Genetic diversity in a germplasm collection of roseroot (Rhodiola rosea) in Norway studied by AFLP

Rhodiola rosea is widely distributed in Norway, but so far limited knowledge exists on the level of genetic diversity. To initiate a selective breeding program, Amplified Fragment Length Polymorphism (AFLP) analysis was used to estimate genetic diversity within the Norwegian R. rosea germplasm collection. AFLP analysis of 97 R. rosea clones using five primer combinations gave a total of 109 polymorphic bands. We detected high percentage of polymorphic bands (PPB) with a mean of 82.3% among the clones of R. rosea. Each of the 97 R. rosea clones could be unambiguously identified based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the Unweighted Pair Group Method with Arithmetic mean (UPGMA). Genetic similarity based on the AFLP data ranged from 0.440 to 0.950 with a mean of 0.631. This genetic analysis showed that there was no close genetic similarity among clones related to their original growing county. No gender-specific markers were found in the R. rosea clones. Analysis of molecular variance (AMOVA) revealed a significantly greater variation within regions (92.03%) than among regions (7.97%). A low level of genetic differentiation (F_ST = 0.043) was observed, indicating a high level of gene flow, which had a strong influence on the genetic structure at different counties. Our results indicate high gene flow among R. rosea clones that might be a result of seed dispersal rather than cross-pollination. Further world-wide studies are required to compare the level of genetic diversity and more studies in R. rosea detailing the consequences of different patterns of gene flow (pollen spread and dispersal of seeds and clonal plants) will be useful for characterization of roseroot.     

Genetic structure in cultivated and wild carrots (Daucus carota L.) revealed by AFLP analysis

Genetic variation within and among five Danish populations of wild carrot and five cultivated varieties was investigated using amplified fragment length polymorphism (AFLP). Ten AFLP primer combinations produced 116 polymorphic bands. Based on the marker data an UPGMA-cluster analysis and principal component analysis (PCA) separated the Daucus collections into three groups, consisting of the wild populations, the old varieties, and the recently bred varieties. The genetic distance between the wild populations reflected the physical distance between collection sites. Analysis of genetic diversity showed that the old varieties released between 1974 and 1976 were more heterogeneous than the newly developed F₁ hybrid varieties. The analysis of molecular variation (AMOVA) showed that the major part of the genetic variation in the plant material was found within populations/varieties. The presence of markers specific to the cultivated carrot makes it possible to detect introgression from cultivated to wild types. Received: 6 October 1999 / Accepted: 4 November 1999     

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.     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001     

Characterization of a genetic resource collection for Miscanthus (Saccharinae,Andropogoneae, Poaceae) using AFLP and ISSR PCR

Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat markers were employed to characterize a genetic resource collection of Miscanthus, a grass under trial in Europe as a biomass crop. The 26 polymorphic markers produced by two ISSR fingerprinting primers were able to discriminate taxa and identify putative clones. AFLP fingerprints were fully reproducible and produced a larger number of markers for the three primer pairs tested, of which 998 were polymorphic (representing 79.3% of all bands). AFLP markers distinguished species, infra-specific taxa (varieties and cultivars) and putatively clonal material. They were also used to assess the inter-relationships of the taxa, to investigate the origin of important hybrid plants and to estimate the overall level of genetic variation in the collection. They were useful for assessing the species status of certain taxa such as M. transmorrisonensis, an endemic from Taiwan that was clearly distinct from M. sinensis; whereas other taxa of disputed species status, such as M. condensatus and M. yakushimanum were not genetically distinct from M. sinensis. The AFLP markers detected a high degree of infra-specific variation and allowed subdivisions of the genetic resource collection to be made, particularly within M. sinensis.     

Dynamic grapevine clones��an AFLP-marker study of the Vitis vinifera cultivar Riesling comprising 86 clones

Grapevine cultivars are planted in worldwide viticulture and are asexually propagated. Horticultural clones are asexually derived from a single individual, and clonal variation can only occur through mutations. Molecular markers are an important tool for the differentiation and identification of clones and mutations. For breeding purposes and clonal selection, knowledge upon the variability of a given clone is essential. The aim of this study was to assess amplified fragment length polymorphism (AFLP) markers for classifying mutations in 86 Riesling clones of Vitis vinifera and to enhance our understanding on the dynamic of grapevine clones analysed by AFLP fingerprints. AFLP markers detected 135 polymorphic bands of a total amount of 305 bands. AFLP markers detected two different types of mutations: single-event mutations, only detected once in one clone, displaying the variation of the grape genome and specific loci mutations where the mutation could be found frequently in the set of clones and therefore stand for the stability of grapevine genome. A general grouping of clones according to age, sub-clonal lineage or origin could not be determined by the set of AFLP markers employed.     

