NGS technologies for analyzing germplasm diversity in genebanks

More than 70 years after the first ex situ genebanks have been established, major efforts in this field are still concerned with issues related to further completion of individual collections and securing of their storage. Attempts regarding valorization of ex situ collections for plant breeders have been hampered by the limited availability of phenotypic and genotypic information. With the advent of molecular marker technologies first efforts were made to fingerprint genebank accessions, albeit on a very small scale and mostly based on inadequate DNA marker systems. Advances in DNA sequencing technology and the development of high-throughput systems for multiparallel interrogation of thousands of single nucleotide polymorphisms (SNPs) now provide a suite of technological platforms facilitating the analysis of several hundred of Gigabases per day using state-of-the-art sequencing technology or, at the same time, of thousands of SNPs. The present review summarizes recent developments regarding the deployment of these technologies for the analysis of plant genetic resources, in order to identify patterns of genetic diversity, map quantitative traits and mine novel alleles from the vast amount of genetic resources maintained in genebanks around the world. It also refers to the various shortcomings and bottlenecks that need to be overcome to leverage the full potential of high-throughput DNA analysis for the targeted utilization of plant genetic resources.     

Molecular characterization of apricot germplasm from an old stone collection

Increasing germplasm erosion requires the recovery and conservation of traditional cultivars before they disappear. Here we present a particular case in Spain where a thorough prospection of local fruit tree species was performed in the 1950s with detailed data of the origin of each genotype but, unfortunately, the accessions are no longer conserved in ex situ germplasm collections. However, for most of those cultivars, an old stone collection is still preserved. In order to analyze the diversity present at the time when the prospection was made and to which extent variability has been eroded, we developed a protocol in apricot (Prunus armeniaca L.) to obtain DNA from maternal tissues of the stones of a sufficient quality to be amplified by PCR. The results obtained have been compared with the results from the profiles developed from apricot cultivars currently conserved in ex situ germplasm collections. The results highlight the fact that most of the old accessions are not conserved ex situ but provide a tool to prioritize the recovery of particular cultivars. The approach used in this work can also be applied to other plant species where seeds have been preserved.     

中国花生核心种质的建立

以中国花生种质资源数据库中记录的6390份花生资源为材料,以其基本数据、特征数据和评价数据为信息,采用分层、层内分组聚类以及随机取样与必选资源相结合的方法,构建了由576份资源组成的花生核心种质,占基础收集品的9.01%。对核心种质的植物学类型组成和遗传多样性指数的分析,以及对各性状特征值、符合率和包含的主要抗病资源抗性等级及重要农艺性状资源的检测结果表明,本研究建立的核心种质是有效的。基础收集品中各种性状的遗传变异在核心种质中均存在,所用15个性状的各种特征值符合率均在90%以上,其中绝大部分性状的符合率达96%以上。     

中国花生核心种质的建立

以中国花生种质资源数据库中记录的6390份花生资源为材料,以其基本数据、特征数据和评价数据为信息,采用分层、层内分组聚类以及随机取样与必选资源相结合的方法,构建了由576份资源组成的花生核心种质,占基础收集品的9.01%。对核心种质的植物学类型组成和遗传多样性指数的分析,以及对各性状特征值、符合率和包含的主要抗病资源抗性等级及重要农艺性状资源的检测结果表明,本研究建立的核心种质是有效的。基础收集品中各种性状的遗传变异在核心种质中均存在,所用15个性状的各种特征值符合率均在90%以上,其中绝大部分性状的符合率达96%以上。     

Is there a future for the Cactaceae genera Copiapoa,Eriosyce and Eulychnia? A status report of a prickly situation

Many of Chile’s iconic cactus species are threatened by human activities and global change. In order to safeguard them from extinction, both in situ and ex situ conservation actions are urgently needed. In this paper, an overview is given of the in situ and ex situ conservation status of the mainly Chilean cactus genera Copiapoa, Eriosyce and Eulychnia, including a worldwide survey of living ex situ collections of the species of these genera. From our results, we can conclude that although the threats to Chile’s remarkable biodiversity are now recognized as an environmental problem, and efforts are underway to protect the threatened endemic flora, many of the most threatened species are currently not protected in situ. Although a higher percentage of living accessions of Copiapoa, Eriosyce and Eulychnia in ex situ collections are of known wild origin compared to results of previous studies on other plant groups, the number of available accessions is insufficient to adequately preserve the genetic diversity of the threatened species. Prospects to upscale both in situ and ex situ conservation of the studied genera are discussed.     

