Genebank Collections Research Articles

Serological detection of seed borne viruses in cowpea regenerated germplasm using protein a sandwich enzyme linked immunorsorbent assay

Cowpea (Vigna unguiculata (L.) Walp) is susceptible to a number of fungal, bacterial and viral diseases. The effects of viruses can be devastating and are a major constraint to the production of cowpea. A study was carried out to detect the presence of seed borne viruses in fourteen cowpea accessions screened from 300 regeneratedcowpea germplasm from the genebank collection of the International Institute of Tropical Agriculture (IITA) in Nigeria. Protein A Sandwich Enzyme linked Immunosorbent Assay (PAS ELISA) was used to detect seed borne viruses in the accessions. Out of 36 accessions of TVu 033 indexed, 31 were infected with Cowpea aphid-borne mosaic virus genus Potyvirus, which is of great economic importance in germplasm conservation and exchange between countries as it causes great loss. Southern bean mosaic virus was not detected in any of the accessionsevaluated. Seeds were harvested from the infected mother plants at maturity and the embryos excised in vitro onto hormone free medium composed of 4.43 g MS basal salt, 30 g sucrose and 8 g of agar. The seedlings obtained were serologically indexed to detect any seed-borne viruses after acclimatisation to screen house conditions. Theseedlings obtained from excised embryos got from harvested seeds gave a negative ELISA result.

Marker-assisted reduction of redundancy in a genebank collection of cultivated lettuce

To reduce the level of redundancy in a collection of cultivated lettuce, data from 160 amplified fragment length polymorphism (AFLP) fragments and 10 polymorphic microsatellites were used in combination with passport data and morphological data, the latter obtained from an experimental field trial performed for verification purposes. Based on the observed distribution of the number of marker differences between and within accessions, a minimum of three AFLP differences and two microsatellite differences were regarded as levels warranting distinction between accessions in the redundancy analysis. The strategy followed in the redundancy analysis was mainly based on the confirmation of duplication by each of two independently generated data sources. The molecular data were used for the validation as well as the identification of potential duplicates, revealing a total number of 198 redundancies, corresponding to 12.9% of the total collection. Trueness to type, number of characterization and evaluation data, and collection management considerations, such as available seed quantities and germination percentages, were used as primary, secondary and tertiary criteria to decide which accession from duplication groups to maintain in the collection. Removal of accessions showed negligible effects on total collection diversity, as quantified for AFLPs and microsatellites, characterization and evaluation traits and resistance profiles against downy mildew pathotypes, indicating that the applied strategy was effective.

Maize gene bank collections as potentially valuable breeding material

Characterization and evaluation of the genetic resources provide breeders with valuable information on an effective utilization of the genetic resources in breeding programs. In this paper we present the results of different research programs aimed at identification of superior genotypes among MRI gene bank accessions, regarding stress tolerance (drought and herbicides), better nutritional quality (phosphorus) and specific traits (cytoplasmic male sterility - CMS). Fifty-two genotypes were identified as a potential source for drought tolerance. Considering herbicide tolerance only genotypes with resistance to the Pivot were found. Within 100 sources of CMS in the collection S cytoplasm was identified as the predominant type. Phytate analysis of 60 maize populations identified three groups of populations - with low (8), intermediate (25) and high (27) phytate content. The results of these researches, which are a part of pre-breeding activities, will be included in MRI breeding programs, with the aim of developing new genotypes with improved traits important in commercial maize breeding and seed production.

Exploring the Natural Biodiversity of Potato for Late Blight Resistance

This paper gives an overview of the activities in Sub-project 4 of the BIOEXPLOIT programme. The European genebank collections of potato need to be narrowed down into (customized) core collections by focusing on loci associated with disease resistance. Therefore, new and more efficient molecular methods are being developed to identify these loci, extensive genotyping of resistance to Phytophthora infestans and signalling loci is being performed, and genotyped selections of accessions are phenotypically analysed for their resistance specificities. The results of these activities are being assembled in an integrated database.

Genetic diversity assessment of sub-samples of cacao, Theobroma cacao L. collections in West Africa using simple sequence repeats marker

Knowledge of genebank and on-farm genetic diversity, particularly in an introduced crop species, is crucial to the management and utilization of the genetic resources available. Microsatellite markers were used to determine genetic diversity in 574 accessions of cacao, Theobroma cacao L., representing eight groups covering parental populations in West Africa, genebank, and farmers’ populations in Nigeria. From the 12 microsatellite markers used, a total of 144 alleles were detected with a mean allelic richness of 4.39 alleles/locus. The largest genetic diversity was found in the Upper Amazon parent population (Hnb = 0.730), followed by the 1944 Posnette’s Introduction (Hnb = 0.704), and was lowest in the Local parent population (Hnb = 0.471). Gene diversity was appreciably high in the farmers’ populations (Hnb = 0.563–0.624); however, the effective number of alleles was lower than that found in the genebank’s Posnette’s population. Fixation index estimates indicated deficiency of heterozygotes in the Upper Amazon and the Local parent populations (Fis = 0.209 and 0.160, respectively), and excess of heterozygotes in the Trinitario parent population (Fis = −0.341). The presence of inbreeding in the Local parent populations and substructure (Wahlund effect) in the Upper Amazon were suggested for the deficiency of heterozygotes observed. Non-significant genetic differentiation observed between the genebank’s and farmers’ populations indicated significant impact of national breeding programs on varieties grown in farmers’ plantations. From this study, we showed that appreciable genetic diversity was present in on-farm and field genebank collections of cacao that can be exploited for crop improvement in West Africa. Suggestions for future conservation of on-farm genetic diversity and local landraces are further discussed.

