带统计结果根据等位变异的片段大小数字化, 用自行编制的ID Analysis 1.0软件进行数据分析。结果表明, 仅需9对引物(Satt100、Sat_218、Satt514、Satt551、Satt380、Satt193、Satt191、Satt442、Sat_084)可将83份参试大豆品种完全区分开。构建了一套黑龙江省大豆品种的分子ID。     

Purity control of F1-hybrid tomato cultivars by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were applied in purity control of hybrid seed production of tomato (Lycopersicon esculentum Mill.). DNA from three commercial F₁-hybrid cultivars and their parental lines was subjected to RAPD screening with 50 primers. Two of four primers which detected polymorphism between the parents tested, generated paternal-specific RAPDs, enabling a clear distinction to be made between hybrids and their maternal parents. In addition, combination of the polymorphic DNA products generated by these primers exhibited hybrid-specific patterns, enabling each cultivar to be identified. This result indicates the practical usefulness of RAPD markers in hybrid-tomato-seed purity-control tests and cultivar identification. The approach is advantageous in its rapidity and simplicity, particularly as an alternative for those cultivars for which lengthy and costly phenotypic tests are currently used.     

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.