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.     

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.     

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.     

Strawberry-cultivar Identification using Randomly Amplified Polymorphic DNA (RAPD) Markers

Accurate and rapid cultivar identification is important for breeders'-rights protection, especially for vegetatively propagated plants. The objective of this study was to test the feasibility of developing cultivar-specific RAPD markers in commercial strawberries (Fragaria ananassa Dutch). Efforts were focused on distinguishing between two newly developed Volcani cultivars, ‘Ofra’ and ‘Dorit’, and six other cultivars, ‘Douglas’, ‘Chandler’, ‘Oso Grande’, ‘Dover’, ‘Nurit’ and ‘Parker’. Reproducible RAPD fingerprints were generated, each containing at least one polymorphic DNA product. A combination of 10 polymorphic DNA products exhibited cultivar-specific patterns enabling the distinction between closely related varieties, such as ‘Ofra’ (which is the progeny of ‘Dorit’ and ‘Parker’) and ‘Dorit’ (which is the progeny of ‘Nurit’ and ‘Dover’). This study shows that RAPD markers can help in the protection of breeders’ rights to strawberry cultivars.     

RAPD analysis of genetic variation in natural populations of Betula alnoides from Guangxi, China

There is an urgent need for the developmentof early identification techniques inolive-trees due to the economic importanceof cultivar identification in periods ofexpansion like now. We have been able toidentify 22 olive-tree cultivars using only10 different, specific, repeatable markers.These markers were designed by the cloningof significant RAPD bands obtained in PCRperformed on bulked DNA to retain thegenetic variability of each cultivar.Clones were partially or totally sequencedand new primers derived from thesesequences were used to obtain SequenceCharacterised Amplified Region (SCAR)fragments. We have demonstrated that theuse of the 10 SCAR markers is enough toprovide a simple, cheap, and reliableprocedure to identify 22 geographicallyrelated olive-tree cultivars.     

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.     

Stability of RAPD markers for determining cultivar specific DNA profiles in rye (Secale cereale L.)

Summary Cultivar specific DNA profiles in rye were revealed by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA (RAPD) sequences. Ten base primers were used for the amplification of genomic DNA of rye cultivars by PCR. RAPD analysis was found to be reproducible among samples between PCR runs. When amplification profiles of different rye cultivars were compared using various primers, the overall profiles were cultivar specific. However, not all primers revealed polymorphisms. These primers appear to amplify conserved sequences in all rye cultivars. Intracultivar studies were conducted on two of the cultivars. In the cultivar Imperial, no polymorphisms were observed among ten plants analyzed with five primers. In the cultivar Balboa, polymorphisms were observed among fifty plants with four of the ten primers analyzed. Despite the small amount of intracultivar variability, RAPD analysis has the potential to be a rapid and reliable method of cultivar identification in this outcrossing species.     

The use of microsatellite polymorphisms for the identification of Australian breeding lines of rice (Oryza sativa L.)

Ten microsatellite loci were analysed for 43 cultivars or breeding lines of rice. Polymorphism-Information-Content values ranged from 0.62 to 0.92. The microsatellite markers were found to be useful for cultivar identification and assessment of genetic relationships. Most of the cultivars could be uniquely identified by at least one microsatellite marker. Genetic heterogeneity was detected within rice samples by amplification of microsatellites from DNA extracted from multiple individual plants and also from bulked DNA preparations. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Genetic variability measures among Bromus catharticus Vahl. populations and cultivars with RAPD and AFLP markers

The objectives of this study were to optimize RAPD and AFLP techniques in B. catharticus, and to determine the genetic variability of populations and commercial prairie grass cultivars with the aforementioned molecular markers. Two populations with contrasting morphological characteristics were evaluated from individual and bulked DNA samples using RAPD markers. Both analyses showed a similar information about inter population variability. Each accession was sampled by a single leaf bulk of 10 plants. Accession similarities were established with 276 RAPD and 714 AFLP bands using Jaccard similarity coefficient. The dendrogram of the accessions using RAPD markers showed that they shared high similarity values (>94%). A similar result was obtained with AFLP markers (similarity values >98%), revealing a narrow genetic basis in the analyzed accessions. Consequently, molecular characterization of germplasm should be considered in addition to morphological criteria, to choose the parental genotypes for breeding programs of this forage crop. The AFLP technique was more efficient to detect DNA polymorphism in our experiments and unique fingerprints were detected for all the accessions. RAPD is a simple and non expensive technique, suitable to estimate genetic similarity. This revised version was published online in August 2006 with corrections to the Cover Date.     

