Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Allelic ladders and reference genotypes for a rigorous standardization of poplar microsatellite data

A correct identification of members of the poplar hybrid complex Populus × canadensis is essential in breeding programs and studies in introgressive gene flow. Molecular marker protocols have been developed for this purpose. However, due to missing standards, these techniques have so far not been suited to the transfer of results between different laboratories. We present here a powerful system of nuclear microsatellite DNA (nSSR) fingerprints, standardized by allelic ladders and reference genotypes. Seven nSSR loci provided fingerprints of 65 commercial poplar clones. Their alleles were used to construct allelic ladders. Thus, a first standardized register of poplar clones is now available. All procedures were optimized according to simplified DNA extraction protocols, multiplexed PCR and electrophoresis procedures. Corresponding data originating from two different electrophoretic platforms in different laboratories were congruent when the allelic ladder was used. Unambiguous differentiation of the clones was based on a very low probability of identity (PI) of 1.95 × 10⁻⁸. Our results revealed discrepancies between clone denotations and genetic fingerprints. This suggests that, potentially, members of the clone collection could have been mixed up, thus confirming the demand for rigorous standards. The protocol presented can be exploited in a manifold way, e.g. to enlarge the present clonal molecular data base, or to use it for purposes of certification and control. Furthermore, the allelic ladders are recommended for use in poplar population genetic studies across different laboratories. The allelic ladders and single sample reference genotypes can be obtained on demand.     

Genetic identification of 91 poplar cultivars based on SSR markers

Accurate genetic identification and relationship analysis of poplar cultivars is necessary to establish commercial poplar plantations and select suitable breeding strategies. In this study, 91 poplar cultivars belonging to four sections (Aigeiros, Tacamahaca, Populus and Turanga) and inter/intra-sectional hybrids were genotyped using 18 polymorphic simple sequence repeat (SSR) markers. In total, 222 alleles were amplified with an average of 12.3 alleles per marker. The mean polymorphic information content and power of discrimination were 0.706 and 0.813, respectively. Five SSR markers (ORPM_103, ORPM_247, GCPM_1048, GCPM_1255 and LG_X_19) constituted a core fingerprint and were sufficient to identify all the tested cultivars. With some notable exceptions, cultivars of the same species generally clustered together in cluster (UPGMA) and ordination (PCO) analyses. Flow cytometry indicated that 11 poplar cultivars were triploid. Among these, seven had three alleles at some loci, suggesting that SSR markers could indicate the ploidy level to some extent. This study provides useful genetic information for the identification and protection of poplar cultivars in China and offers a guideline for the selection of poplar crossing parents based on ploidy level and genetic relationships.     

Screening of ESTs from Septoria musiva (teleomorph Mycosphaerella populorum) for detection of SSR and PCR–RFLP markers

The development of an expressed sequence tag (EST) collection for the poplar pathogen Septoria musiva presented an opportunity for the development of locus‐specific polymorphic markers. Five microsatellites simple sequence repeat (SSR) and six polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) markers were identified and tested on three S. musiva populations collected in the north‐central and northeastern regions of North America. The results obtained support previously published random amplified polymorphic DNA (RAPD) data.     

Molecular characterization of Lebanese olive germplasm

Lebanon, an East Mediterranean country, does not have a comprehensive reference national olive collection. A report published 30 years ago indicated the presence of four Lebanese varieties, but the confusion regarding these varieties was discussed then and is still prevalent today hindering prospects for conserving and exploring the national germplasm. This study assessed the existing in situ diversity of old Lebanese olive groves using simple sequence repeat (SSR) and amplified fragment length polymorphisms (AFLP). Trees (140) were collected from 14 groves located in the four traditional olive areas. Analysis was based on 22 polymorphic alleles produced from six SSR primers, and on 135 polymorphic AFLP fragments. SSR and AFLP did not yield consistent results in terms of level of polymorphism, with SSR detecting higher variation than AFLP. On the other hand, both clustered trees based on geographic provenance. AFLP coefficient of similarity between trees ranged between 0.70 and 0.99 indicating the possible presence of varieties since some values fall within reported intervarietal ranges of 0.65 to 0.84. SSR unveiled the presence of nine different genotypes: four corresponding each to a provenance and five consisting of single trees characterized by the presence of less frequent alleles with an average of 0.33. Results also revealed a high incidence of clones greater than 90% in three of the four provenances. The findings of this study point for the need to plan for a sampling strategy that takes into consideration geographic provenances.     

