The origin of cultivated <Emphasis Type="Italic">Coffea arabica</Emphasis> L. varieties revealed by AFLP and SSR markers

Molecular markers were used to assess polymorphism between and within the genetic bases of coffee (i.e. Typica and Bourbon) spread from Yemen since the early 18th century that have given rise to most arabica cultivars grown world-wide. Eleven Coffea arabica accessions derived from the disseminated bases were evaluated by amplified fragment length polymorphism (AFLP) using 37 primer combinations and simple-sequence repeats (SSRs) produced by six microsatellites. Four cultivars growing in Yemen and 11 subspontaneous accessions collected in the primary centre of diversity of the species were included in the study in order to define their relationship with the accessions derived from the genetic bases of cultivars. One hundred and seven AFLP markers were used to calculate genetic distances and construct a dendrogram. The accessions derived from the disseminated bases were grouped separately, according to their genetic origin, and were distinguished from the subspontaneous accessions. The Yemen cultivars were classified with the Typica-derived accessions. Except for one AFLP marker, all AFLP and SSR markers present in the cultivated accessions were also detected in the subspontaneous accessions. Polymorphism among the subspontaneous accessions was much higher than among the cultivated accessions. It was very low within the genetic bases, confirming the historical documentation on their dissemination. The results enabled a discussion of the genetic diversity reductions that successively occurred during the dissemination of C. arabica from its primary centre of diversity.     

Identification of duplicates for the optimization of carrot collection management

Molecular markers have proved their efficiency for the identification of duplicate accessions in genetic resources collections. Partners of the GENRES Carrot project decided to evaluate the use of molecular markers for the identification of carrot accession duplicates. As a model analysis, 21 presumed duplicate accessions of Jaune du Doubs were selected. Only accessions that were not distinguished on a morphological basis were subjected to molecular analysis. The crucial question was to determine the threshold required to declare whether accessions were duplicates or not. We used a strategy based on the comparison between intravarietal and intervarietal genetic distances. DNA extractions were made on 4–8 bulks of five individuals per accession, and the bulks were analysed using 75 AFLP markers. An additional set of 7 bulks was extracted from one accession to provide true control replicates. With the exception of the true duplicates, all the accessions were clearly differentiated. Based on these results, a general strategy for the identification of carrot duplicates is proposed.     

Hybridization in the section Mentha (Lamiaceae) inferred from AFLP markers

The amplified fragment length polymorphism (AFLP) method was used to evaluate genetic diversity and to assess genetic relationships within the section Mentha in order to clarify the taxonomy of several interspecific mint hybrids with molecular markers. To this end, genetic diversity of 62 Mentha accessions from different geographic origins, representing five species and three hybrids, was assessed. Three EcoRI/MseI AFLP primer combinations generated an average of 40 AFLP markers per primer combination, ranging in size from 50 to 500 base pairs (bp). The percentage of markers polymorphic ranged from 50% to 60% across all accessions studied. According to phenetic and cladistic analysis, the 62 mint accessions were grouped into two major clusters. Principal coordinates analysis separated species into well-defined groups, and clear relationships between species and hybrids could be described. Our AFLP analysis supports taxonomic classification established among Mentha species by conventional (morphological, cytological, and chemical) methods. It allows the assessment of phenetic relationships between species and the hybrids M. spicata and M. × piperita, largely cultivated all over the world for their menthol source, and provides new insights into the subdivision of M. spicata, based for the first time on molecular markers.     