A new DNA marker for sex identification in purple asparagus

Garden asparagus (Asparagus officinalis L.) is an economically-important perennial crop. This plant is dioecious, as there are both male and female individuals; male individuals are preferred over females for agricultural production. To reduce the time required for garden asparagus breeding, various male-specific DNA markers are utilized. Male-specific DNA markers, such as Asp1-T7sp and MSSTS710, are currently available for sex identification in many asparagus cultivars. In the current study, we found that these markers are not suitable for sex identification in the purple asparagus cultivar ‘Pacific Purple’, as male-specific amplification of this marker was detected in some male individuals of this cultivar but not in other males. The Asp1-T7sp marker is suitable for use in sex identification in various Asparagus species related to A. officinalis, indicating that the region around this marker is conserved among these species. Thus, we isolated a DNA fragment around this marker by inverse PCR and produced a new DNA marker, MspHd, based on this sequence. However, like Asp1-T7sp and MSSTS710, MspHd was not suitable for sex identification in the cultivar ‘Pacific Purple’. Since all ‘Pacific Purple’ males have morphologically similar male flowers with functional stamens, we produced a new male-specific marker based on the sex determination gene, MSE1/AspMYB35/AspTDF1, which is responsible for stamen development. This marker, named AspMSD, is suitable for sex identification in ‘Pacific Purple’. In addition, this marker can be utilized for sex identification in various asparagus cultivars and some related Asparagus species.     

DNA标记在植物品种鉴定上的应用现状

为了全面地了解DNA指纹在品种鉴定上的利用现状,更重要的是促进DNA指纹技术切实有效的应用于植物品种鉴定,本研究归纳了目前开发出的可用于植物品种鉴定的DNA指纹技术,指出已有的DNA指纹品种鉴定技术的优势与不足,总结了DNA指纹应用于品种鉴定的分析措施,并认为受限于缺乏可以将DNA指纹转化为植物鉴定信息的理想措施,所以DNA标记还未真正有效地应用于生产实践中,因此提出一种基于DNA指纹的人工绘制品种鉴定简图(MCID)法是很重要的,本研究也重点阐述了MCID法的实施过程。     

DNA分子标记在银杏中的应用

DNA分子标记广泛用于生物研究的许多方面。本文简要介绍了RFLP、RAPD、AFLP、SSR及ISSR等几种目前常用分子标记的原理,归纳总结了分子标记在银杏中的应用研究进展。(1)获得了2个雄性特有的RAPD标记和2个雌性特有的AFLP标记,为银杏的早期性别鉴定及相关基因克隆奠定了基础;(2)采用RAPD标记和ISSR标记对我国部分栽培品种进行了分子鉴别和分类的研究,编制了一些品种的DNA指纹检索表;(3)利用RAPD和ISSR标记对一些群体、个体及栽培品种或变异类型进行了遗传分化和遗传多样性研究,结果发现银杏具有较高的遗传多样性,群体间、个体间、栽培品种及类型间都存在不同程度的遗传分化;(4)利用ISSR标记对一些个体的遗传杂合性进行了研究,结果显示个体的平均杂合率为43.53%;(5)构建了包含62个RAPD标记、19个连锁群的银杏分子遗传图谱;(6)探索了银杏优先保护种群的确定。分子标记在银杏其它方面的应用还很少。今后,除了继续对上述方面进行深入系统的研究外,还应充分运用DNA分子标记技术,开展银杏的分子标记辅助选择育种、种质评价与鉴别及保育生物学等方面的研究。     

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.     

Identification of two RAPD markers genetically linked to a recessive allele of a Fusarium wilt resistance gene in pigeonpea (Cajanus cajan L. Millsp.)