Conversion of AFLP bands into high-throughput DNA markers

The conversion of AFLP bands into polymorphic sequence-tagged-site (STS) markers is necessary for high-throughput genotype scoring. Technical hurdles that must be overcome arise from genome complexity (particularly sequence duplication), from the low-molecular-weight nature of the AFLP bands and from the location of the polymorphism within the AFLP band. We generated six STS markers from ten AFLP bands (four AFLPs were from co-dominant pairs of bands) in soybean (Glycine max). The markers were all linked to one of two loci, rhg1 on linkage group G and Rhg4 on linkage group A2, that confer resistance to the soybean cyst nematode (Heterodera glycines I.). When the polymorphic AFLP band sequence contained a duplicated sequence or could not be converted to a locus-specific STS marker, direct sequencing of BAC clones anchored to a physical map generated locus-specific flanking sequences at the polymorphic locus. When the polymorphism was adjacent to the restriction site used in the AFLP analysis, single primer extension was performed to reconstruct the polymorphism. The six converted AFLP markers represented 996 bp of sequence from alleles of each of two cultivars and identified eight insertions or deletions, two microsatellites and eight single-nucleotide polymorphisms (SNPs). The polymorphic sequences were used to design a non-electrophoretic, fluorometric assay (based on the TaqMan technology) and/or develop electrophoretic STS markers for high-throughput genotype determination during marker-assisted breeding for resistance to cyst nematode. We conclude that the converted AFLP markers contained polymorphism at a 10- to 20-fold higher frequency than expected for adapted soybean cultivars and that the efficiency of AFLP band conversion to STS can be improved using BAC libraries and physical maps. The method provides an efficient tool for SNP and STS discovery suitable for marker-assisted breeding and genomics.     

Primula farinosa in Denmark; genetic diversity and population management

During the past centuries Danish populations of Primula farinosa have seriously declined in number. We investigated the genetic structure and genetic diversity of plants of seven populations from two different regions, Zealand and Bornholm in Denmark, using three AFLP markers. Two populations from nearby Scania, Sweden were included as reference. We found 54 unambiguously polymorphic loci. The genetic structure analysis suggested division of the 268 plants into three distinct groups, to a large extent matching the geographical distribution of the populations. Analysis of molecular variance (AMOVA) indicated significant genetic differentiation of 67% within populations and 33% among the populations. Our results suggest that genetic differentiation among regions and unique local genetic diversity should carefully be considered in future conservation attempts if we are to maintain as much genetic variation as possible. We present a historical overview of the decline in Danish populations and discuss conservation management and restoration strategies.     

Diversity in fertility potential and organo-sulphur compounds among garlics from Central Asia

Extending the collection of garlic (Allium sativum L.) accessions is an important means that is available for broadening the genetic variability of this cultivated plant, with regard to yield, quality, and tolerance to biotic and abiotic traits; it is also an important means for restoring fertility and flowering. In the framework of the EU project Garlic and Health, 120 garlic accessions were collected in Central Asia – the main centre of garlic diversity. Plants were documented and thereafter maintained in field collections in both Israel and The Netherlands. The collection was evaluated for biological and economic traits. Garlic clones vary in most vegetative characteristics (leaf number, bulb size and structure), as well as in floral scape elongation and inflorescence development. A clear distinction was made between incomplete bolting and bolting populations; most of the accessions in the latter populations produced flowers with fertile pollen and receptive stigma. Wide variations were recorded with regard to differentiation of topsets, their size, number and rapidity of development. Furthermore, significant variation in organo-sulphur compounds (alliin, isoalliin, allicin and related dipeptides) was found within garlic collections and between plants grown under differing environmental conditions. Genetic fingerprinting by means of AFLP markers revealed three distinct groups within this collection, differing also in flowering ability and organo-S content.     

Genetic diversity of sorghum accessions resistant to greenbugs as assessed with AFLP markers.

Sorghum, Sorghum bicolor (L.) Moench, is the fifth most important cereal crop grown worldwide and the fourth in the United States. Greenbug, Schizaphis graminum (Rondani), is a major insect pest of sorghum with several biotypes reported to date. Greenbug biotype I is currently the most prevalent and most virulent on sorghum plants. Breeding for resistance is an effective way to control greenbug damage. A successful breeding program relies in part upon a clear understanding of breeding materials. However, the genetic diversity and relatedness among the greenbug biotype I resistant accessions collected from different geographic origins have not been well characterized, although a rich germplasm collection is available. In this study, 26 sorghum accessions from 12 countries were evaluated for both resistance to greenbug biotype I and genetic diversity using fluorescence-labeled amplified fragment length polymorphism (AFLP). Twenty-six AFLP primer combinations produced 819 polymorphic fragments indicating a relatively high level of polymorphism among the accessions. Genetic similarity coefficients among the sorghum accessions ranged from 0.69 to 0.90. Cluster analysis indicated that there were two major groups based on polymorphic bands. This study has led to the identification of new genetic sources of sorghum with substantial genetic variation and distinct groupings of resistant accessions that have the potential for use in the development of durable greenbug resistant sorghum.     

