Vine-1 retrotransposon-based sequence-specific amplified polymorphism for Vitis vinifera L. genotyping

The sequence‐specific amplification polymorphism (S‐SAP) method, derived from the amplified fragment length polymorphism (AFLP) technique, produces amplified fragments containing retrotransposon long terminal repeat ( LTR ) sequence at one end and a host restriction site at the other. The development and application of this procedure to the LTR of the Vine‐1 element from grapevine is reported. Two primers derived from one of the LTR sequences flanking the retrotransposon were used in combination with MseI degenerated primers on 15 grapevine accessions. S‐SAP results were compared with AFLP data. The heterozygosity and gene diversity values were higher for S‐SAP than for the AFLP procedure. Results show that S‐SAP amplification is effective in identifying polymorphisms and defining genetic distances among cultivars, and could be used for fingerprinting and for ‘Traminer’ clone identification. To the contrary Vine‐1 retrotransposon‐based S‐SAP was not able to distinguish ‘Pinot’ clones.     

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.     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.     

Amplified fragment length polymorphism analysis in diploid cultivars of rhodesgrass

The amplified fragment length polymorphism (AFLP) technique was applied to detect genetic variation in a sample of 47 plants representing 12 diploid cultivars of rhodesgrass. In this analysis, 50±91 easily scorable fragments could be detected in a single reaction. Each of the individual plants was uniquely identified by a combination of three primer pairs and an 80.2% level of polymorphism was obtained. Large amounts of genetic variation were present within all the cultivars. The results showed that AFLPs could be a robust technique for genome analysis in rhodesgrass with a promising potential as a breeding tool.     

哺鸡竹亲缘关系的AFLP和SRAP分析

本研究采用AFLP和SRAP分子标记技术对9个哺鸡竹类竹种(含2个变型)的亲缘关系进行分析。研究结果表明:12对AFLP引物共扩增出359条带,多态性条带比率为49.7%,相似系数变化范围在0.779～0.978之间;24对SRAP引物共扩增出258条带,多态性条带比率为64.0%,相似系数变化范围在0.721～0.958之间。UPGMA聚类分析表明,AFLP和SRAP标记均将哺鸡竹分成2大类:第Ⅰ类为富阳乌哺鸡竹、花哺鸡竹、黄纹竹、黄秆乌哺鸡竹、毛壳花哺鸡竹、乌哺鸡竹、白哺鸡竹;第Ⅱ类为云和乌哺鸡竹、红哺鸡竹。对这两种标记相似性系数进行相关性分析,AFLP与SRAP的结果呈极显著相关(r=0.934),但SRAP标记比AFLP标记可在竹类及其种下等级检测到更大的遗传变异。     

甘蔗种质遗传基础的AFLP分析

采用AFLP分子标记技术对54份甘蔗种质（14份祖亲种、40份栽培品种或品系）的遗传基础进行了分析。利用筛选出的4对多态性较强的引物组合（M+CAG/E+ACG,M+CTC/E+ACT,M+CTG/E+ACC,M+CTG/E+ACG）,构建了甘蔗54份种质的AFLP指纹图谱,这4对引物组合共扩增出396条谱带,其中多态带390条,占98.5%。54份种质的遗传相似系数变化     

AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass. This revised version was published online in August 2006 with corrections to the Cover Date.     

基于AFLP标记技术的三翅槭与三角枫亲缘关系分析

为了从分子水平上揭示三翅槭与三角枫的亲缘关系,明确三翅槭在槭属中的分类地位,笔者利用AFLP分子标记技术对三翅槭1份材料和三角枫4份材料进行亲缘关系检测,并采用UPGMA法进行聚类分析。结果表明：8对引物组合共获得202条谱带,其中99条带具有多态性,多态性比率为49.01%,说明AFLP标记技术适用于三翅槭和三角枫亲缘关系研究。聚类分析表明,在遗传相似系数为0.71时,可将三翅槭和三角枫划分成2个相互独立的类群,说明三翅槭与三角枫在分子水平上的分类与形态学上的分类一致,此外三翅槭与三角枫之间的遗传相似系数为0.71也表明它们之间的亲缘关系较近。通过AFLP分子标记,成功分析了三翅槭与三角枫亲缘关系,为证明三翅槭是槭属新种提供了分子证据。     