An SSR-based linkage map of Capsicum annuum

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.     

Genetic stability of cryopreserved shoot tips of <Emphasis Type="Italic">Rubus</Emphasis> germplasm

Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars (“Hillemeyer” and ‘Silvan’), and one raspberry cultivar (“Mandarin”) as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from “Marion” and “Meeker” microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and ‘Silvan’ displayed the same AFLP fingerprints as their corresponding mother plants. Only long-cultured in vitro shoot tips displayed polymorphisms in vitro, and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability.     

Natural hybridisation between <Emphasis Type="Italic">Populus nigra</Emphasis> L. and <Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">x canadensis</Emphasis> Moench. Hybrid offspring competes for niches along the Rhine river in the Netherlands

Black poplar (Populus nigra L.) is a major species for European riparian forests but its abundance has decreased over the decades due to human influences. For restoration of floodplain woodlands, the remaining black poplar stands may act as source population. A potential problem is that P. nigra and Populus deltoides have contributed to many interspecific hybrids, which have been planted in large numbers. As these Populus x canadensis clones have the possibility to intercross with wild P. nigra trees, their offspring could establish themselves along European rivers. In this study, we have sampled 44 poplar seedlings and young trees that occurred spontaneously along the Rhine river and its tributaries in the Netherlands. Along these rivers, only a few native P. nigra L. populations exist in combination with many planted cultivated P. x canadensis trees. By comparison to reference material from P. nigra, P. deltoides and P. x canadensis, species-specific AFLP bands and microsatellite alleles indicated that nearly half of the sampled trees were not pure P. nigra but progeny of natural hybridisation that had colonised the Rhine river banks. The posterior probability method as implemented in NewHybrids using microsatellite data was the superior method in establishing the most likely parentage. The results of this study indicate that offspring of hybrid cultivated poplars compete for the same ecological niche as native black poplars. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

A fast and simple genetic survey reveals the spread of poplar hybrids at a natural Elbe river site

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. × canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. × canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P.  × canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.     

Development and characterization of polymorphic microsatellite markers from Fragaria viridis,a wild diploid strawberry

To date, the development of microsatellite (SSR) markers in the genus Fragaria has focused on F. vesca. However, further species are thought to have contributed to the complex allo‐octoploid genome of the cultivated strawberry, F.×ananassa. Here, we present 22 new SSR markers developed from the diploid species F. viridis. Twenty‐one of the primer pairs amplified polymorphisms in six F. viridis accessions, with an average of 4.95 alleles per primer pair and an average expected heterozygosity of 0.68. Fourteen of these primer pairs, and a locus monomorphic in F. viridis, amplified polymorphic alleles in the parents of a F. vesca mapping population.     

Suppressed recombination around the MXC3 locus, a major gene for resistance to poplar leaf rust

A positional cloning strategy is being implemented in Populus for the isolation of the dominant MXC3 allele, which confers resistance to poplar leaf rust caused by Melampsora×columbiana (pathotype 3). AFLP markers were used to saturate the chromosomal region around the MXC3 locus in a large (n=1,902) Populus trichocarpa×P. deltoides (T×D) mapping pedigree segregating 1:1 for rust resistance and susceptibility. The high-resolution linkage map developed around the MXC3 locus contains 19 AFLP markers and spans a genetic distance of 2.73 cM. Of the 19 AFLP markers, seven were found to co-segregate with the locus. One co-segregating AFLP marker, CCG.GCT_01, was converted to an STS marker (BVS1) and used to identify a physical contig of overlapping BAC clones from the MXC3 region. Genetic and physical mapping of markers isolated from the BAC contig failed to delimit the MXC3 locus within a 300-kb interval defined by the overlapping BAC clones. This result indicates a >25-fold reduction in recombination frequency in the MXC3 region compared to the average rate of recombination for the Populus genome. Received: 8 December 2000 / Accepted: 1 March 2001     

Identification of a major quantitative trait locus (QTL) for yellow spot (<Emphasis Type="Italic">Mycovellosiella koepkei</Emphasis>) disease resistance in sugarcane

