Assembling a core collection from the flax world collection maintained by Plant Gene Resources of Canada

Between 1998 and 2008, numerous projects were conducted by the Canadian national genebank, Plant Gene Resources of Canada, for the regeneration, characterization and evaluation of the whole flax (Linum usitatissimum L.) germplasm collection. The whole collection comprised 3378 accessions and, according to the passport data, several of these were probably genetically very similar or even identical. Therefore, a subset of 381 accessions was selected that represented the diversity found in the whole collection. Sampling accessions from the whole collection was made using characterization and evaluation data and followed six different methods: (1) For seven qualitative characters, each unique combination of character expression was represented by three accessions; (2) for quantitative characters, a fixed number of accessions representing the lowest and highest observed values was included; (3) for stem fibre content, disease ratings, seed vigour and drought tolerance, a fixed number of accessions with desirable performance was included; (4) a subset of the 57 most distinct accessions based on RAPD markers was included; (5) a subset of 40 pure lines that were created based on extreme low and high values for 1000 seed weight, seed oil content and fatty acid profiles was included; (6) a subset of fibre flax cultivars of known relevance in European flax breeding and another subset of flax cultivars of known relevance for North American linseed breeding were included. The goal was to maximize the diversity available in a limited number of flax accessions by preserving the range of variation present in the whole collection, while improving evenness. The core collection was assembled in response to requests by flax breeders. This paper compares distribution parameters in the whole and core collections.     

Evaluation of the discriminance capacity of RAPD, isoenzymes and morphologic markers in apple (Malus x domestica Borkh.) and the congruence among classifications

Twenty-one apple accessions were characterised and classified taxonomically with randomly amplified polymorphic DNA (RAPD). The results were compared with morphological and isoenzymatic classifications of the same material by calculating the congruence between similarity matrices. The results confirmed the greater discrimination capacity of RAPD compared with isoenzymes, although the taxonomic clusters obtained with both types of markers were congruent. The genetic relationships calculated using RAPD and isoenzymes showed little congruence with the morphological relationships among accessions. Isoenzymatic markers are useful in germplasm banks to evaluate the diversity of newly introduced accessions. The use of RAPD markers are especially beneficial to discriminate between material that is genetically similar, to evaluate genetic variability within a collection and to choose the components of the core collection.     

Marker-assisted sampling of the cultivated Andean potato Solanum phureja collection using RAPD markers

The potato crop originated in the Andean highlands where numerous farmer's varieties and non-cultivated wild species exist. An Andean potato collection is held in trust at the International Potato Center (CIP) to preserve the biodiversity of this crop and ensure the supply of germplasm for potato improvement worldwide. A core collection representing the biodiversity of the Andean potato germplasm is under construction using morphological, molecular, and geographic data. One of the eight cultivated potato species, Solanum phureja, has been genotyped using the RAPD technique. A protocol suitable for large germplasm collection genotyping has been developed to process numerous samples at reasonable costs. From 106 RAPD primers evaluated, we have selected 12 primers yielding 102 polymorphic markers, which unambiguously discriminated all 128 accessions but 2 that are possible duplicates. The S. phureja germplasm collected throughout the Andean countries appears to have a homogeneous genetic constitution. There was no clear geographic pattern as indicated by cluster analysis of the RAPD data. A sub-group of 20 accessions has been identified on the basis of the marker data and selected to maximize molecular (RAPD) variance and polymorphism. The probability of capturing equal amounts of marker polymorphism in this sub-group of 20 accessions by random sampling is less than 40%. This set accessions represents our first group of accessions that may constitute a core of the S. phureja collection. This tentative core will be challenged for diversity content by alternate markers and agronomic traits. Hence, the methodology for sampling less than 10% of the base collection, proposed for core collections by Brown (1989), can be based on molecular marker data provided cost-efficient fingerprints are developed.     