马铃薯品种遗传多样性的AFLP分析

利用AFLP分子标记技术对19份克新系列马铃薯品种进行了遗传多样性分析,用30对引物组合进行了初筛,选出7对有多态性的引物组合进行了详细研究。每对AFLP引物组合扩增出54~90条带,共获得495条带,其中多态性条带为302条。AFLP分析表明19个材料的遗传距离介于0.2091~0.7679之间,平均值为0.4811。聚类分析将19份材料划分为4类,其中第2类包括14个品种,占总数73.86%,表明多数品种亲缘关系较近,但有少数品种亲缘关系较远,说明克新系列马铃薯的遗传基础有所拓宽。研究表明：AFLP指纹分析技术具有很高的分辨率,适于进行马铃薯遗传多样性研究     

RAPD和AFLP标记分析中国马铃薯主要品种的遗传多样性

采用RAPD 和AFLP两种方法分析71份中国各地马铃薯主要品种,均可将其完全区分,并可对其进行分子鉴定;证明中国马铃薯主要品种遗传组成上差异小,遗传多样性差。由于标记方法的原理差异和栽培马铃薯遗传组成复杂性,用2种方法分类的结果有所差异。AFLP标记检测获得的Shannon-weaver指数和Simpson指数均高于RAPD标记检测的结果,AFLP标记检测多态性的能力远高于RAPD标记。AFLP标记平均每个引物组合检测到100.1个位点,其中54.9条为多态性位点,而RAPD标记的相应数据分别为12.5和9.8个。不同的标记方法在马铃薯遗传多样性研究中存在差异,聚类结果从分子水平反映了中国现有主要马铃薯品种遗传基础的狭窄。     

用RAPD、ISSR和AFLP标记分析系谱关系明确的甘薯品种的亲缘关系

用RAPD、ISSR和AFLP标记对系谱关系明确的7个甘薯品种进行了亲缘关系分析。24个RAPD引物、14个ISSR引物和9对AFLP引物分别扩增出173、174和168条多态性带。3种分子标记在检测甘薯品种间遗传差异上相关程度高，其中RAPD与ISSR之间的相关系数最大为0.9328。用ISSR标记估计的品种间遗传距离为0.1286~1.0932，平均0.4883，大于其余2个标记的估计值。3种分子标记皆可揭示甘薯品种的亲缘关系，其中ISSR标记产生的聚类图与系谱图最吻合，认为ISSR标记更适于分析甘薯品种的亲缘关系。     

甘蔗（Saccharum officinarum L.）品种遗传差异的AFLP分子标记分析

采用AFLP技术,从64对引物中筛选出5对带型分布均匀、多态性丰富且分辨能力强的引物,对两个在生产上广泛推广应用的甘蔗品种的AFLP指纹图谱进行分析,计算了两品种间的遗传相似性和遗传距离。结果表明,5对引物在2个甘蔗品种间均存在显著的差异,其中多态性位点占总扩增位点的10.2%,区分率达100%。这对应用AFLP技术鉴定甘蔗     

Amplified fragment length polymorphism as a tool for molecular characterization of almond germplasm: genetic diversity among cultivated genotypes and related wild species of almond,and its relationships with agronomic traits

Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient method for producing DNA fingerprints and molecular characterization. Our objectives were to: estimate genetic similarities (GS), marker indices, and polymorphic information contents (PICs) for AFLP markers in almond cultivars; assess the genetic diversity of almond cultivars and wild species, using GS estimated from AFLP fingerprints and molecular characterization; and facilitate the use of markers in inter-specific introgression and cultivar improvement. The genetic diversity of 45 almond cultivars from Iran, Europe, and America, were studied assaying 19 primer combinations. In addition, several agronomic traits were evaluated, including flowering and maturity times, self-incompatibility, and kernel and fruit properties. Out of the 813 polymerase chain reaction fragments that were scored, 781 (96.23%) were polymorphic. GS ranged from 0.5 to 0.96, marker indices ranged from 51.37 to 78.79, and PICs ranged from 0.56 to 0.86. Results allowed the unique molecular identification of all assayed genotypes. However, the correlation between genetic similarity clustering as based on AFLP and clustering for agronomic traits was low. Cluster analysis based on AFLP data clearly differentiated the genotypes and wild species according to their origin and pedigree, whereas, cluster analysis based on agronomic data differentiated according the pomological characterization. Our results showed the great genetic diversity of the almond cultivars and their interest for almond breeding.     