Microsatellite-aided detection of genetic redundancy improves management of the International Cocoa Genebank, Trinidad

Cacao (Theobroma cacao L.), the tree from which cocoa butter and chocolate is derived, is conserved in field genebanks. The largest of these ex situ collections in the public domain is the International Cocoa Genebank, Trinidad (ICG,T). Reduction of genetic redundancy is essential to improve the accuracy and efficiency of genebank management. This study examined the pedigree and genetic diversity in a subset of 387 accessions in this collection. Sibship reconstruction of this subset revealed 56 full-sib families nested within 189 half-sib families. Sixteen centers of interconnectivity were identified, which suggested a high level of genetic redundancy in the collection. Generally, consistent phylogenetic trees were obtained using different genetic distance measures. However, a principal coordinate analysis of the D _est differentiation measure elicited the best representation of accession group clustering, and we recommend this approach when probing fine-scale genetic differentiation among cacao accessions. The composite genetic diversity of 414 cacao accessions was contained in a core set of 59 unique accessions. These results have significant implications in the conservation of genetic resources of the ICG,T and other cacao genebanks. The approach developed in this study is recommended as a strategy to curators in guiding conservation management practices of cacao and other similar ex situ genebanks.     

Genetic diversity and population structure in cultivated sunflower and a comparison to its wild progenitor, <Emphasis Type="Italic">Helianthus annuus</Emphasis> L

Crop germplasm collections are valuable resources for ongoing plant breeding efforts. To fully utilize such collections, however, researchers need detailed information about the amount and distribution of genetic diversity present within collections. Here, we report the results of a population genetic analysis of the primary gene pool of sunflower (Helianthus annuus L.) based on a broad sampling of 433 cultivated accessions from North America and Europe, as well as a range-wide collection of 24 wild sunflower populations. Gene diversity across the cultivars was 0.47, as compared with 0.70 in the wilds, indicating that cultivated sunflower harbors roughly two-thirds of the total genetic diversity present in wild sunflower. Population structure analyses revealed that wild sunflower can be subdivided into four genetically distinct population clusters throughout its North American range, whereas the cultivated sunflower gene pool could be split into two main clusters separating restorer lines from the balance of the gene pool. Use of a maximum likelihood method to estimate the contribution of the wild gene pool to the cultivated sunflower germplasm revealed that the bulk of the cultivar diversity is derived from two wild sunflower population genetic clusters that are primarily composed of individuals from the east-central United States, the same general region in which sunflower domestication is believed to have occurred. We also identified a nested subset of accessions that capture as much of the allelic diversity present within the sampled cultivated sunflower germplasm collection as possible. At the high end, a core set of 288 captured nearly 90% of the alleles present in the full set of 433, whereas a core set of just 12 accessions was sufficient to capture nearly 50% of the total allelic diversity present within this sample of cultivated sunflower.     

Could taxonomic misnaming threaten the ex situ conservation and the usage of plant genetic resources?

Ex situ conservation of plant germplasm, especially seed banking, is a favourable and widely used method for the conservation of plant genetic resources (PGR). The long-term conservation of these resources is fundamental for food security and plant breeding in order to stem the losses in agrobiodiversity and to meet the global challenges that agriculture is facing. The conservation and accessibility of PGR relies on their correct taxonomic labelling and on the building of a searchable database that links ex situ collections together. In the current study, we analysed the impact of taxonomic misnaming in the two major PGR databases (Genesys PGR, EURISCO), listing accessions conserved worldwide. The aim was to understand if taxonomic misnaming issues prevent PGR conservation. We chose as a case-study seed collections of accessions of the genus Citrullus (watermelon genepool), the taxonomy and nomenclature of which have been largely revised in recent times. We observed that taxonomic misnaming greatly limits PGR conservation with only 3% of the accessions listed in the databases correctly named; moreover, 28% were affected by taxonomic errors that prevent the establishment of the accessions’ taxonomic identity, with consequences on their conservation and exploitation. The existence of the problem was also confirmed by the experimental propagation of three misnamed accessions. We suggest herein a series of actions that, put in place, could solve the extant misnaming issues in the databases and prevent their reoccurrence, allowing the correct conservation and the usability by the stakeholders of all the accessions.     

