PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.     

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。     

Effects of domestication on genetic diversity in Chimonanthus praecox: Evidence from chloroplast DNA and amplified fragment length polymorphism data

Chimonanthus praecox (L.) Link is a widely cultivated endemic winter-flowering plant in China that has a long cultivation history. Genetic diversity and genetic structure were compared between wild and cultivated groups to reveal the geographic origin of the cultivated genotypes using chloroplast DNA (cpDNA) sequences and amplified fragment length polymorphism (AFLP) markers. Nine haplotypes were identified using three combined chloroplast fragments. Based on chloroplast data, the wild group showed greater genetic variation and genetic differentiation and a lower measure of gene flow compared to the cultivated group. The AFLP markers also supported this trend. More than 40% of the cpDNA haplotypes were shared between wild and cultivated groups, with shared haplotypes originating from multiple wild populations, suggesting multiple origins of cultivated plants. Moreover, principal coordinate analysis, UPGMA, and structure analysis of AFLP markers revealed that two wild populations clustered with most of the cultivated populations of Ch. praecox, suggesting that most of the cultivated populations mainly originated from these two populations. The combined cpDNA and AFLP results indicated that modern cultivated Ch. praecox experienced multiple events of origin involving two geographic origins, eastern China (Tianmu Mountain) and southwestern China (the border of Hunan–Guangxi–Sichuan–Guizhou).     

DNA fingerprinting of Leonurus cardiaca L. germplasm in Iran using amplified fragment length polymorphism and inter-retrotransposon amplified polymorphism

In the present study, the extent of inter and intra-population genetic variation was evaluated in Leonurus cardiaca accessions naturally growing in Iran by AFLP and IRAP markers. The fingerprints corresponding to AFLP and IRAP markers revealed high levels of heterozygosity, indicating that L. cardiaca is predominantly an out-crossing species. The average percentage polymorphism was detected as 58% and 90.8% on utilizing AFLP and IRAP data, respectively. Gene diversity values within populations varied 0.14 to 0.20 for AFLP and 0.12 to 0.21 for IRAP. The overall levels of genetic variation present in the L. cardiaca germplasm in Iran were finally determined by combining the AFLP and IRAP datasets to ensure wide genome coverage. The phenogram depicted that the accessions of Dargaz population were genetically distinct from other populations. Based on AFLP and IRAP analysis, it is concluded that L. cardiaca maintains high levels of genetic variation at inter and intra-population level.     

Genetic differentiation analysis of African cassava (Manihot esculenta) landraces and elite germplasm using amplified fragment length polymorphism and simple sequence repeat markers

Molecular‐marker‐aided evaluation of germplasm plays an important role in defining the genetic diversity of plant genotypes for genetic and population improvement studies. A collection of African cassava landraces and elite cultivars was analysed for genetic diversity using 20 amplified fragment length polymorphic (AFLP) DNA primer combinations and 50 simple sequence repeat (SSR) markers. Within‐population diversity estimates obtained with both markers were correlated, showing little variation in their fixation index. The amount of within‐population variation was higher for landraces as illustrated by both markers, allowing discrimination among accessions along their geographical origins, with some overlap indicating the pattern of germplasm movement between countries. Elite cultivars were grouped in most cases in agreement with their pedigree and showed a narrow genetic variation. Both SSR and AFLP markers showed some similarity in results for the landraces, although SSR provided better genetic differentiation estimates. Genetic differentiation (Fst) in the landrace population was 0.746 for SSR and 0.656 for AFLP. The molecular variance among cultivars in both populations accounted for up to 83% of the overall variation, while 17% was found within populations. Gene diversity (H_e) estimated within each population varied with an average value of 0.607 for the landraces and 0.594 for the elite lines. Analyses of SSR data using ordination techniques identified additional cluster groups not detected by AFLP and also captured maximum variation within and between both populations. Our results indicate the importance of SSR and AFLP as efficient markers for the analysis of genetic diversity and population structure in cassava. Genetic differentiation analysis of the evaluated populations provides high prospects for identifying diverse parental combinations for the development of segregating populations for genetic studies and the introgression of desirable genes from diverse sources into the existing genetic base.     