Molecular characterization of novel resynthesized rapeseed (Brassica napus) lines and analysis of their genetic diversity in comparison with spring rapeseed cultivars*

Resynthesized (RS) rapeseed generated from interspecific hybridization between suitable forms of Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and B. oleracea L. (CC, 2n = 18) represents a potentially important resource to expand genetic diversity in the narrow gene pool of oilseed rape (B. napus L., AACC, 2n = 38). In this study 165 RS rapeseed lines originating from crosses between an Indian Yellow Sarson (B. rapa ssp. trilocularis) and five different cauliflower (B. oleracea convar. botrytis) cultivars were studied using amplified fragment length polymorphism (AFLP) markers and their genetic diversity was compared in relationship to an assortment of 40 diverse spring oilseed and fodder rape varieties. Using three AFLP primer combinations, a total of 467 polymorphic bands were scored. Cluster analysis allowed differentiation among the different RS lines, which, as expected, were genetically highly divergent from the cultivars. The genetic diversity of the material is discussed in relation to its morphological variability with a view to the implementation of RS lines in oilseed rape breeding.     

Italian rice varieties: historical data, molecular markers and pedigrees to reveal their genetic relationships

Two molecular marker approaches [amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR)] were employed to study genomic relationship among 96 rice cultivars. These included most of the best reputed Italian accessions. AFLP produced 461 fragments, 248 (53%) of which were polymorphic, SSR produced four to 11 alleles in the 12 genomic loci investigated. Genomic similarity was estimated independently for the two molecular marker techniques. Both AFLP and SSR dendrograms agree in splitting the cultivars into two main clusters: a small one, comprising four exotic accessions, and a larger one which could be split into four subgroups. These were also analysed on the basis of historical and pedigree information. This is the first report on the application of DNA polymorphism analysis to reveal genomic relationship among cultivated Italian rice germplasm. Results will be useful for breeding programmes.     

A genetic linkage map of rhodesgrass based on an F1 pseudo-testcross population

The linkage relationships between 164 polymorphic amplified fragment length polymorphism (AFLP) and 25 restriction fragment length polymorphism (RFLP) fragments assayed in a pseudo‐testcross population generated from the mating of single genotypes from two divergent cultivars were used to construct female, ‘Katambora’ (‘Kat’) and male, ‘Tochirakukei’ (‘Toch’) parental genetic maps for rhodesgrass. The ‘Kat’ genetic map consists of 84 marker loci (72 AFLP and 12 RFLP markers) distributed on 14 linkage groups and spans a total length of 488.3 cM, with an average distance of 7.8 cM between adjacent markers. The ‘Toch’ genetic map consists of 61 marker loci (52 AFLP and nine RFLP) mapped on 12 linkage groups spanning a total length of 443.3 cM, with an average spacing of 9.0 cM between adjacent markers. About 23% of the markers remained unassigned. The level of segregation distortion observed in this cross was 11.1%. In both maps, linked duplicated RFLP loci were found. These linkage maps will serve as a starting point for linkage studies in rhodesgrass with potential application for marker‐assisted selection in breeding programmes.     

Identification of two SCAR markers co-segregated with the dominant Ms and recessive ms alleles in onion (Allium cepa L.)

Cytoplasmic genetic male-sterility is used to produce hybrid onion (Allium cepa L.) seeds worldwide. In this paper, we present the results of research aimed toward identifying PCR-based markers linked to the Ms locus through amplified fragment length polymorphism (AFLP). After screening 512 AFLP primer combinations, only one AFLP fragment was identified as being flanking linked to the dominant Ms allele. Subsequently, the AFLP marker was converted into a sequence-characterized amplified region (SCAR) marker, designated as DNF-566, co-segregated with the dominant Ms allele in first backcross (BC₁) segregated populations. Furthermore, we designed another molecular marker (RNS-357) co-segregated with the ms allele to identify different genotypes (i.e., MsMs, Msms, or msms). Both markers could be used for evaluating onion lines with different genetic backgrounds (including male-sterile lines, maintainer lines, male-fertile lines, and commercial based F₁ hybrid cultivars). The results of this study indicate that maintainer plants could be directly selected by using these 2 SCAR markers in the onion breeding process, and this may contribute significantly toward breeding onion F₁ hybrid cultivars.     