Molecular characterization of an international cacao collection using microsatellite markers

Plant germplasm collections invariably contain varying levels of genetic redundancy, which hinders the efficient conservation and utilization of plant germplasm. Reduction of genetic redundancies is an essential step to improve the accuracy and efficiency of genebank management. The present study targeted the assessment of genetic redundancy and genetic structure in an international cacao (Theobroma cacao L.) collection maintained in Costa Rica. A total of 688 cacao accessions maintained in this collection were genotyped with 15 simple sequence repeat (SSR) loci, using a capillary electrophoresis genotyping system. The SSR markers provided a high resolution among the accessions. Thirty-six synonymously labeled sets, involving 135 accessions were identified based on the matching of multilocus SSR profiles. After the elimination of synonymous sets, the level of redundancy caused by closely related accessions in the collection was assessed using a simulated sampling scheme that compared allelic diversity in different sample sizes. The result of the simulation suggested that a random sample of 113 accessions could capture 90% of the total allelic diversity in this collection. Principal Coordinate Analysis revealed that the Trinitario hybrids from Costa Rica shared a high similarity among groups as well as among individual accessions. The analysis of the genetic structure illustrated that the within-country/within-region difference accounted for 84.6% of the total molecular variation whereas the among-country/among-region difference accounted for 15.4%. The Brazilian germplasm contributed most to this collection in terms of total alleles and private alleles. The intercountry/interregion relationship by cluster analysis largely agreed with the geographical origin of each germplasm group and supported the hypothesis that the Upper Amazon region is the center of diversity for cacao. The results of the present study indicated that the CATIE International Cacao Collection contains a high level of genetic redundancy. It should be possible to rationalize this collection by reducing redundancy and ensuring optimal representation of the genetic diversity from distinct germplasm groups. The results also demonstrated that SSR markers, together with the statistical tools for individual identification and redundancy assessment, are technically practical and sufficiently informative to assist the management of a tropical plant germplasm collection.     

Genetic diversity assessment and ex situ conservation strategy of the endangered Dendrobium officinale (Orchidaceae) using new trinucleotide microsatellite markers

Dendrobium officinale (Orchidaceae) is an endangered plant species with important medicinal value. To evaluate the effectiveness of ex situ collection of D. officinale genetic diversity, we developed 15 polymorphic trinucleotide microsatellite loci of D. officinale to examine the genetic diversity and structure of three D. officinale germplasm collections comprising 120 individuals from its germplasm collection base and their respective wild populations consisting of 62 individuals from three provinces in China. The three germplasm collections showed reductions in gene diversity and average number of alleles per locus, but an increase in average number of rare alleles (frequency?≤?0.05) per locus in comparison to their wild populations. However, the differences in gene diversity between the germplasm collections and wild populations were not statistically significant. The analysis using STRUCTURE revealed evident differences in genetic composition between each germplasm collection and its wild population, probably because the D. officinale individuals with distinct genotypes in each wild population were unevenly selected for establishing its germplasm collection. For conservation management plans, we propose that D. officinale individuals with rare alleles need to be conserved with top priority, and those individuals with the most common alleles also should be concerned. The 15 new microsatellite loci may be used as a powerful tool for further evaluation and conservation of the genetic diversity of D. officinale germplasm resources.     

The genetic diversity of the Vigna angularis complex in Asia.