Molecular genecology of temperature response in Lolium perenne: 2. association of AFLP markers with ecogeography

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker-assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan-European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.     

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.     

Genetic diversity among oat varieties of worldwide origin and associations of AFLP markers with quantitative traits

One hundred and fourteen oat (Avena sativa L.) varieties of worldwide origin were evaluated for genetic diversity based on 77 molecular polymorphisms produced by eight selective AFLP primer combinations. Genetic similarity, calculated using the DICE coefficient, was used for cluster analysis and principal component analysis was applied. In addition population structure was explored to identify discrete subpopulations based on allele frequency. Although clustering and population structure showed relationships with region and country of origin, there was no obvious relationship to hull presence or hull colour. Oat varieties originating from European breeding programs showed less diversity than varieties originating from North and South America. Associations between AFLP markers and agronomic traits (grain yield, groat yield, panicle emergence, plant height, and lodging) as well as kernel quality traits (kernel weight, test weight, screening percent and groat percent) were also investigated. Marker-trait associations were tested using a naïve simple regression model and five additional models that account for population structure. Significant associations were found for 23 AFLP markers, with many of these affecting multiple traits. This study demonstrates that diversity can be significantly enhanced using a global collection, and provides evidence for marker-trait associations that can be validated in segregating populations and exploited through marker-assisted selection.     

Assessment of genomic imprinting of SLC38A4, NNAT, NAP1L5, and H19 in cattle

Background

Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia), and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP).

Results

Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA). This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied.

Conclusion

We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres. Future germplasm collection strategies should focus on sampling a large number of plants. Covering many diversity centres is less important because each centre represents a major part of the total diversity in sesame, Central Asia centre being the only exception. The same recommendation holds for the choice of parents for segregant populations used in breeding projects. The traditional assumption that selecting genotypes of different geographical origin will maximize the diversity available to a breeding project does not hold in sesame.     

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, <Emphasis Type="Italic">Ficus carica</Emphasis> L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.     

Molecular genetic diversity of the Turkish national hazelnut collection and selection of a core set

European hazelnut (Corylus avellana L.) is an economically and nutritionally important nut crop with wild and cultivated populations found throughout Europe and in parts of Asia. This study examined the molecular genetic diversity and population structure of 402 genotypes including 143 wild individuals, 239 landraces, and 20 cultivars from the Turkish national hazelnut collection using simple sequence repeat (SSR) markers. A total of 30 SSR markers yielded 407 polymorphic fragments. Diversity analysis of the Turkish hazelnut genotypes indicated that they fell into three subpopulations according to ad hoc statistics and neighbor-joining algorithm. Although all cultivars clustered together, they overlapped with the wild accessions and landraces. Thus, the dendrogram, principal coordinate, and population structure analyses suggest that they share the same gene pool. A total of 78 accessions were selected as a core set to encompass the molecular genetic and morphological diversity present in the national collection. This core set should have priority in preservation efforts and in trait characterization.     

Genetic diversity and differentiation in Eryngium alpinum L. (Apiaceae): comparison of AFLP and microsatellite markers