Relationship between heterosis and genetic distance based on morphological traits and AFLP markers in pepper

Genetic diversity is considered as one of the criteria for the selection of parents for hybrid breeding. The present study was undertaken to evaluate genetic divergence among seven pepper cultivars and to assess the relationship between heterosis and parental genetic distance. Twenty‐one F₁ hybrids and seven parents were evaluated for 15 morphological characters in a greenhouse and in the field. The parents were examined for DNA polymorphisms using six amplified fragment length polymorphism (AFLP) primer combinations. Cluster analysis using two genetic distance measures grouped the seven parents differently. Mid‐parent and high‐parent heterosis was observed for most characters. Most hybrids outperformed the parental lines for fruit yield, earliness and plant height. Morphological and AFLP‐based distance measurements were efficient enough to allocate pepper genotypes into heterotic groups. The correlations of morphological distances with mid‐parent heterosis were significant for days to flowering and maturity, suggesting earliness can be predicted from morphological distances of parental lines. However, the correlations of AFLP‐measured genetic distances with mid‐ and high‐parent heterosis were non‐significant for all characters, except for fruit diameter, and proved to be of no predictive value.     

Evaluating genetic relationships in seed impatiens, Impatiens walleriana, using AFLP profiling

Although seed impatiens is the leading bedding plant in the US, little work has been conducted on evaluating the genetic variability present in this crop. In this study, amplified fragment length polymorphism technology was utilized to determine the level of polymorphism present across 20 commercial cultivars of seed impatiens, to assess their genetic variability, and to investigate their genetic relatedness. A total of eight EcoRI and MseI primer combinations were used for polymerase chain reaction amplification. Fluorescence‐labelled amplification products were subjected to electrophoresis and then analysed using an automated sequencer. High levels of polymorphism were detected among all 20 cultivars for all primer combinations tested. Gower's Genetic Dissimilarity estimates for the entire set of cultivars ranged from 1.000 to 0.316. The dendogram generated from these dissimilarity data revealed a number of groupings, including one major division which placed all of the cultivars into one of two groups. The implications of these results on genetic variability, genotypic relationships and genetic diversity in seed impatiens is discussed.     

Application of the TRAP technique to lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) genotyping

Summary To demonstrate the applicability of the target region amplification polymorphism (TRAP) marker technique to lettuce genotyping, we fingerprinted 53 lettuce (Lactuca sativa L.) cultivars and six wild accessions (three from each of the two wild species, L. saligna L. and L. serriola L.). Seven hundred and sixty-nine fragments from 50 to 900 bp in length were amplified in 10 PCR reactions using 10 fixed primers in combination with four fluorescent labeled arbitrary primers. Three hundred and eighty-eight of these fragments were polymorphic among the 59 Lactuca entries and 107 fragments were polymorphic among the 53 lettuce cultivars and the six wild accessions; 251 fragments were present only in the wild species. These markers not only discriminated all cultivars, but also revealed the evolutionary relationship among the three species: L. sativa, the cultivated species, is more closely related to L. serriola than to L. saligna. Cluster analysis grouped the cultivars by horticultural types with a few exceptions. These results are consistent with previous findings using RFLP, AFLP, and SAMPL markers. The TRAP markers revealed significant differences in genetic variability among horticultural types, measured by the average genetic similarity among the cultivars of the same type. Within the sample set, the leaf type and butterhead types possessed relatively high genetic variability, the iceberg types had moderate variability and the romaine types had the lowest variability. The genetic behavior of TRAP markers was assessed with a mapping population of 45 recombinant inbred lines (RILs) derived from an interspecific cross between L. serriola and L. sativa. Almost all the markers segregated in the expected 1:1 Mendelian ratio and are being incorporated into the existing lettuce linkage maps. Our results indicate that the TRAP markers can provide a powerful technique for fingerprinting lettuce cultivars. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.