Patterns of SSR variation in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) seeds under ex situ genebank storage and accelerated ageing

Maintaining seed viability and germplasm integrity is a challenging task in conservation of plant genetic resources, as seeds under storage will lose viability and genetic changes will occur. Attempt was made to analyze the patterns of genetic changes in wheat germplasm under ex situ genebank storage and accelerated ageing treatments. A set of 16 naturally aged wheat accessions under ex situ genebank storage since 1994 were sampled. Four recently regenerated wheat accessions were selected, four random seed samples were chosen from each accession, and three of them were exposed to three different accelerated ageing treatments. These 32 seed samples in two germplasm sets displayed a range of germination rates from 4 to 98 %. Thirty-seven microsatellite markers representing 21 wheat chromosomes were applied to screen 12 seeds of each sample and 449 SSR alleles were scored. Large SSR variation was found in each germplasm set. There was 73.1 % of the total SSR variation present among the naturally aged samples and 78.2 % present among the accelerated ageing samples. Several analyses for genetic association consistently revealed no clear genetic separations among samples of high or low germination rates in both germplasm sets. Samples under different accelerated ageing treatments did not show much genetic differentiations from the original sample of each accession. Mantel tests revealed non-significant associations between SSR variability and sample germination rates for both germplasm sets. These findings are useful for understanding seed deterioration under different ageing conditions and suggest that genome-wide SSR variability may not provide sensitive markers for the monitoring of wheat seed viability.     

Genetic variation in radish (Raphanus sativus L.) germplasm from Pakistan using morphological traits and RAPDs

Genetic diversity of 30 radish (Raphanus sativus L.) accessions was investigated at the phenotypic level with morphological characters and at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Thirty-six morpho-physiological traits were recorded from seedling stage to harvest. The 31 primers used generated 202 RAPD bands, of which 158 (78.2%) were polymorphic. Multivariate procedures were used to classify the germplasm on the basis of phenotypic traits and RAPD fragments. Dendrograms were generated for the Euclidean distance from the morphological data and the Nei's genetic distance from the RAPD markers. Phenotypically, all the accessions were classified into four major groups corresponding to the different forms of cultivated radish. The morphological diversity existing within each of these groups suggested that they should be discriminated into the three botanical convarieties, sativusT (large-rooted), caudatus (pod-type) and oleifer (oilseed-type). Clustering of the accessions did not show any pattern of association between the morphological characters and the collection sites. Instead, landrace groups were associated with their morphological similarities and horticultural uses. On the other hand, the intra-specific genetic relationships of several accessions based on RAPD analysis were related primarily to their collection sites rather than to their phenotypic affinities. The level of polymorphism exhibited by the various convarieties could be exploited in genetic mapping populations to tag economically important traits. These genotypes also could serve as a useful germplasm source for root, leaf, pod and seed. This preliminary study of traditional radish landraces from Pakistan provides useful information regarding their horticultural potential.     

Genetic diversity in tomato (Solanum lycopersicum L.) germplasm using fruit variation implemented by tomato analyzer software based on high throughput phenotyping

The decrease of genetic variation in crops like tomato (Solanum lycopersicum L.) due to domestication, breeding, and population bottle necks for fresh market consumptions has increased the need for this cash crop’s conservation and characterizations. To study the genetic variation of this valuable genetic resource in the National Plant Gene Bank of Iran, a collection of 589 tomato accessions from different regions was characterized using semi-automatic and high-throughput techniques. Based on the fruit shape, the studied accessions were classified into ten groups and significant differences for all the fruit characteristics studied indicated an abundant diversity within the germplasm collected from different regions. Studied tomato germplasm was grouped into nine main clusters based on hierarchical cluster analysis. More than 83% of data variation was explained by seven components in which the first two components explained for 50% of the variation where fruit shape index and proximal/distal fruit end shape showed a high contribution in the variation of the first component. This unique genetically divergent germplasm could be utilized to select tomato breeding lines of interest. Also, accessions belonged to the heterotic clusters could be selected for hybridization and conservation purpose as well as enrichment of pre-breeding programs of the tomato germplasm.

     

Pattern analysis of the diversity of morphological plant attributes and herbage yield in a world collection of white clover (trifolium repens L.) germplasm characterised in a summer moisture stress environment of Australia