In this study we used amplified fragment length polymorphism (AFLP) and microsatellite (short sequence repeat or SSR) markers to identify a major quantitative trail locus (QTL) for yellow spot (Mycovellosiella koepkei) disease resistance in sugarcane. A bi-parental cross between a resistant variety, M 134/75, and a susceptible parent, R 570, generated a segregating population of 227 individuals. These clones were evaluated for yellow spot infection in replicated field trials in two locations across two consecutive years. A χ²-test (χ² at 98% confidence level) of the observed segregation pattern for yellow spot infection indicated a putative monogenic dominant inheritance for the trait with a 3 (resistant):1(susceptible) ratio. The AFLP and SSR markers identified 666 polymorphisms as being present in the resistant parent and absent in the susceptible one. A genetic map of M 134/75 was constructed using 557 single-dose polymorphisms, resulting in 95 linkage groups containing at least two markers based on linkages in coupling. QTL analysis using QTLCartographer v1.17d and MAPMAKER/QTL v1.1 identified a single major QTL located on LG87, flanked by an AFLP marker, actctc10, and an SSR marker, CIR12284. This major QTL, which was found to be linked at 14 cM to an AFLP marker, was detected with LOD 8.7, had an additive effect of −10.05% and explained 23.8% of the phenotypic variation of yellow spot resistance.     

A genetic map of pineapple (Ananas comosus (L.) Merr.) including SCAR, CAPS, SSR and EST-SSR markers

Despite the paramount importance of pineapple (Ananas comosus L.) in world production and trade of tropical fruits, the genomics of this crop is still lagging behind that of other tropical fruit crops such as banana or papaya. A genetic map of pineapple was constructed using an F2 segregating population obtained from a single selfed F1 plant of a cross A. comosus var. comosus (cv. Rondon, clone BR 50) × A. comosus var. bracteatus (Branco do mato, clone BR 20). Multiple randomly amplified markers (RAPD, ISSR and AFLP) were brought together with SSR and EST-SSR markers identified among sequences uploaded to public databases and with sequence-specific markers (SCAR, SSR and CAPS) derived from random amplified markers. Sixty-three randomly amplified markers (RAPD, ISSR and AFLP) were selected and cloned, resulting in 71 sequences which were used to generate sequence-specific SCAR and CAPS markers. The present map includes 492 DNA markers: 57 RAPD, 22 ISSR, 348 AFLP, 20 SSR, 12 EST-SSR, 25 SCARs, 8 CAPS, and the morphological trait locus “piping”, gathered into 33 linkage groups that integrate markers inherited from both botanical varieties, four linkage groups with markers only from var. comosus and three linkage groups with markers exclusively from var. bracteatus. The relatively higher mapping efficiency of sequence-specific markers derived from randomly amplified markers (50.7%) versus SSR (31.4%) and EST-SSR (28.9%) markers is discussed. Spanning over 80% of the 2,470 cM estimated average length of the genome, the present map constitutes a useful research tool for molecular breeding and genomics projects in pineapple and other Bromeliaceae species.     

基于磁珠富集法开发并筛选西川红景天的微卫星标记

利用FIASCO磁珠富集法,开发和筛选青藏高原特有珍贵植物西川红景天(Rhodiola alsia)多态性微卫星标记。结果表明:用(AG)15和(AC)15两种微卫星标记探针构建富集文库,共获得阳性克隆约2500个;从中随机挑取1200检测,发现具有多态性的阳性克隆达400个;随机挑取200多态性的阳性克隆进行测序,从中获得105个SSR位点,用在线软件primer3-2.3.4成功设计得SSR引物105对;其中45对可以成功扩增,而13对所扩增的片段在相距较远的4个自然居群的24个个体中显示较高的遗传多态性。用4个居群的80个个体检测这13对引物发现,平均等位基因数(A)约为9.192,观测杂合度(Ho)和期望杂合度(He)均值分别约为0.712和0.734,如此高的多态性已经满足后期研究的需要;数个位点在某些居群中显著偏离哈迪温伯格平衡(P0.01),这可能是实际研究的居群并不能达到哈迪—温伯格定律所假设的无限大等理想状态所致。结合此前基于表达序列标签(Expressed Sequence Tag,EST)序列开发的SSR多态性位点,该研究结果为今后利用SSR位点开展西川红景天的居群遗传结构分析和其他研究提供了一组有效工具。     

