Genetic diversity and phylogenetic relationships among accessions of hop, Humulus lupulus, as determined by amplified fragment length polymorphism fingerprinting compared with pedigree data

DNA fingerprinting using amplified fragment length polymorphisms (AFLPs) was successfully employed to detect genetic relationships and variability among 90 hop cultivars and breeding lines comprising a collection of the world's hop germplasm. Seven AFLP primer combinations produced a total of 347 fragments of which 151 (43.5%)) were polymorphic. One‐hundred and thirty informative, highly reproducible DNA polymorphisms were used to estimate the genetic similarity (GS) which varied between 1.0 (e.g. ‘Saazer’ vs. ‘Tettnanger’) and 1.17 (‘Columbus’ vs. ‘Tettnanger’, ‘Spalter’ and ‘Saazer’). UPGMA (unweighted pair‐group method with arithmetic averages) clustering revealed two main clusters, reflecting the two main sources of origin and the two main breeding objectives: one cluster of mainly European origin representing the aroma pool and a second cluster associating accessions with European germplasm infiltrated by wild American genes with less aroma quality, but a higher bittering potential. Each main branch was composed of four or three subclusters with subgroups, respectively. Assignment of almost all genotypes in the dendrogram was consistent with the pedigree data as far as they are known. Consequently, AFLPs are shown to be suitable for assessing the genetic variability in hop germplasm and are useful for describing the genetic relationships among cultivars and accessions, which allows phylogenetic questions to be addressed.     

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.     

A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data

Nineteen of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the UnitedStates and Canada were examined for AFLP markerpolymorphisms. For the AFLP reactions, the EcoRI-ACC primer was used in combination with fourMseI primers (MseI-CAC, MseI-CAG,MseI-CAT, or MseI-CTT). Each set ofprimers produced 46–66 scorable fragments ranging insize between 50 and 500 bp. The polymorphic fragmentsproduced from each set of primers were more thansufficient to distinguish among all the cultivars,demonstrating the usefulness of AFLP markers forcultivar identification. Similarity coefficients werecalculated based on data from 228 AFLP markers anddata from 15 previously characterized RAPD markers. The RAPD markers had been specifically selected forfingerprinting purposes because they succesfullydistinguish 41 strawberry cultivars, including the 19cultivars analyzed in this study. Separatedendrograms were constructed based on analysis of theAFLP and RAPD marker data using a neighbor-joiningalgorithm. The dendrograms were compared and found tobe very different. Correlations between similaritycoefficients calculated from AFLP marker data,similarity coefficients calculated from RAPD markerdata, and coefficients of coancestry calculated frompedigree information were evaluated. Interestingly,a better correlation with the coefficients ofcoancestry was observed with the RAPD marker data thanwith the AFLP marker data.     

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.     

Heterogeneity of random amplified polymorphic DNA sequences in individual seedlings and bulked samples of four cultivars of Brassica napus

Using four different random amplified polymorphic DNA (RAPD) primers, a qualitative and quantitative assessment was made of the level of DNA sequence heterogeneity present in the seedlings of four representative Australian rapeseed cultivars. It was found that, depending upon the primer/cultivar combination, the seedlings diverged from total homogeneity to almost complete heterogeneity. The increase or decrease of sample-specific RAPD sequences was evaluated in proportional mixtures of DNA from individual seedlings. These results were then compared with those obtained from bulked DNA samples containing DNA from all the seedlings of a cultivar. From these comparisons, it was found that for a specific RAPD to be detectable in a bulked sample, the particular polymorphism had to be present in at least 15% of the individual seedlings. Even so, the bulked samples produced cultivar-specific RAPD banding patterns with all four primers, showing that any of these primers could be used to identify the different rapeseed cultivars. In contrast to the cultivars ‘Oscar’, ‘Dunkeld’ and ‘Narendra’, the cultivar ‘Rainbow’ was found to be highly heterogeneous—as shown by a diversity of RAPD combinations rather than the presence of differing length RAPDs—and it is suggested that this heterogeneity may be related to the improved tolerance of this cultivar to blackleg infection.     

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.     