Information on the variation available for different plant attributes has enabled germplasm collections to be effectively utilised in plant breeding. A world sourced collection of white clover germplasm has been developed at the White Clover Resource Centre at Glen Innes, New South Wales. This collection of 439 accessions was characterised under field conditions as a preliminary study of the genotypic variation for morphological attributes; stolon density, stolon branching, number of nodes, number of rooted nodes, stolon thickness, internode length, leaf length, plant height and plant spread, together with seasonal herbage yield. Characterisation was conducted on different batches of germplasm (subsets of accessions taken from the complete collection) over a period of five years. Inclusion of two check cultivars, Haifa and Huia, in each batch enabled adjustment of the characterisation data for year effects and attribute-by-year interaction effects. The component of variance for seasonal herbage yield among batches was large relative to that for accessions. Accession-by-experiment and accession-by-season interactions for herbage yield were not detected. Accession mean repeatability for herbage yield across seasons was intermediate (0.453). The components of genotypic variance among accessions for all attributes, except plant height, were larger than their respective standard errors. The estimates of accession mean repeatability for the attributes ranged from low (0.277 for plant height) to intermediate (0.544 for internode length).Multivariate techniques of clustering and ordination were used to investigate the diversity present among the accessions in the collection. Both cluster analysis and principal component analysis suggested that seven groups of accessions existed. It was also proposed from the pattern analysis results that accessions from a group characterised by large leaves, tall plants and thick stolons could be crossed with accessions from a group that had above average stolon density and stolon branching. This material could produce breeding populations to be used in recurrent selection for the development of white clover cultivars for dryland summer moisture stress environments in Australia. The germplasm collection was also found to be deficient in genotypes with high stolon density, high number of branches, high number of rooted nodes and large leaves. This warrants addition of new germplasm accessions possessing these characteristics to the present germplasm collection.     

Morphological patterns in a world collection of <Emphasis Type="Italic">Cleome gynandra</Emphasis>

Cleome gynandra L. is a leafy vegetable native to sub-Saharan Africa and Asia and cultivated on dry areas. The plant plays an important role in the food and nutrition security of local communities. The objective of this study was to illustrate variation and diversity in the Cleome collection held at the World Vegetable Center. In total, 242 accessions were cultivated and morphologically characterized and analyzed. The gynophore and filament, both important organs of the flower, played a key role in taxonomical identification. High morphological variation was detected in traits including plant height, pod length, leaf size, flower color, and earliness. Distinct morphological differentiation was detected between Asian and African accessions. On average, the African accessions were larger, less uniform and later in flowering and seed maturation than the Asian accessions. The results were used to establish a core collection of 49 Cleome accessions. Gaps in the current collection were detected. High within-accessions diversity challenges the existing ex situ conservation system; to maintain diversity, seed should be collected from a large number of plants, both during the collection mission and in ex situ regeneration. The results are relevant for germplasm collection strategies and regeneration protocols for good genebank practices.     

Determination of genetic variability by RAPD markers in cauliflower,cabbage and kale local cultivars from France

RAPD markers were used to analyse the genetic variability among a cole crop cultivar collection from France and to identify possible duplicate accessions for facilitating the germplasm multiplication. We surveyed 24 cauliflower, 24 cabbage and 48 kale populations using 62, 100 and 89 RAPD markers respectively on 40 seed bulk samples per accession. Genetic distances were calculated on the basis of these markers. Using the phylogenetic package PAUP we compared the genetic clusters obtained for the RAPD markers with the groups based on morphologic and agronomic data. For cauliflowers and cabbages a drastic selection by growers led to an extensive differentiation of morphologic and precocity traits related to geographic origin, which matches the grouping based on RAPD markers. Furthermore, RAPDs detected in some cases misclassification of accessions in the collection. Kales formed an homogeneous group probably due to extensive genetic exchanges leading to a continuous diversity. For the other crops, different horticultural types were separated in several subgroups by the RAPD markers suggesting different genetic origins. RAPD markers provide a rapid and informative approach to analyze the genetic variability in a collection. It can be applied to autogamous (spring cauliflower) or allogamous (winter cauliflower) crops, even in cases where the intra-population diversity is extensive (cabbages).     

Identification of intra-accession genetic diversity in selfing crops using AFLP markers: implications for collection management