An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP

Ganoderma lucidum is one of the most important medicinal materials and plant pathogens. Because of its specific interhybridization, the genetic background, however, is relatively unclear. It made identification of Ganoderma strains, especially closely related strains difficulty. Amplified fragment length polymorphism (AFLP) using 14 primer combinations and internal transcribed spacer (ITS) PCR-RFLP were used in a comparative study which was designed to investigate the closely related Ganoderma strains genetic relations at molecular level. The analysis of 37 Ganoderma strains showed there were 177 polymorphic AFLP markers and 12 ITS PCR-RFLP markers, and all accessions could be uniquely identified. Among the Ganoderma accessions, similarity coefficients ranged from 0.07692 to 0.99194 in AFLP. The Ganoderma strains formed a tight cluster in nine groups in AFLP whereas seven groups in ITS PCR-RFLP. The cluster analysis revealed that the taxonomical system of subgenus Ganoderma is composed of Sect. Ganoderma and Sect. Phaeonema, and the strain 22 should be a variant form of strain 21. All methods delineated the Ganoderma strains from the different regions seeming to show a greater level of genetic diversity. It indicated that the genotype study at molecular level is a useful complement method to the current classification system of Ganoderma strains based on morphological traits. The congruency of the experiments was analyzed using the biostatistical software DPS V3.01.     

甜瓜(Cucumis melo L.)AFLP标记的多态性及聚类分析研究

利用 AFLP 技术对30个甜瓜材料进行多态性和聚类分析研究。从120对 MseI 和 PstI 引物中筛选出25对扩增效果好的引物,共扩增出262条多态性带。聚类分析结果新疆甜瓜中的夏甜瓜类型,新疆甜瓜中的冬甜瓜类型、美国粗皮甜瓜类型、日本甜瓜类型、梨瓜类型各聚为一组。上述材料的聚类分析结果与依据生态类型和地理起源的分类结果基本吻合,表明 AFLP 用于甜瓜种内不同材料间的遗传变异性分析是可行的。     

Genetic diversity in traditional Ethiopian highland maize accessions assessed by AFLP markers and morphological traits

In the highland regions of Ethiopia the heterogeneity of the land, the climate, and soil favors the presence of a large number of landraces. We analyzed a representative sample of 62 traditional Ethiopian highland maize accessions, using amplified fragment length polymorphism (AFLP^®) markers and morphological traits with the aim to group the accessions based on the their genetic profiles and morphological traits, to study agroecological variation and to assess the level of correlation between phenotypic and genetic distances. Eight EcoRI/MseI primer combinations and 15 morphological traits were used. The accessions varied significantly for all of the measured morphological traits. Of a total of 650 AFLP markers that were scored, 89.5% were polymorphic. Pair-wise genetic distance estimates based on AFLP data revealed dissimilarity coefficients ranging from 0.32 to 0.69 (mean of 0.57). Cluster analysis of the AFLP data grouped most accessions collected from the Northern highlands into one major cluster. It, however, failed to separate the Western and Southern accessions into different clusters. Regardless of the large variation in environmental conditions between agroecologies, only 9% of the total genetic variation was found between agroecologies, whereas 91% was found within agroecologies in Ethiopia. This finding may be explained by long distance seed exchange, continuous seed introduction and gene flow between agroecologies. The relationship between morphological and AFLP-based distances was significant and positive. Based on this study, three groups of highland accessions, with distinctive genetic profiles and morphological traits were identified. This information will be useful for further collections and conservation of the unique diversity included in the highland maize landraces of Ethiopia.     

Patterns of AFLP variation in a core subset of cultivated hexaploid oat germplasm

Many core collections have been developed from large collections of crop germplasm, but most of these have not been characterized, particularly using molecular techniques, for germplasm management and utilization. We have attempted to characterize a structured sample representing a world collection of 11,622 cultivated hexaploid oat accessions in the hope of understanding the genetic structure of the world collection. The amplified fragment length polymorphism (AFLP) technique was applied to screen 670 accessions representing 79 countries and one group of uncertain origin. For each accession, 170 AFLP polymorphic bands detected by five AFLP primer pairs were scored. Analyses of the AFLP data showed the effectiveness of the stratified sampling applied in capturing country-wise AFLP variation. The frequencies of polymorphic bands ranged from 0.11 to 0.99, with an average of 0.72. The majority (89.9%) of the AFLP variation resided within accessions of each country, and only 6.2% of the AFLP differences existed among accessions of major geographic regions. Accessions from the Mediterranean region were the most distinct, while those from Russia and the USA were the most diverse. The two distinct groups that were observed were separated largely on the basis of common oat and red oat. Red oat was genetically more diverse than its common and hull-less counterparts, and hull-less oat was more related to common oat than red oat. Landrace and non-landrace accessions displayed similar AFLP variation patterns. These patterns are significant for understanding the domestication of cultivated oat and are useful in classifying the intraspecific diversity of oat germplasm, developing specific core subsets of the oat collection, and exploring new sources of genes for oat improvement.     