福建地区火龙果种质资源调查及ISSR分析

为摸清福建地区的火龙果种质资源分布情况,了解其亲缘关系,对福建地区火龙果种植情况进行调查并取样。用ISSR技术对收集的20份火龙果材料进行遗传多样性分析。从40条ISSR随机引物中筛选出11条适合的引物,扩增获得142个位点,多态性比率为74.65%。Jaccard聚类分析显示,20份种质间相似系数（GS值）在0.487~0.937,平均值为0.646,当GS值为0.559时可分为3类。初步揭示了福建地区火龙果种质资源的分布情况和亲缘关系,‘白巨龙’、‘洛红’和‘红仙蜜1号’适合在福建推广,为火龙果栽培选种和育种工作提供参考价值。     

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.     

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.     

Molecular characterization and genetic relatedness among walnut (Juglans regia L.) genotypes based on RAPD markers

The potential use of the Randomly Amplified Polymorphic DNA (RAPD) technique for characterization and assessment of genetic relationships was investigated in nineteen walnut (Juglans regia L.) genotypes used as parents or released as cultivars from the breeding program of the University of California at Davis. Most of the 72 decamer primers used yielded scorable amplification patterns based on discernable bands. The results obtained produced a unique fingerprint for each of the walnut genotypes studied. Cluster analysis separated the 19 walnut genotypes into two main groups whose differences were related to their pedigree. Genotypes sharing common parents tend to group together and with at least one of the parents. Thus, RAPD markers can detect enough polymorphism to differentiate among walnut genotypes, even among closely related genotypes, and the genetic similarity based on RAPDs appears to reflect the known pedigree information. RAPD technology can be useful in current walnut breeding programs, allowing the identification of new cultivars as well as the assessment of the genetic similarity among genotypes which will help in selecting the best parents to obtain new genetic combinations. This revised version was published online in July 2006 with corrections to the Cover Date.     

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 Relationships in Malus Evaluated by RAPD 'Fingerprinting' of Cultivars and Wild Species

The potential use of RAPD markers for taxonomic studies in Malus was investigated using 18 accessions of wild species and 27 apple cultivars. 29 preselected random decamer primers were applied to three sets of Malus genotypes. Random amplified polymorphic DNA (RAPD) ‘fingerprints’ were analysed for polymorphic amplification fragments, and coefficients estimating genetic similarity were calculated on the basis of about 50 polymorphic RAPD loci in each set of genotypes. Cluster analysis by an unweighted pair-group method with arithmetic averages (UPGMA) revealed that, in the cultivars, the molecular classification was in good agreement with the known lineage. A dendrogram generated for the wild species gave relationships that were, in principle, in accordance with the known phylogenetic information. Closely related species from section I were clearly distinguishable from those of sections III and IV. On the molecular level, a high degree of genetic diversity was found among both different apple cultivars and wild species of the genus Malus. The results gave additional evidence for the hypothesis that M. pumila and M. sylvestris were involved in the origin of the cultivated apples.     

8份剑麻种质亲缘关系的ISSR和RAPD分析

为了揭示剑麻栽培品种的遗传多样性,利用ISSR和RAPD分子标记技术对8份剑麻种质的亲缘关系进行分析。结果表明,筛选后选用的8条ISSR引物和8条RAPD引物,分别产生了53条和66条扩增条带,其中多态性条带分别为44条和61条,多态性条带百分率分别为83.02%和92.42%。根据2种标记的扩增结果,用UPGMA法对8份剑麻种质进行聚类分析,供试材料之间具有较高的遗传多样性,其品种间遗传相似系数分别为0.59~0.80和0.52~0.76。2个标记的聚类结果基本一致,但有点差异,可将供试的8份剑麻种质划分为2类群,而且2个标记聚类结果呈显著相关性,相关系数为0.70。可见,剑麻种质资源的遗传多样性丰富。     

Genetic diversity of Cannabis sativa germplasm based on RAPD markers

Random amplified polymorphic DN A (RAPD) markers were generated from 13 cultivars and accessions of Cannabis sativa L. Approximately 200 fragments generated by 10 primers of arbitrary sequence were used to assess the level of DNA variation. Statistical analysis was performed using the Dice coefficient of similarity and principal coordinate analysis. The grouping of the accessions according to the cluster analysis was in good agreement with their origin and lines with common ancestors were grouped together. Principal coordinates 1 and 2 revealed a clear separation of Italian and Hungarian germplasm and a third group, including a mixture of genotypes coming from different places; the third coordinate separated the Korean group which is probably the most divergent germplasm. Variability within the two cultivars ‘Carmagnola’ and ‘Fibranova1’ was also shown, suggesting good possibilities for long–term selection work. RAPD markers provide a powerful tool for the investigation of genetic variation in cultivars/accessions of hemp.     