Characterisation of genetic diversity in potential biomass willows (Salix spp.) by RAPD and AFLP analyses.

The genus Salix (willow) contains a number of species which have great potential value as biomass crops in short rotation coppice (SRC). Efforts to improve biomass willows by breeding are currently hampered by the limited information available on genetic diversity and on genetic relationships within and among species, clones, and hybrids in the gene pool. Hybridisation occurs commonly in nature and the relatedness of many clones is unclear. Molecular markers were used to assess genetic diversity in a reference set of willows maintained within the U.K. National Collection and 16 elite clones currently being evaluated in field trials at several European sites. The two marker systems tested, RAPDs and AFLPs, were equally informative for revealing relationships within the reference set of clones. No differences were observed when alternative similarity coefficients were compared or when analysis was restricted to the use of polymorphic bands only. Good agreement with available knowledge of the clonal origins was obtained and one instance of duplicate clones was identified. AFLPs revealed more genetic diversity and discriminated between closely related clones. A difference in the relationships revealed was observed with one AFLP primer combination. RAPDs were more problematic, both in terms of reproducibility and scorability.     

Molecular Polymorphism of the Wheat Leaf Rust Fungus in Morocco Using Amplified Fragment Length Polymorphism

The genetic variability and collection structure of the wheat leaf rust fungus Puccinia recondita collected from four agro‐ecological areas of Morocco, Abda‐doukala, Chaouia‐Tadla, Gharb and Tangérois were investigated by amplified fragment length polymorphism (AFLP) markers. A set of five AFLP primers combinations which generated 253 polymorphic loci were used. Hierarchical partitioning revealed that bread wheat collections of Puccinia recondita form a single collection. No significant variation was observed between durum wheat collections of Puccinia recondita; they maintained most of the genetic variability within rather among collections. The distribution pattern of genetic variation of Puccinia recondita collections seems to be the result of high gene flow and the mixed reproduction system.     

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.     

我国西北春麦区小麦育成品种遗传多样性的AFLP分析

对我国西北春麦区56份小麦育成品种应用扩增片段长度多态性(Amplified Fragment Length Polmorphics,简称AFLP)分子标记技术进行遗传多样性分析。共用24对引物组合进行扩增,每对引物组合的平均多态性条带为14．7,多态性百分率为24．4,而多态性信息指数PIC范围为0．11～0．44,平均0．22。结合品种的系谱亲缘关系分析,得知依据AFLP数据的类群划分结果与品种的亲缘系谱关系基本一致,表明AFLP技术用于种质鉴定和遗传多样性研究是有效的、可取的;同时。对如何合理应用AFLP数据中的多态性带和共有带进行聚类分析,及如何正确对待小麦核心种质构建中的形态和农艺性状数据与分子数据的问题作了进一步的探讨。仅用多态性谱带产生的相似系数矩阵与用所有扩增谱带产生的相似系数矩阵之间的相关系数r=0．86,表明在利用AFLP进行品种间遗传关系分析时,利用所有扩增产物的信息是必要的;如果仅仅是为了鉴剐品种或压缩样品,完全可以只考虑多态性扩增产物。利用AFLP分子数据和田间数据对56份材料进行主成分分析(PCO),发现用田间数据产生的PCO图,材料之间分散,遗传关系不很明了,进一步压缩样品难度较大;而分子数据产生的PCO图,可将材料分成明显的五类,聚类结果与品种系谱基本相吻合,为进一步压缩样品提供了科学依据。形态数据与分子数据聚类的结果差异较大,相关系数仅为0．310因此,在利用田间数据的基础上,必须兼顾和利用DNA数据,才能保证所建立核心种质的代表性。这也是一条比较科学、经济和可行的途径。     

Genetic relationships and clonal identity in a collection of commercially relevant poplar cultivars assessed by AFLP and SSR

A collection of 66 poplar commercial clones widely cultivated in Italy, China and in other countries of southern Europe and belonging to various poplar species and hybrids, have been fingerprinted using both amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) techniques. Three AFLP primer combinations and six SSRs unambiguously genotyped the analysed poplar collection, with the exception of three groups of six, four and two individuals, which turned out to be indistinguishable even if they met the standards currently applied for distinctness, uniformity and stability (DUS) testing when registered. High levels of variation were detected with both molecular techniques; a total of 201 AFLP bands were amplified of which 96% turned out to be polymorphic and up to 15 SSR alleles were identified at a single locus, with a mean of 9.3 alleles per locus in the case of Populus × canadensis. The probability of matching fortuitously any two genotypes at all the SSR loci in the case of P. × canadensis was less then 7.5×10^–9. The AFLP-derived dendrogram and principal coordinate analysis (PCOORDA) clustered the clones with respect to their taxonomic classification, and allowed their genetic interrelationships to be established. Correct identification of poplar varieties is essential for ensuring the effective correspondence between the real and the declared identity of a clone, to avoid commercial frauds, and to establish breeding programmes. Molecular markers may play a major role to satisfy all these needs.     