AFLP diversity in the common vetch ( Vicia sativa L.) on the world scale

The Vavilov Institute of Plant Industry (VIR) keeps a living seed collection of about 700 accessions of landraces and local cultivars of common vetch ( Vicia sativa L.) that have been collected over a period of more than 50 years throughout the former USSR. Much of the material is available nowhere else. The collection of this economically important fodder crop is well adapted to the various growing regions of Russia and serves as a basis for the all domestic vetch breeding programs. Using AFLP as a DNA fingerprinting method we investigated 673 accessions from the VIR and compared their genetic variability with that of the worldwide vetch collection of the Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), 450 accessions. The analysis is a first assessment of the intra-specific diversity of V. sativa stored ex situ on a scale of more than 1,000 accessions. Six primer combinations, which gave clear polymorphic amplification products with 96 test samples, were chosen from 111 primer combinations tested. The selected AFLP primers used to analyse the V. sativa intra-specific diversity resulted in 70 unequivocally recognizable polymorphic fragments. We found that all of the AFLP fragments generated can be detected with varying frequency throughout the entire distribution area of V. sativa. The difference in frequency of some AFLP fragments between the regions may amount to 90%. The arrangement of most of the accessions in all dendrograms reflects their geographical origin, with a differentiation between Russia, Western Europe, Turkey and Bulgaria, and the Mediterranean. The "Russian" genepool stored at the IPK is a limited and biased sample of the available diversity when compared to the material stored at the VIR. Approximately 10-15% of the accessions in each geographical group showed AFLP patterns that clustered with members of other groups. This appreciable overlap raises several questions: (1) to which degree is an AFLP pattern representative of the overall genetic similarity of the samples; (2) to which degree are samples collected at a site adaptively limited to that site? Since our data identify accessions with very similar AFLP patterns from very diverse geographic origins, a comparison of the agronomic performance of these accessions (possibly in the two regions) will provide important information for the utilization of ex situ germplasm collections.     

AFLP,RAPD, and ISSR analysis of intraspecific polymorphism and interspecific differences of allotetraploid species <Emphasis Type="Italic">Aegilops kotschyi</Emphasis> Boiss. and <Emphasis Type="Italic">Aegilops variabilis</Emphasis> Eig

To evaluate genetic variation, 27 accessions of allotetraploid species Aegilops kotschyi and Ae. variabilis with the US genome were analyzed using the AFLP, RAPD, and ISSR methods. A total of 316 polymorphic RAPD fragments, 750 polymorphic AFLP fragments, and 234 polymorphic ISSR fragments were obtained. It was demonstrated that the analyzed species were characterized by a considerable level of nuclear genome variation. According to the data of ISSR and RAPD analysis, the average value of the Jaccard similarity coefficient for the accessions of Ae. variabilis from different geographical regions was slightly lower than that for the accessions of Ae. kotschyi. At the same time, AFLP analysis showed no considerable differences in the levels of intraspecific variation of the studied species. Analysis of the summarized RAPD, ISSR, and AFLP marking data in the Structure software program showed that most of the analyzed accessions with high degree of probability could be assigned to one of two groups, the first of which corresponded to Ae. kotschyi and the second corresponded to Ae. variabilis, thereby confirming the species independence of Ae. kotschyi and Ae. variabilis. Accessions k900, k907, k908, and v90 could not with a sufficiently high degree of probability be assigned to one of the species, which possibly was the result of interspecific hybridization. Analysis of the species diversity using different molecular markers made it possible to identify the accessions that were notably different from other accessions of its species.     