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.     

Genetic maps of RAPD, AFLP and ISSR markers in Ananas bracteatus and A. comosus using the pseudo-testcross strategy

Genetic maps of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP) and inter simple sequence repeats (ISSR) markers in pineapple (2n = 2x = 50) are reported for the first time. On the basis of a segregating population of 46 F1 individuals from a cross Ananas comosus x A. bracteatus, genetic maps of these two species were constructed using the two‐way pseudo‐testcross approach. The A. bracteatus map consists of 335 markers (60 RAPDs, 264 AFLPs and 11 ISSRs) assembled into 50 linkage groups, 26 of them with at least four markers. The A. comosus map consists of 157 markers (33 RAPDs, 115 AFLPs, eight ISSRs and the ‘piping’ trait locus) organized into 30 linkage groups, 18 of them with at least four markers. These maps cover, respectively, 57.2% of the A. bracteatus genome estimated as 3693 cM long, and 31.6% of the A. comosus genome calculated as 4146 cM. A rough estimate of 120 and 127 kbp/ cM on average was found for the relationship between physical and genetic distance for A. bracteatus and A. comosus, respectively.     

A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm

Several DNA marker systems and associated techniques are available today for fingerprinting plant germplasm but information on their relative usefulness in particular crops is limited. The study investigated PCR based DNA fingerprinting in a set of 39 potato cultivars using RAPDs (20 primers), ISSRs (6 primers), AFLPs (2 primers) and SSRs (5 primer pairs). Results show that each of the four techniques can on their own, individually identify each cultivar, but that techniques differ in the mean number of profiles generated per primer (or primer pair) per cultivar, referred to as Genotype Index (GI). The order of merit based on this criterium and in this material was AFLPs (GI = 1.0), a multi-locus SSR (GI = 0.77),RAPDs (GI = 0.53), ISSRs (GI = 0.47) and single locus SSRs (GI = 0.36). Problems in relating banding patterns to individual loci and alleles for polyploid genomes, using these techniques as they are currently employed, are also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

石斛种质资源遗传多样性的RAPD分析

利用RAPD技术对10种石斛种质资源的遗传多样性及亲缘关系进行了分析。从100条10bp的RAPD引物中筛选获得17条多态性引物,对石斛属的10个种的基因组DNA进行扩增。共获得200条多态性带,平均每个引物产生11.8个多态性条带。材料间遗传相似系数变化范围为0.356~0.676。根据RAPD标记的结果,采用UPGMA法进行聚类分析,将石斛属的10个种区分开来,划分为4类。结果表明：RAPD标记技术较好地从分子水平上揭示石斛种质资源的遗传背景、亲缘关系。     

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.     

Male and female genetic linkage map of hops, Humulus lupulus

A male and female linkage map of hop has been constructed using 224 DNA polymorphisms (106 amplified fragment length polymorphisms (AFLPs), three random amplified polymorphic DNAs (RAPDs), one RAPD‐sequence‐tagged‐site (STS), and three microsatellite (STSs) segregating in an F₁ population of the English cultivar ‘Wye Target’‐the German male breeding line ‘85/54/15’. Linkage between these loci was estimated using JOINMAP Version 2.0. The final map for the female parent consisted of 110 loci assigned to eight linkage groups covering a distance of 346.7 cM. For the male map, 57 loci could be mapped on nine linkage groups spanning over 227.4 cM. One of these male linkage groups (Gr09‐M) presumably represents the Y chromosome, since all markers assigned (10 AFLPs, three RAPDs and one STS) were closely linked to the male sex (M). Because of their sex‐specific segregation, 10 doubly heterozygous AFLPs spanning a distance of 18.7 cM could be identified as markers describing the X chromosome, which is part of the male and female map. Three STMSs, which had already proved useful in hop genotyping, could be integrated as codominant locus‐specific markers and thus allowed to produce reliable allelic bridges between the female and male counterparts.