Summary Fusarium wilt (Fusarium udum Butler) is a soil borne disease of pigeonpea which causes substantial yield losses. The disease can occur at any stage of plant development, from the young seedling to the pod filling stage. Though resistance is simply inherited, transfer to locally adapted cultivars has been difficult due to linkage drag and difficulty in accurate phenotyping, except in sick plots. An attempt was made to identify RAPD markers associated with wilt phenotype by using F2 populations derived from contrasting parents; GSl (susceptible) ‘ICPL87119 (resistant) and GS1’ ICP8863 (resistant). Parents and F₂s were grown in a national _Fusarium sick-plot at Gulbarga, India and phenotyped as resistant or susceptible during the entire crop growth period. In both the crosses, resistance to wilt segregated as a monogenic dominant character. DNA samples extracted from sick plot grown, early seedling stage plants of parents and 254 F₂ plants of GS1 × ICPL87119 were held separately for marker identification. PCR reactions using 340 random decamer primers with genomic DNA of parents resulted in detection of 45 polymorphic amplicons from 39 primers. PCR testing of bulked DNA from subsets of resistant and susceptible plants revealed the presence of two amplicons at 704 bp and 500 bp (OPM03704 and OPAC11500) with susceptibility. Analysis of individual F₂ plants showed a segregation ratio of 3: 1 for the presence: absence of the amplicon in both crosses. Considering the wilt reaction and susceptibility-linked RAPD marker, it was possible to deduce genotype of every F₂ plant and the genotypic ratio for wilt reaction was 1RR: 2Rr: 1rr, as expected.     

Bulked AFLP analysis for the assessment of genetic diversity in white clover (Trifolium repens L.)

The use of bulked leaf samples from individual plants for amplified fragment length polymorphism (AFLP) analysis was evaluated as a tool for assessment of genetic diversity in white clover (Trifolium repens L.). Bulking of leaf samples produced slightly simpler AFLP profiles compared to the combined profiles of individual plants from the same cultivar. Approximately 90% of bands which were present in individual plants were present in bulked samples of the same cultivar. The majority of those absent were rare bands, shared by less than 25% of individual plants. Replicate bulk samples gave almost identical banding patterns, demonstrating the robustness of the bulked AFLP technique. Cluster analysis of AFLP data derived from individual plants resulted in a phenogram similar to that produced from data derived from bulked samples of the same plants. AFLP analysis of bulked samples detected significant amounts of genetic variability among 52 cultivars and accessions with genetic similarity values ranging from 0.42 to 0.92. However, cluster analysis of AFLP data only partially reflected the geographic origin of cultivars and accessions and was not congruent with cluster analysis based on variation for morphophysiological characters. Bulked AFLP analysis provides a powerful tool for rapid assessment of genetic variability in white clover and may also be used for cultivar identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

以DNA位点纯合率评价小麦品种的一致性和稳定性

为了解小麦杂交组合高世代株系和我国小麦品种的DNA位点纯合率,及其评价小麦品种一致性和稳定性的可行性,采用172对SSR、99对EST-SSR和76对AFLP-SCAR引物,检测了10个F₄代株系、10个F₅代株系和511个品种的DNA位点纯合率。结果表明,F₄代和F₅代株系的DNA位点纯合率分别为82.1%~94.5%和95.7%~99.4%。根据F₅代500个单株的DNA纯合位点比率,推测出F₆代株系的DNA位点纯合率为98%~100%。在511个品种中,有10%的品种其DNA位点纯合率低于95%。通过比较证明,DNA位点纯合率越高品种的一致性和稳定性越好。对2006—2009连续3个年度国家冬小麦区域试验品种的检测证明,DNA位点纯合率高于95%的大多数品种和DNA位点纯合率为90%~95%的少数品种具备一致性和稳定性;DNA位点纯合率低于90%的品种不具备一致性和稳定性。说明可以将DNA位点纯合率作为评价小麦品种一致性和稳定性的辅助标准。并提出了以20个个体为样本、检测50个DNA位点的小麦品种DNA位点纯合率检测方法以及检测方法中需注意的细节。     

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.     

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.