A selected set of accessions of components of the azuki bean (Vigna angularis) complex comprising 123 cultivated accessions and 23 wild or weedy accessions from Bhutan, China (including Taiwan), India, Japan, Korea, and Nepal was analyzed using amplified fragment length polymorphism (AFLP) methodology. Using 12 AFLP primer pairs, 580 unambiguous bands were generated, 313 (53.9%) of which were polymorphic among azuki bean accessions. All 580 bands were used to assess phenotypic (band) and genetic (nucleotide) diversity among the 146 azuki bean accessions. The results indicate five major groups of azuki bean germplasm primarily associated with geographic origin of accessions and their status: wild, weedy, or cultivated. These five groups are (i) Himalayan wild, (ii) Nepal-Bhutan cultivated, (iii) Chinese wild, (iv) Taiwan wild - Bhutan cultivated, and (v) northeast Asian accessions. Within the northeast Asian accessions, three subgroups are present. These consist of (v1) Japanese complex - Korean cultivated, (v2) Japanese cultivated, and (v3) Chinese cultivated accessions. The results suggest domestication of azuki bean occurred at least twice, once in the Himalayan region of southern Asia and once in northeast Asia. The remarkable diversity of azuki bean germplasm in the Himalayan region compared with other regions suggests this is a rich source of germplasm for plant breeding. The results suggest there are important gaps in the germplasm collections of azuki bean and its close relatives from various parts of Asia and that specific collecting missions for Vigna germplasm related to azuki bean in the highlands of subtropical Asia are needed.     

Indian Plant Germplasm on the Global Platter: An Analysis

Food security is a global concern amongst scientists, researchers and policy makers. No country is self-sufficient to address food security issues independently as almost all countries are inter-dependent for availability of plant genetic resources (PGR) in their national crop improvement programmes. Consultative Group of International Agricultural Research (CGIAR; in short CG) centres play an important role in conserving and distributing PGR through their genebanks. CG genebanks assembled the germplasm through collecting missions and acquisition the same from national genebanks of other countries. Using the Genesys Global Portal on Plant Genetic Resources, the World Information and Early Warning System (WIEWS) on Plant Genetic Resources for Food and Agriculture and other relevant databases, we analysed the conservation status of Indian-origin PGR accessions (both cultivated and wild forms possessed by India) in CG genebanks and other national genebanks, including the United States Department of Agriculture (USDA) genebanks, which can be considered as an indicator of Indian contribution to the global germplasm collection. A total of 28,027,770 accessions are being conserved world-wide by 446 organizations represented in Genesys; of these, 3.78% (100,607) are Indian-origin accessions. Similarly, 62,920 Indian-origin accessions (8.73%) have been conserved in CG genebanks which are accessible to the global research community for utilization in their respective crop improvement programmes. A total of 60 genebanks including 11 CG genebanks have deposited 824,625 accessions of PGR in the Svalbard Global Seed Vault (SGSV) as safety duplicates; the average number of accessions deposited by each genebank is 13,744, and amongst them there are 66,339 Indian-origin accessions. In principle, India has contributed 4.85 times the number of germplasm accessions to SGSV, in comparison to the mean value (13,744) of any individual genebank including CG genebanks. More importantly, about 50% of the Indian-origin accessions deposited in SGSV are traditional varieties or landraces with defined traits which form the backbone of any crop gene pool. This paper is also attempting to correlate the global data on Indian-origin germplasm with the national germplasm export profile. The analysis from this paper is discussed with the perspective of possible implications in the access and benefit sharing regime of both the International Treaty on Plant Genetic Resources for Food and Agriculture and the newly enforced Nagoya Protocol under the Convention on Biological Diversity.     