Seed conservation in ex situ genebanks—genetic studies on longevity in barley

Recognizing the danger due to a permanent risk of loss of the genetic variability of cultivated plants and their wild relatives in response to changing environmental conditions and cultural practices, plant ex situ genebank collections were created since the beginning of the last century. World-wide more than 6 million accessions have been accumulated of which more than 90% are stored as seeds. Research on seed longevity was performed in barley maintained for up to 34 years in the seed store of the German ex situ genebank of the Leibniz-Institute of Plant Genetics and Crop Plant Research in Gatersleben. A high intraspecific variation was detected in those natural aged accessions. In addition three doubled haploid barley mapping populations being artificial aged were investigated to study the inheritance of seed longevity. Quantitative trait locus (QTL) mapping was based on a transcript map. Major QTLs were identified on chromosomes 2H, 5H (two) and 7H explaining a phenotypic variation of up to 54%. A sequence homology search was performed to derive the putative function of the genes linked to the QTLs.

LET'S GO LOCAL INITIATIVE IN POHNPEI, MICRONESIA FOR PROMOTING UNDERUTILIZED CROPS

Pohnpei State, Federated States of Micronesia, located in the Northern Pacific Ocean, has a great diversity of plants, including 48 giant swamp taros, 55 bananas, 133 breadfruits, and 171 yam cultivars among the staple food crops. However, since the 1970s, traditional food crops have been neglected with the shift to imported processed foods. Although nutritional status was previously good, dietary and lifestyle changes have led to serious nutrition-related health problems. Over half of the preschool children suffer from vitamin A deficiency, adults are commonly obese, and approximately 20% of adults are diabetic. There is concern about loss of traditional knowledge. Thus, the Island Food Community of Pohnpei, a non-governmental organization, initiated activities to increase production and consumption of island foods for the many benefits these offer (food security, health, economic, environment, and culture). Four strategic areas of work were established: innovative awareness activities (including posters presenting the Varieties Message relating to the value of yellow-fleshed cultivars, provitamin A carotenoid content and health benefits; proclamation of 'Karat' as the Pohnpei State Banana; national 'Karat' postal stamps; Going Yellow video; Go Local billboards and email network); conservation of rare carotenoid-rich cultivars in gene bank collections (including Pandanus, an atoll island food); small-scale processing of local foods; and research (including nutrient analysis, documentation of the traditional food system as part of a global health project, and dietary/health assessments). Many traditional carotenoid-rich cultivars (i.e., banana, taro, pandanus) having the potential to protect against vitamin A deficiency, diabetes, cancer, and heart disease were identified. Measurable impact from the campaign through marketing and dietary studies was clearly shown, e.g., 'Karat' was previously not marketed but is now regularly available at local markets. Dietary improvement has been shown. The Let's Go Local initiative, with its inter-agency, participatory, ethnographic approach, has become well-known in many Micronesian communities. Further work, including greater support and resources in order to bring the awareness message to more communities, developing small-scale processing of local crops, and increasing availability of planting materials, is needed to expand the program to achieve full impact. Due to the initial response to our program, it is expected that this approach will be successful. Some areas of this program may be relevant to other communities where similar local foods have been neglected and underutilized.

Duplication assessments in Nordic Avena sativa accessions at the Canadian national genebank

This study applied two methods to assess intra-collection duplication within 339 Nordic oat (Avena sativa L.) accessions preserved by Plant Gene Resources of Canada (PGRC). Putative duplicates, that is accessions carrying a similar accession name, were grouped into 52 duplication groups and included 230 of the 339 Nordic oat accessions. A field assessment based on visual inspection of field plots was conducted during two growing seasons to detect distinct phenotypes within each duplication group. Simultaneously, a descriptor assessment using seven characters with altogether sixteen character states was used in both years for the same purpose. The combined results of both assessments and both years indicated that among the 230 accessions in duplication groups only 118 could be identified as distinct. This would allow for a reduction of 33% of the Nordic oat accessions at PGRC. The field assessment method detected fewer (75%) distinct accessions than the descriptor assessment (84%), when considering all accessions identified as distinct as 100%. Repeatability between years was higher in the field assessment (70%) than in the descriptor assessment (64%). The field assessment requires an experienced germplasm evaluator, but allows for handling large numbers of germplasm accessions and for detecting functional, fitness related, and user-relevant diversity. Combining field assessment with descriptor assessment and more sophisticated methods on selected subgroups may be the most efficient method for determination of internal duplication in genebank collections. Bulking phenotypically similar accessions within duplication groups is preferable to eliminating duplicate accessions when collection rationalization is required, as it reduces the risk of loosing diversity.