Genetic diversity and structure of 12 populations of Eryngium alpinum L. were investigated using 63 dominant amplified fragment length polymorphism (AFLP) and seven codominant microsatellite (48 alleles) markers. Within-population diversity estimates obtained with both markers were not correlated, but the microsatellite-based fixation index Fis was correlated with both AFLP diversity indices (number of polymorphic bands and Nei's expected heterozygosity). Only AFLP diversity indices increased with the size of populations, although they did not significantly differ among them (Kruskall-Wallis test). The discrepancy between AFLPs and microsatellites may be explained by a better coverage of the genome with numerous AFLPs, the higher mutation rates of microsatellites or the absence of significant difference among within-population diversity estimates. Genetic differentiation was higher with AFLPs (theta=0.40) than with microsatellites (theta=0.23), probably due to the higher polymorphism of microsatellites. Thus, we considered global qualitative patterns rather than absolute estimates to compare the performance of both types of markers. On a large geographic scale, the Mantel test and multivariate analysis showed that genetic patterns were more congruent with the spatial arrangement of populations when inferred from microsatellites than from AFLPs, suggesting higher homoplasy of AFLP markers. On a small spatial scale, AFLPs managed to discriminate individuals from neighboring populations whereas microsatellites did not (multivariate analysis), and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with microsatellites. However, dominant AFLPs cannot be used to study heterozygosity-related topics. Thus, distinct molecular markers should be used depending on the biological question and the geographical scale investigated.     

Genetic diversity in colonial bentgrass (Agrostis capillaris L.) revealed by EcoRI-MseI and PstI-MseI AFLP markers.

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI-MseI and 6 PstI-MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI-MseI) and 0.63 (PstI-MseI). Greater genetic diversity was detected by the EcoRI-MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.     

Comparative analysis of population genetic structure in Athyrium distentifolium (Pteridophyta) using AFLPs and SSRs from anonymous and transcribed gene regions

To examine the performance and information content of different marker systems, comparative assessment of population genetic diversity was undertaken in nine populations of Athyrium distentifolium using nine genomic and 10 expressed sequence tag (EST) microsatellite (SSR) loci, and 265 amplified fragment length polymorphism (AFLP) loci from two primer combinations. In range-wide comparisons (European vs. North American populations), the EST-SSR loci showed more reliable amplification and produced more easily scorable bands than genomic simple sequence repeats (SSRs). Genomic SSRs showed significantly higher levels of allelic diversity than EST-SSRs, but there was a significant correlation in the rank order of population diversities revealed by both marker types. When AFLPs, genomic SSRs, and EST-SSRs are considered, comparisons of different population diversity metrics/markers revealed a mixture of significant and nonsignificant rank-order correlations. However, no hard incongruence was detected (in no pairwise comparison of populations did different marker systems or metrics detect opposingly significant different amounts of variation). Comparable population pairwise estimates of F(ST) were obtained for all marker types, but whilst absolute values for genomic and EST-SSRs were very similar (F(ST) = 0.355 and 0.342, respectively), differentiation was consistently higher for AFLPs in pairwise and global comparisons (global AFLP F(ST) = 0.496). The two AFLP primer combinations outperformed 18 SSR loci in assignment tests and discriminatory power in phenetic cluster analyses. The results from marker comparisons on A. distentifolium are discussed in the context of the few other studies on natural plant populations comparing microsatellite and AFLP variability.     

High level of genetic diversity among spelt germplasm revealed by microsatellite markers.

The genetic diversity of spelt (Triticum aestivum (L.) Thell. subsp. spelta (L.) Thell.) cultivated presently is very narrow. Although the germplasm collections of spelt are extensive, the related genetic knowledge is often lacking and makes their use for genetic improvement difficult. The genetic diversity and structure of the spelt gene pool held in gene banks was determined using 19 simple sequence repeat (SSR) markers applied to 170 spelt accessions collected from 27 countries and 4 continents. The genetic distances (1 - proportion of shared alleles) were calculated and an unweighted pair-group method with arithmetic averaging (UPGMA)-based dendrogram was generated. The genetic diversity was high: 259 alleles were found and the mean interaccession genetic distance was 0.782 +/- 0.141. The dendrogram demonstrated the much higher genetic diversity of spelt held in germplasm collections than in the currently used genotypes. Accessions with the same geographical origin often tended to cluster together. Those from the Middle East were isolated first. All but one of the Spanish accessions were found in a unique subcluster. Most accessions from eastern Europe clustered together, while those from northwestern Europe were divided into two subclusters. The accessions from Africa and North America were not separated from the European ones. This analysis demonstrates the extent of genetic diversity of spelts held in germplasm collections and should help to widen the genetic basis of cultivated spelt in future breeding programs.     