小麦地方品种繁殖更新过程中的遗传多样性变化分析

采用分别保存于长期库及中期库的3个小麦地方品种的6份材料,进行了9项农艺性状及35个与农艺性状相关的微卫星标记检测,每份材料选取30个单株进行遗传多样性与遗传结构分析。结果表明:(1)更新前,3个小麦地方品种均为遗传异质性群体,在SSR位点上的异质度分别为57.14%、48.57%和5.71%。(2)在农艺性状表现上,只有温泉小麦3在株高和穗粒数上更新后比更新前显著增加,其他材料无显著差异。(3)在SSR位点多态性表现上,3个品种在更新后均发生了遗传多样性变化,8个与粒重、产量、生育期性状相关位点存在等位位点丢失现象,其中2个与粒重、生育期相关位点频率变化显著。(4)综合农艺性状调查与SSR分子标记检测结果发现,3个品种更新前后在多样性指数上无显著差异,遗传分化系数Gst分别为0.0269、0.0324和0.0380,即更新前后遗传差异分别为2.69%、3.24%和3.80%。上述结果建议,经繁殖更新的小麦种质资源能够比较完好地保持其遗传多样性和遗传结构。对于遗传异质性小麦地方品种在繁殖更新后存在遗传多样性丢失的危险,为了保证更新前后的遗传完整性,建议在繁殖更新过程中每个品种至少应保持300个单株的群体。     

Is It Still Necessary to Continue to Collect Crop Genetic Resources in the Mediterranean Area? A Case Study in Catalonia

Crop genetic resources have been extensively collected in Europe in the last century, creating large, publicly available ex situ collections. While this huge genetic diversity is often underutilized, in recent decades, several initiatives have emerged at the local level to collect germplasm cultivated on farm. Uncoordinated actors often carry out these collecting missions without considering previously collected data. To explore whether new collecting missions are likely to be worthwhile, we studied the crop genetic resources conservation network in Catalonia by analyzing the passport data and geographical distribution of germplasm stored in seed banks. Moreover, to determine whether this germplasm was representative of the diversity cultivated on farm, we performed new collecting missions in four randomly selected areas in the European Union’s Natura 2000 network and compared the results with the ex situ databases. Seed banks currently hold a large germplasm collection (2931 accessions), although most materials are conserved in private collections without regulated systems for seed regeneration and are not present as duplicates in the National Inventory. One important shortcoming of the ex situ network is that the germplasm conserved ex situ shows a low geographical coverage, representing only 35.3% of the municipalities in Catalonia. Our new missions allowed us to collect 234 accessions, mostly tomatoes (17.5%) and beans (16.2%). The ecological indicators’ richness (both at species (S) and variety (V) levels), total abundance (A), and the Shannon-Weaver diversity index calculated at species (H2, considering the different accessions of each variety as a single population) and variety levels (H3, considering the intra-varietal genetic diversity) were higher in the newly collected germplasm than in the ex situ collections, suggesting that seed banks do not accurately represent the genetic diversity still cultivated on farm. Moreover, some important landraces from each area were absent or underrepresented in the ex situ collections. Thus, it is necessary to continue to devote efforts to collecting germplasm; better organization between actors and targeting specific species/varieties can increase the efficiency of new collecting missions. As a conclusion, we propose different criteria to guide new missions and to improve the network’s conservation activities.     

Diversity of wild and cultivated pearl millet accessions (Pennisetum glaucum [L.] R. Br.) in Niger assessed by microsatellite markers

Genetic diversity of crop species in sub-Sahelian Africa is still poorly documented. Among such crops, pearl millet is one of the most important staple species. In Niger, pearl millet covers more than 65% of the total cultivated area. Analyzing pearl millet genetic diversity, its origin and its dynamics is important for in situ and ex situ germplasm conservation and to increase knowledge useful for breeding programs. We developed new genetic markers and a high-throughput technique for the genetic analysis of pearl millet. Using 25 microsatellite markers, we analyzed genetic diversity in 46 wild and 421 cultivated accessions of pearl millet in Niger. We showed a significantly lower number of alleles and lower gene diversity in cultivated pearl millet accessions than in wild accessions. This result contrasts with a previous study using iso-enzyme markers showing similar genetic diversity between cultivated and wild pearl millet populations. We found a strong differentiation between the cultivated and wild groups in Niger. Analyses of introgressions between cultivated and wild accessions showed modest but statistically supported evidence of introgressions. Wild accessions in the central region of Niger showed introgressions of cultivated alleles. Accessions of cultivated pearl millet showed introgressions of wild alleles in the western, central, and eastern parts of Niger.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Cedric Mariac and Viviane Luong have contributed equally to this work.     