羊草种质基因组DNA的AFLP多态性研究

羊草是禾本科牧草之王 ,在当前我国西部生态建设和草原畜牧业发展中发挥着重要作用。用AFLP方法对2 7份我国不同地区分布的羊草 (Leymuschinensis (Trin .)Tzvel)材料进行了基因组DNA多态性分析 ,8对AFLP引物组合在 2 7个不同羊草基因型中共扩增出 5 37条带 ,产生出的DNA片段大小分布在 75bp - 5 30bp之间。其中单态性带 89条 ,占 16 .6 % ,多态性带 32 9条 ,占 6 1.3%。平均每对引物组合扩增的DNA带数为 6 6 .13,总的多态性比率为 78.84%。AFLP多态信息含量PIC值分布于 0 .0 - 0 .5之间 ,平均PIC值为 0 .2 16 ,出现的PIC最大值 (0 .5 )约占AFLP标记的 8.5 % ,说明羊草基因组DNA的多态性比较丰富。以 5 37个AFLP标记为原始数据 ,根据Nei和Li的方法对 2 7份羊草材料进行遗传变异和聚类分析的结果表明 :羊草种内有高频率的遗传变异发生 ,且与地理分布和生态环境密切相关 ;2 7份羊草不同基因型被划分为四大类群 ,不同类群相互间的遗传距离相对较大 ,在树状图中表现为较远的亲缘关系。对羊草种内遗传变异发生的原因和品种的形成进行了初步讨论。     

Genetic diversity in tetraploid switchgrass revealed by AFLP marker polymorphisms

Switchgrass (Panicum virgatum) is a perennial warm-season grass native to North America that has been identified as a dedicated cellulosic biofuel crop. We quantified genetic diversity in tetraploid switchgrass germplasm collected at Oklahoma State University and characterized genetic relatedness among the collections from distinct regions. Fifty-six tetraploid accessions, including seven upland and 49 lowland genotypes from throughout the US, were examined. The amplified fragment length polymorphism (AFLP) procedure was utilized to generate DNA profiling patterns that were scored visually. Sixteen selective AFLP primer combinations were used to amplify 452 polymorphic bands. The accessions' genetic similarity coefficients, UPGMA (unweighted pair-group method with arithmetic averaging) cluster analysis and principle coordinate analysis, were performed. The upland and lowland accessions clustered according to ecotypes, with one exception (TN104). Genetic similarity coefficients among the accessions ranged from 0.73 to 0.95. Analysis of molecular variance (AMOVA) was performed, showing significant differences between the upland and lowland genotypes. The trnL marker confirmed that TN104 was a lowland genotype, but the trnL marker identification of upland and lowland genotypes was not consistent with the AFLP analysis in two germplasms (Miami and AR4).     

Genetic diversity of different Tunisian fig (Ficuscarica L.) collections revealed by RAPD fingerprints

The genetic diversity in Tunisian fig (Ficus carica L.) was studied using RAPD markers. Thirty-five fig cultivars originating from diverse geographical areas and belonging to three collections were analysed. Random decamer primers were screened to assess their ability to detect polymorphisms in this crop. Forty-four RAPD markers were revealed and used to survey the genetic diversity and to detect cases of mislabelling. As a result, considerable genetic diversity was detected among the studied F. carica accessions. The relationships among the 35 varieties were studied by cluster analysis. The dendrogram showed two main groups composed of cultivars with similar geographic origin. Moreover, the male accessions (caprifigs) were clustered indistinctively within the female ones, suggesting a narrow genetic diversity among these accessions. Our data proved that RAPD markers are useful for germplasm discrimination as well as for investigation of patterns of variation in fig. Since this designed procedure has permitted to establish a molecular database of the reference collections, the opportunity of this study is discussed in relation to the improvement and rational management of fig germplasm.     