Germplasm conserved as seeds in genebanks requires regular regeneration. In this process, selection and genetic drift may cause loss of genetic diversity from accessions. In the case of selfing crops, separation of distinct lines into different accessions may be an efficient strategy to avoid these negative effects. In order to evaluate the applicability of this method for collection management, knowledge about the level of intra-accession genetic diversity is required. By means of AFLP analysis intra-accession variation was investigated in two cultivars, two landraces and two wild populations of ex situ conserved barley germplasm. In the total sample of 216 individuals analysed (36 per accession), 22 genotypes were observed based on 104 polymorphic loci. The number of genotypes detected ranged from 1 to 3 per accession, except for a Nepalese landrace that revealed 12 genotypes. An UPGMA cluster analysis grouped the genotypes unambiguously into the accession they belonged to and genotypes within accessions were generally found to be closely related. In order to determine the repeatability of the results obtained, 11 individuals belonging to 4 genotypes from the Nepalese landrace were scored for a second set of AFLP markers. Matrices of genetic distances calculated for the two AFLP datasets were found to be highly correlated (r = 0.9346, P < 0.001). Separation of genotypes into different accessions was considered a relevant option only for the Nepalese landrace. Analysis of molecular variance indicated that this accession could be well divided into 8 distinct lines. Further implications of the results for genebank practices are discussed.     

Viability of Oryza sativa L. seeds stored under genebank conditions for up to 30 years

Germination ability, equilibrium relative humidity (eRH), and moisture content of ‘control’ seed samples representing 183 rice accessions stored in the active (2–4 °C) and base (?10 °C until 1993, then ?20 °C) collections of the T. T. Chang Genetic Resources Center were determined after storage for 20.5–30.5 years. Germination of seeds that had been stored in the base collection was generally high (>70 %), whereas germination was more variable for seeds stored in the active collection. Samples with lower viability after storage in the active collection were likely to have lower viability after storage in the base collection. There were significant differences in the moisture content-eRH relationship of the seeds depending on whether the seeds had been stored in the active or base collection. Based on re-test data for regular seed samples regenerated in 1979–1980 and stored in the active collection for up to 31 years, estimates of the time for ability to germinate to fall to 50 % (p ₅₀) ranged from 54 to 997 years. For the same seed samples stored in the base collection for approximately 31 years, ability to germinate has been maintained and germination increased due to improved procedures. The ability to germinate of base collection samples was also generally higher than that of ‘safety duplicate’ samples of the same seed lots that had been sent to the National Center for Genetic Resources Preservation, USA in 1981 and stored at ?18 °C. This may have been due to uptake of moisture either during processing for dispatch or as a consequence of poor packaging material. The results are discussed in relation to long-term seed storage and genebank management.     

Exploring the population genetics of genebank and historical landrace varieties

Landrace accessions have long been recognized as an important source of genetic diversity for crop species, and landraces are stored in genebanks across the world as genetic resources for future crop development. Landraces are also an important part of the human cultural heritage and as such they have been used for genetic studies to make inferences about historical agriculture. However, surprisingly little is known about the within-accession diversity of landrace crops of different species. In order to evaluate the diversity of Swedish landraces we used microsatellite markers to genotype accessions of four species (barley, pea, oats and rye), both extant genebank material and 114-year-old seed samples of similar geographic origin and type. We found consistently high levels of within-population genetic diversity in the historical material, but varying and often lower diversity levels in the genebank accessions. We also make tentative conclusions about how representative the genebank material is to what was originally cultivated in its reported area of origin and suggest that the true identity of the genebank accessions is unclear and that historical seed collections should be a more appropriate material for the study of historical agriculture.     

The Garlic and Shallot Core Collection image database of IPK presenting two vegetatively maintained crops in the Federal ex situ genebank for agricultural and horticultural crops at Gatersleben, Germany

Garlic and shallot are important vegetable and spice plants, garlic is also a medicinal crop widely used throughout the world. Both belong to the genus Allium, which has been a main target of the taxonomical research at the IPK for many years. Therefore, the IPK’s living collection of Allium is one of the world’s largest special collections of this genus with garlic and shallot being essential parts of this collection. Furthermore, they are subject to special activities to preserve the material in vitro and in cryopreservation. Both methods help protecting valuable germplasm from abiotic and biotic threats and reduce the maintenance costs in the long term. At present, the garlic collection maintained at the IPK incorporates germplasm of 509 accessions in total including all safety duplicates not offered for exchange. Of shallot, 114 accessions are present as well. Passport data as well as phenotypic data of 159 accessions of garlic, one of great headed garlic and 16 of shallot are included in the Garlic and Shallot Core Collection Database (GSCC). An additional part of the database is a comprehensive collection of images illustrating morphological characteristics of the accessions. Exploration of the information is supported by a web-based application. The GSCC is available at http://www.ipk-gatersleben.de/databases/gscc.     