Culture-induced variation in plants of Coffea arabica cv. caturra rojo,regenerated by direct and indirect somatic embryogenesis

Amplified fragment-length polymorphism (AFLP) was used to evaluate the stability of DNA in regenerated plantlets of Coffea arabica obtained by direct (DSE) and indirect somatic embryogenesis (ISE). Cluster analysis using the unweighted pair-group method (UPGMA), showed no specific grouping pattern related to the type of embryogenesis. These results suggest that the somatic embryogenesis (SE) process has a mechanism for the selection of normal and competent cells. Bulked DNA from regenerated plants obtained by DSE and ISE, and from the mother plants, was used to characterize specific AFLP fragments associated with each SE process. Twenty-three primer combinations were tested. A total of 1446 bands were analyzed, with 11.4% being polymorphic and 84% being specific for regenerated plants. Furthermore, specific bands were detected for DSE, ISE, and the mother plants. These results indicate that the SE process induces rearrangements at the DNA level and demonstrates discrepancies between the mechanisms involved in each SE process. Coffea arabica breeding programs that involve DSE and ISE can use AFLP as an additional tool for assessing DNA stability.     

Evaluation of genetic diversity and germ plasm identification of 44 species, clones, and cultivars from 5 sections of the genusPopulus based on amplified fragment length polymorphism analysis

The genusPopulus L. (Salicaceae) can be divided into 5 sections with distribution throughout the world. Accurate identification ofPopulus clones and species is essential for effective selection, breeding, and management of genetic resources. In this study, amplified fragment length polymorphism (AFLP) analysis, which was reported as a reliable technique with high efficiency in detecting polymorphism, was used to conduct analyses of genetic diversity and variety identification of 44 species, clones, and cultivars ofPopulus that represent a wide range of breeding and commercially available germplasms. Cluster analysis of the 44 samples was carried out, and a dendrogram of genetic relatedness was developed on the basis of the AFLP data. DNA fingerprints of the 44 samples were developed from 12 selected bands amplified with 2 primer combinations (M-CAG/E-TA and M-CAG/E-TC). Each sample has its unique fingerprint pattern and can be distinguished from the others. Furthermore, 1 specific AFLP band of the cultivarPopulus canadensis cl. Guariento coming from fragments amplified by primer combination M-CTC/E-AG was successfully converted into a sequence-characterized amplified region (SCAR) marker. The results indicate that AFLP analysis should be considered as the preferred technique for the study of polymorphism inPopulus. This research is the first report concerning the use of AFLP analysis in genetic diversity and germplasm identification among all sections ofPopulus.     

AFLP markers reveal high polymorphic rates in ryegrasses (Lolium spp.)

An evaluation was performed of the potential use of AFLP markers to reveal polymorphisms among Lolium perenne plants with different degrees of kinship. Radioactive and fluorescent detection techniques were applied. The use of a fluorescent detection approach contributed greatly to the speed and ease of conducting and interpreting the AFLP patterns. The great discriminative power of AFLP markers and their capacity to represent genetic relationships among ryegrass plants was shown. Despite the high polymorphic value of the AFLP markers, standard statistical tests could not differentiate between two gene pools derived from different breeding programmes. It proved also impossible to correlate fodder and turf phenotypes with AFLP distance data. A very important point revealed by our data is the high degree of genetic diversity within commercial ryegrass varieties. Our findings are relevant to any outcrossing crop with a breeding strategy based on the production of synthetic populations.     

Accumulation of the chalcone isosalipurposide in primary leaves of barley flavonoid mutants indicates a defective chalcone isomerase

Mutants defective in flavonoid biosynthesis have become increasingly useful in elucidating the potential functions of these compounds in plants. To define the role of flavonoids as UV-B protectants in barley, we have screened part of the collection of proanthocyanidin-free barley mutants at the Carlsberg Research Laboratory, Copenhagen, Denmark. The four mutants ant 30–245, ant 30–272, ant 30–287 and ant 30–310 showed drastically reduced flavonoid levels in the primary leaf as compared to their corresponding parent varieties, and in addition accumulated a new mutant-specific phenolic compound which was identified as the chalcone glucoside isosalipurposide. Results from diallelic crosses indicate that all four mutants belong to the same new complementation group, which is designated as the Ant 30 locus. This gene has not earlier been described in barley. The data presented suggest a defective chalcone isomerase gene for the observed flavonoid pattern in leaves of ant 30 mutants.