Genetic diversity analysis of common beans based on molecular markers

A core collection of the common bean (Phaseolus vulgaris L.), representing genetic diversity in the entire Mexican holding, is kept at the INIFAP (Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico) Germplasm Bank. After evaluation, the genetic structure of this collection (200 accessions) was compared with that of landraces from the states of Oaxaca, Chiapas and Veracruz (10 genotypes from each), as well as a further 10 cultivars, by means of four amplified fragment length polymorphisms (AFLP) +3/+3 primer combinations and seven simple sequence repeats (SSR) loci, in order to define genetic diversity, variability and mutual relationships. Data underwent cluster (UPGMA) and molecular variance (AMOVA) analyses. AFLP analysis produced 530 bands (88.5% polymorphic) while SSR primers amplified 174 alleles, all polymorphic (8.2 alleles per locus). AFLP indicated that the highest genetic diversity was to be found in ten commercial-seed classes from two major groups of accessions from Central Mexico and Chiapas, which seems to be an important center of diversity in the south. A third group included genotypes from Nueva Granada, Mesoamerica, Jalisco and Durango races. Here, SSR analysis indicated a reduced number of shared haplotypes among accessions, whereas the highest genetic components of AMOVA variation were found within accessions. Genetic diversity observed in the common-bean core collection represents an important sample of the total Phaseolus genetic variability at the main Germplasm Bank of INIFAP. Molecular marker strategies could contribute to a better understanding of the genetic structure of the core collection as well as to its improvement and validation.     

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.     

Simple sequence repeat (SSR) markers in the ectomycorrhizal fungus Laccaria bicolor for environmental monitoring of introduced strains and molecular ecology applications

Simple sequence repeat (SSR) markers were developed from SSR-enriched genome libraries for the ectomycorrhizal basidiomycete Laccaria bicolor. Seven markers were single-locus and amplified unambiguously in L. bicolor. The seven SSR markers were further characterized using an array of 15 L. bicolor strains representative of diverse origins worldwide. The observed number of alleles per locus varied from 5–9 and the values of observed heterozygosity from 0.167 to 0.667. The seven SSR loci could be amplified from DNA extracted from root tips of L. bicolor inoculated pine seedlings. All the L. bicolor ectomycorrhizas analysed exhibited the same SSR multi-locus profile as that detected for the UAMH8232 inoculant strain. The set of markers described represents a potent tool for the monitoring of introduced strains of L. bicolor and for molecular ecology applications.     

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     

Ex-situ conservation of Black poplar in Europe: genetic diversity in nine gene bank collections and their value for nature development

Populus nigra L. is a pioneer tree species of riparian ecosystems that is threatened with extinction because of the loss of its natural habitat. To evaluate the existing genetic diversity of P. nigra within ex-situ collections, we analyzed 675 P. nigra L. accessions from nine European gene banks with three amplified fragment length polymorphism (AFLP) and five microsatellite [or simple sequence repeat (SSR)] primer combinations, and 11 isozyme systems. With isozyme analysis, hybrids could be detected, and only 3% were found in the gene bank collection. AFLP and SSR analyses revealed effectively that 26% of the accessions were duplicated and that the level of clonal duplication varied from 0% in the French gene bank collection up to 78% in the Belgian gene bank collection. SSR analysis was preferred because AFLP was technically more demanding and more prone to scoring errors. To assess the genetic diversity, we grouped material from the gene banks according to topography of the location from which the accessions were originally collected (river system or regions separated by mountains). Genetic diversity was expressed in terms of the following parameters: percentage of polymorphic loci, observed and effective number of alleles, and Neis expected heterozygosity or gene diversity (for AFLP). Genetic diversity varied from region to region and depended, to some extent, on the marker system used. The most unique alleles were identified in the Danube region (Austria), the Rhône region (France), Italy, the Rijn region (The Netherlands), and the Ebro region (Spain). In general, the diversity was largest in the material collected from the regions in Southern Europe. Dendrograms and principal component analysis resulted in a clustering according to topography. Material from the same river systems, but from different countries, clustered together. The genetic differentiation among the regions (F_st/G_st) was moderate.Communicated by H.F. LinskensAFLP is a registered trademark of Keygene