利用分子标记和数量性状基因型值构建作物核心种质库的研究

提出了一种基于分子标记数据及数量性状基因型值构建作物种质资源核心种质库的方法.采用包括基因型与环境互作的遗传模型及相应的混合线性模型统计分析方法,无偏预测各材料的基因型值,分别用基因型值和分子标记数据计算个体间的相似系数,加权得到最终的相似距离.采用不加权类平均法（UPGMA）进行系统聚类,用多次聚类随机取样法构建核心种质库.以水稻DH群体111个基因型8个农艺性状、175个分子标记位点的数据为实例,按四种抽样比率（25%,20%,15%,10%）构建了四个核心种质库,比较了核心种质库与整个群体的分子标记多样性及数量性状的遗传变异,评价了所用方法的有效性。     

Comparative study of different molecular markers for classifying and establishing genetic relationships in Coffea canephora

The genetic variability characterization of the accessions of the germplasm collection, using molecular markers, is being applied as a complementary strategy to the traditional approaches to redefine the plant genetic resources. In this study, we compared the informativeness and efficiency of the molecular markers RAPD, AFLP and SSR in the analysis of 94 accessions of Coffea canephora germplasm held by the breeding program of the Brazilian Agricultural Research Corporation (Embrapa), Rondônia State, Brazil. For this, we considered the marker’s discriminatory power and level of polymorphism detected and also the genetic relationships and clustering (dendrogram) analysis. The RAPD marker yielded low-quality data and problems in the discrimination of some accessions, being less recommended for genetic studies of C. canephora. The SSRs had a higher level of information content and yielded high-quality data, while AFLP was the most efficient marker system because of the simultaneous detection of abundant polymorphism markers per few reactions. Our results indicate that AFLP and SSR, allies to the intrinsic characteristics of each technique, are the most suitable molecular markers for genetic studies of C. canephora. However, the choice of AFLP or SSR in the species characterization should be made in agreement with some characteristics that are discussed in this work.     

Molecular genetic diversity and association mapping of nut and kernel traits in Slovenian hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis>) germplasm

European hazelnut (Corylus avellana L.), cultivated in several areas of the world including Europe, Anatolia, and the USA, is an economically important nut crop due to its high mineral, oleic acid, amino acid, and phenolic compound content and pleasant flavor. This study examined molecular genetic diversity and population structure of 54 wild accessions and 48 cultivars from the Slovenian national hazelnut collection using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Eleven AFLP primer combinations and 49 SSR markers yielded 532 and 504 polymorphic fragments, respectively. As expected for a wind-pollinated, self-incompatible species, levels of genetic diversity were high with cultivars and wild accessions having mean dissimilarity values of 0.50 and 0.60, respectively. In general, cultivars and wild accessions clustered separately in dendrogram, principal coordinate, and population structure analyses with regional clustering of the wild material. The accessions were also characterized for ten nut and seven kernel traits and some wild accessions were shown to have breeding potential. Morphological principal component analysis showed distinct clustering of cultivars and wild accessions. An association mapping panel composed of 64 hazelnut cultivars and wild accessions had considerable variation for the nut and kernel quality traits. Morphological and molecular data were associated to identify markers controlling the traits. In all, 49 SSR markers were significantly associated with nut and kernel traits [P < 0.0001 and LD value (r ²) = 0.15–0.50]. This work is the first use of association mapping in hazelnut and has identified molecular markers associated with important quality parameters in this important nut crop.     

Molecular genetic diversity of the pea (Pisum sativum L.) from the Vavilov Research Institute collection detected by the AFLP analysis

In this work, a set of pea accessions obtained from the Vavilov Research Institute (VIR) collection comprising 83 P. sativum samples, including representatives of three subspecies, was studied using the AFLP method. Local cultivars for different uses with maximum ecological and geographical diversity (including those from the centers of origin of the species) were predominantly chosen for the study; a number of their morphological and biological characteristics were evaluated. We obtained 382 polymorphic AFLP fragments; each sample was characterized by a unique set of fragments. The genetic diversity of the studied material was evaluated, and a wide range of genetic differences in the investigated samples (0.07–0.27) was demonstrated. The affiliation of the samples to the certain subspecies was not confirmed by the obtained data; the ecogeographical differentiation of the samples was not reflected by the data. Factor analysis allowed us to identify the sample groups of European and Asian origin and the intermediate nature of most of the samples from the studied set of pea accessions.     

Nucleotide-binding site (NBS) profiling of genetic diversity in durum wheat.