A new collection of wild populations of Capsicum in Mexico and the southern United States

An exploration and collection mission for wild populations of Capsicum was carried out in the fall of 2006 and 2007, in 13 Mexican states and in the U.S. states of Arizona and Texas. The aim of this collection was to expand the number of accessions of wild chile pepper (Capsicum annuum var. glabriusculum and Capsicum frutescens) that are publicly available for research in plant improvement and for subsequent use in an inquiry into the domestication of C. annuum. While Mexico and the United States National Plant Germplasm System both have germplasm repositories INIFAP—Instituto Nacional de Investigaciones Forestales—Agrícolas y Pecuarias and USDA GRIN—United States Department of Agriculture’s Genetic Resources Information Network) with accessions of C. annuum var. glabriusculum, the very limited number available, their age, and/or validity of the information attached to many accessions do not allow for extensive research. Four hundred and sixty-six plants were sampled over two field seasons, of which copies of the collection reside in both UAA and at UC Davis. Given the current environment with the intellectual property of varieties of crop plants and, particularly, the extreme restrictions affecting explorations and the official procuring and sharing of germplasm across national borders, this U.S.—Mexico collaboration is one of the few examples of joint U.S.—Mexico germplasm collection efforts.     

Characterisation of germplasm accessions of Napier grass (pennisetum purpureum and P. purpureum×P. glaucum Hybrids) and comparison with farm clones using RAPD

Fifty six germplasm accessions of the important East African fodder crop Napier grass, Pennisetum purpureum, and its hybrids with P. glaucum, were characterised using 67 random amplified polymorphic DNA (RAPD) fragments. No or very low intra-accession variation was found for 49 of the accessions examined, confirming field observations that this species is predominantly clonally propagated. Comparison of intra and inter-accession variation identified several groups of identical/similar accessions that could be targeted if the collection is to be rationalised, and also highlighted two misplantings of germplasm material during transfer to a field trial site. A neighbour joining dendrogram of Jaccard's similarity estimates, clearly separated 50 accessions of P. purpureum from three P. glaucum individuals, and placed six hybrid accessions in an intermediate position. These groupings were well supported by a nested AMOVA (P<0.001; 29.5% of total variance due to taxonomic delineation). The main group of P. purpureum individuals could be further differentiated into five sub-groups (designated East Africa, Southern Africa, USA1, USA2 and Miscellaneous, to reflect the majority membership of sub-groups) and examination of the within P. purpureum component of the nested AMOVA, found them to be significantly different (P<0.001; 18.8% of variance). Genetic diversity across all accessions was found to be fairly high (Shannon's diversity index 0.306) and thus the collection probably represents a wide genetic base for this species. In addition to germplasm accessions, 25 Kenyan farm clones were also analysed. A principal coordinate analysis found that all but one of the clones clustered with the main P. purpureum group of accessions, indicating that the majority are probably not interspecific hybrids. The origin and pedigree of clones is discussed based on genetic similarity amongst clones and to germplasm accessions.     

Seeds capture the diversity of genetic resource collections of <Emphasis Type="Italic">Malus sieversii</Emphasis> maintained in an orchard

Many species in genebank collections are maintained vegetatively either as in vitro cultures or as plants in the field or greenhouse. In these cases, individual genotypes (cultivars) are the focus of conservation. In crop wild relatives, where the value of an accession is in the heterogeneity of segregating genotypes, seeds may be the most representative form of conservation. We used two sets of field-planted Malus sieversii apple trees that originated from seeds collected from two wild populations in Kazakhstan as a model system to determine if seeds from hand-pollinated crosses could be used to represent the allelic diversity of the parent trees in a genebank orchard. A crossing design was developed that used each of the selected trees from the two Kazakhstan collection sites as mothers and also as fathers in the form of bulked pollen pools. The sets of offspring from these crosses were genotyped using microsatellite markers. Paternity analyses revealed the percentage of offspring that was derived from each of the parent trees. We also determined the contribution of each parent to the fertilization events from the pollen pools. Although there were some unintended pollination events (fertilization by trees in unexpected pollen pools) and unequal contribution of pollen parents within pollen pools, we demonstrated that the seedlings effectively captured the allelic frequency and diversity of the parents. A seed collection strategy is the most efficient way to conserve the diversity of crop wild relatives even in collections typically conserved as individual clones.