东亚——北美东部间断透骨草属的扩增片段长度多态性

透骨草属(Phryma)是一个单种属,间断分布于东亚与北美东部.尽管东亚与北美东部居群形态差异非常小,但分子变异却非常明显.本研究进一步运用AFLP两对引物来衡量透骨草属的遗传多样性并评估其形态保守性.结果发现透骨草的遗传差异主要存在于两大洲的居群之间.聚类与PCA分析显示透骨草分成两大支与其地理分布相吻合,一支全部来自东亚,另一支则是北美东部的居群.我们的结果强烈支持透骨草东亚--北美东部居群存在明显的遗传分化和形态保守.     

AFLP markers for the study of rice biodiversity

 AFLP was used as a DNA fingerprinting technique in rice (Oryza sativa L.) germplasm analysis. The high efficiency and random coverage of AFLP markers were established. With only five combinations of primers and RFLP anchors, a framework linkage map was constructed. This map demonstrated that the AFLP markers from a limited number of primers were not confined to any particular regions or chromosomes in the rice genome. To analyse the biodiversity of 57 rice germplasm accessions, we examined 179 polymorphic AFLP markers generated from four primer combinations. Both principal component analysis and cluster analysis were used, and three groups were clearly identified which corresponded to genotypes of Isozyme Groups I, II and VI. The number of markers needed for robust classification of rice germplasm and the diversity between/within the groups was established. Received: 15 July 1997 / Accepted: 29 October 1997     

Evaluation of genetic diversity of Panicum turgidum Forssk from Saudi Arabia

The genetic diversity of 177 accessions of Panicum turgidum Forssk, representing ten populations collected from four geographical regions in Saudi Arabia, was analyzed using amplified fragment length polymorphism (AFLP) markers. A set of four primer-pairs with two/three selective nucleotides scored 836 AFLP amplified fragments (putative loci/genome landmarks), all of which were polymorphic. Populations collected from the southern region of the country showed the highest genetic diversity parameters, whereas those collected from the central regions showed the lowest values. Analysis of molecular variance (AMOVA) revealed that 78% of the genetic variability was attributable to differences within populations. Pairwise values for population differentiation and genetic structure were statistically significant for all variances. The UPGMA dendrogram, validated by principal coordinate analysis-grouped accessions, corresponded to the geographical origin of the accessions. Mantel’s test showed that there was a significant correlation between the genetic and geographical distances (r = 0.35, P < 0.04). In summary, the AFLP assay demonstrated the existence of substantial genetic variation in P. turgidum. The relationship between the genetic diversity and geographical source of P. turgidum populations of Saudi Arabia, as revealed through this comprehensive study, will enable effective resource management and restoration of new areas without compromising adaptation and genetic diversity.     

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.     

二色胡枝子遗传多样性AFLP分析

利用AFLP对二色胡枝子14个居群的161个个体的遗传多样性进行分析,5对引物共扩增出253条带,多态带百分率（P）为96.8%,二色胡枝子种群的平均位点等位基因数（Ao）为1.968,平均有效等位基因数（Ae）为1.643,Nei’s基因多样性指数（H）为0.369,Shannon信息指数（I）为0.544。14个种源的平均多态性条带数为159条,平均多态带百分率为62.9%,平均Nei’s基因多样性指数为0.257,I＝0.373。二色胡枝子遗传多样性的76.68%分布于种源内（Φst＝0.233 2）,各种源间的差异显著。聚类结果表明,二色胡枝14个种源可划分为三个大类,居群的遗传多样性参数与其地理、生态因子均不显著相关,遗传多样性无明显地域分布格局。