Molecular markers are effective tools to investigate genetic diversity for resistance to pathogens. NBS (nucleotide-binding site) profiling is a PCR (polymerase chain reaction)-based approach to studying genetic variability that specifically targets chromosome regions containing R-genes and R-gene analogues. We used NBS profiling to measure genetic diversity among 58 accessions of durum wheat. Mean polymorphism rates detected using MseI and AluI as restriction enzymes were 34% and 22%, respectively. Mean number of polymorphisms per enzyme-primer combination was equal to 23.8 +/- 5.9, ranging from 13 to 31 polymorphic bands. In total, 96 markers over 190 indicated a good capacity to discriminate between accessions (the polymorphic index content ranging from 0.30 to 0.50). The results obtained with NBS profiling were compared with simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) data of the same set of accessions. The genetic distances computed with 190 NBS profiling markers were in close agreement with those obtained with AFLP and SSR markers (r = 0.73 and 0.76, respectively). Our results indicate that NBS profiling provides an effective means to investigate genetic diversity in durum wheat.     

Genetic relationships within Brassica rapa as inferred from AFLP fingerprints

Amplified fragment length polymorphism (AFLP) markers were employed to assess the genetic diversity amongst two large collections of Brassica rapa accessions. Collection A consisted of 161 B. rapa accessions representing different morphotypes among the cultivated B. rapa, including traditional and modern cultivars and breeding materials from geographical locations from all over the world and two Brassica napus accessions. Collection B consisted of 96 accessions, representing mainly leafy vegetable types cultivated in China. On the basis of the AFLP data obtained, we constructed phenetic trees using mega 2.1 software. The level of polymorphism was very high, and it was evident that the amount of genetic variation present within the groups was often comparable to the variation between the different cultivar groups. Cluster analysis revealed groups, often with low bootstrap values, which coincided with cultivar groups. The most interesting information revealed by the phenetic trees was that different morphotypes are often more related to other morphotypes from the same region (East Asia vs. Europe) than to similar morphotypes from different regions, suggesting either an independent origin and or a long and separate domestication and breeding history in both regions.     

Genetic variability of cultivated Chaenomeles speciosa (Sweet) Nakai based on AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis was performed to evaluate genetic relationships among 52 Chaenomeles speciosa accessions grown in China. A total of 208 polymorphic markers were generated from eight selective primer pair combinations. Genetic variations were remarkably observed in a wide range of dissimilarities, percent polymorphisms, and average polymorphism information. Genetic similarity values were calculated using Jaccard's coefficient between individuals of different flowering quince accessions; these values ranged from 0.296 to 0.931 with a mean of 0.597. Cluster analysis and principal coordinate analysis were then performed on the basis of AFLP profiles. In our generated dendrogram, accessions were clustered into seven major groups. Mantel's test was performed to determine cophenetic correlation (r = 0.9); this result indicated a very good fit of the dendrogram. We conducted analysis of molecular variance and found greater variations within cultivars than among cultivars. AFLP analysis results further revealed relevant information regarding the genetic background of flowering quince accessions essential for future breeding programs related to plant improvement.     

Molecular identification of new varieties of<Emphasis Type="Italic">Nierembergia linariaefolia</Emphasis> (Graham), a native Argentinean ornamental plant

Six Nierembergia linariaefolia clones were selected for ornamental traits during a native germplasm development program. For fingerprinting diagnosis, 13 anchored inter-simple sequence repeat (ISSR) primers and 6 amplified fragment length polymorphism (AFLP) primer-enzyme combinations were used. Both markers revealed high levels of polymorphism, enabling genetic discrimination of the accessions analyzed by using 443 informative ISSRs and 541 AFLP markers. Both molecular techniques are suitable for monitoring genetic diversity in Nierembergia linariaefolia and, under our experimental conditions, they showed correlation coefficients of 0.629 for similarity matrices and of 0.649 in the cophenetic matrices. These results suggest that ISSRs are a good choice for DNA analysis in N. linariaefolia when simple manipulation and a low budget are required.