Genetic differentiation in relation to seed weights in wild soybean species (Glycine soja Sieb. & Zucc.)

Seed weight is one of the most important botanical and phylogenetic characteristics. The study objective was to understand whether there is genetic difference in different seed weights of wild soybean (Glycine soja Sieb. & Zucc.). A total of 563 wild soybean samples, which belonged separately to genebank germplasm accessions (220 samples), one regional population samples (293 plants) and one natural population (150 plants), were analyzed using microsatellite markers. Of four size classes, the smallest seed size type had the highest coefficient of variation in seed weight; small and large seed types had relatively great genetic differences. In the national genebank germplasm accessions, genetic diversity gradually decreased from quantitatively dominant small and middling seed types to less frequent large seed types. In the regional and natural populations, generally, small to middling seed sizes had higher genetic diversity than the smallest and larger seed sizes. Cluster analysis revealed genetic differences in seed size traits. The semi-wild type (Glycine gracilis Skvortzow) was the most genetically differentiated from other seed sizes. However, it was also clearly shown that the phylogenic genetic differentiation among seed sizes was less than the genetic differentiation among geographical habitat populations in the wild soybean species.     

Strategy for exploiting exotic germplasm using genetic, morphological, and environmental diversity: the Aegilops tauschii Coss. example

Hexaploid bread wheat evolved from a rare hybridisation, which resulted in a loss of genetic diversity in the wheat D-genome with respect to the ancestral donor, Aegilops tauschii. Novel genetic variation can be introduced into modern wheat by recreating the above hybridisation; however, the information associated with the Ae. tauschii accessions in germplasm collections is limited, making rational selection of accessions into a re-synthesis programme difficult. We describe methodologies to identify novel diversity from Ae. tauschii accessions that combines Bayesian analysis of genotypic data, sub-species diversity and geographic information that summarises variation in climate and habitat at the collection point for each accession. Comparisons were made between diversity discovered amongst a panel of Ae. tauschii accessions, bread wheat varieties and lines from the CIMMYT synthetic hexaploid wheat programme. The selection of Ae. tauschii accessions based on differing approaches had significant effect on diversity within each set. Our results suggest that a strategy that combines several criteria will be most effective in maximising the sampled variation across multiple parameters. The analysis of multiple layers of variation in ex situ Ae. tauschii collections allows for an informed and rational approach to the inclusion of wild relatives into crop breeding programmes.     

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.     

巴西植物遗传资源保护与对外交流管理

巴西拥有高等植物55000种,居世界第一。植被分为6个区系,即亚马逊雨林、塞拉多(Cerrado)稀树草原、卡廷加(Caatinga)旱生植被、大西洋雨林、南方森林草原和潘塔纳尔(Pantanal)湿地植被。已建立554个原生境保护区和126个基因库,保存植物遗传资源25万份。对外交流由巴西农业科学院(EMBRAPA)遗传资源与生物技术中心(CENARGEN)统一管理。     

Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus×domestica borkh. core subset collection

 A collection of 66 Malus×domestica Borkh. accessions from the USDA-ARS Plant Genetic Resources Unit’s core collection was screened with a set of eight SSR (simple sequence repeat) primers developed at the PGRU in order to determine genetic identities, estimate genetic diversity, and to identify genetic relationships among these accessions. All eight primer pairs generated multiple fragments when used in amplification reactions with DNA from these accessions. High levels of variation were detected with a mean of 12.1 alleles per locus and a mean heterozygosity across all eight loci of 0.693. The eight primer pairs utilized in this study unambiguously differentiated all but seven pairs of accessions in this collection of 66 M.×domestica Borkh. genotypes. The probability of matching any two genotypes at all eight loci in this study was approximately 1 in 1 billion. The markers detected two misnamed accessions in the collection. Genetic-identity data produced a genetic-relatedness phenogram which was concordant with geographic origins and/or known pedigree information. These SSR markers show great promise as tools for managing Malus ex situ germplasm collections as well as for collection and preservation strategies concerning wild Malus populations in situ. Received: 28 March 1998 / Accepted: 29 April 1998