Assessing diversity in common bean (Phaseolus vulgaris L.) accessions at phenotype and molecular level: a preliminary approach

Assessing diversity across and within crop varieties is relevant to improve the description of collections in genebanks and in on-farm conservation. The differences among and within several common bean (Phaseolus vulgaris L.) landraces, collected from and maintained ex situ in NE Italy, were initially studied using a set of 15 phenotypic descriptors. From a subgroup of five bean accessions, microsatellite genotyping was performed after the extraction of DNA from each of 38–40 individuals. One of the 15 phenotypical descriptors (plant architecture) was of no use. Only ten out of the 23 molecular markers did work, however sufficient to discriminate the accessions. The phenotypic descriptors identified a portion of the within-population variability. A few discrepancies were obtained when observations of phenotype descriptors were run independently in two alpine locations in NE Italy. The genetic approach carried out separately on DNA of about 40 individuals clarified the structure of the five accessions.

Assessment of genetic diversity ofGuizotia abyssinica(L.f.) Cass. (Asteraceae) from Ethiopia using amplified fragment length polymorphism

Seventeen populations of niger (Guizotia abyssinica), representing all regions in Ethiopia where this crop is grown, were investigated using the amplified fragment length polymorphism (AFLP) technique, in order to determine the extent and distribution of its genetic diversity. A total of 539 AFLP loci were scored using seven primer combinations applied to 170 individual plants. Of these, 90% were polymorphic and all the individuals investigated were genetically unique. Despite the fact that most of the variation was within populations, populations were differentiated at a significant level (analysis of molecular variance;P < 0.001). There were no significant differences between populations in relation to the extent and altitude of cultivation. A significant positive correlation was revealed between Nei's standard genetic distance and geographic distance. Cluster analysis and principal coordinate analysis revealed that populations from the same regions were clustered together in most cases. Further collection of niger germplasm from areas underrepresented in gene bank collections is recommended.

AFLP data support the recognition of a new tuber-bearing Solanum species but are uninformative about its taxonomic relationships

Solanum section Petota, containing the cultivated potato and its wild relatives, is a group of around 200 species. Many of these species are morphologically very variable with unclear boundaries, and the group as a whole appears to be somewhat over-classified. Describing a new species in this group should only be undertaken with caution, and molecular data can be used to test the distinctness of any putative new taxon. AFLP markers have shown the ability to reliably distinguish species in several groups within the genus Solanum. We tested the distinctness of a new tuber-bearing Solanum species using morphological and AFLP data, and tried to establish its affiliation to the series within the section. There was clear support for the species status of the material known as Solanum hannemanii in genebank collections, but the AFLP data were inconclusive about its relationships to the other investigated species. Also, the distinction of the series Tuberosa and Megistacroloba, to which these species belong, was not supported.

Using of ITS sequences for the evaluation of taxonomic relations of the representatives of tribe Vicieae (Adans.) Bronn сем. Fabaceae Lindl

ITS1-5.8S-ITS2 nucleotide polymorphism аnalysis of Vicieae species that represent accessions of Russian genebank (VIR) collection have been performed. Nucleotide substitutions in ITS1-5.8S-ITS2-sequences and indels of different length that were species-specific or specific for Lathyrus and Viciaspecies groups have been revealed. New data proving taxonomic status of accessions with uncertain taxonomic position have been obtained.

Marker-assisted optimization of an expert-based strategy for the acquisition of modern lettuce varieties to improve a genebank collection

To regularly improve the composition of the lettuce collection of the Centre for Genetic Resources, the Netherlands (CGN) with modern varieties, feedback from crop experts is used to select approximately 10% of the new material for incorporation in the collection. In the present study, assessments of six experts were compared to microsatellite data of 414 new varieties and 1408 existing accessions. Based on the microsatellite data, the extent to which the genetic diversity of the collection would be enriched (added value) was calculated for specific sets of new varieties. When individual assessments of experts were evaluated, the total added value of expert-based selections was not significantly higher compared to randomly chosen groups, except for a single expert. Unfamiliarity with new varieties was shown to be a crucial factor in the assessment of crop experts. According to the current acquisition protocol that seeks for consensus among experts, varieties are selected based on recommendations from at least three experts. This protocol also did not perform better than randomly chosen groups of new varieties. However, significantly better results were obtained with alternative protocols. It was concluded that breeding value was a more decisive criterion in the current acquisition protocol than maximal extension of the genetic diversity within the collection. A modified protocol addressing both commercial and diversity aspects was suggested in order to meet the demands of plant breeders as well as conservationists.

Perennial kales: collection rationalization and genetic relatedness to other Brassica oleracea crop types

Perennial kale is a rare leafy vegetable and forage crop that is mainly vegetatively propagated and therefore expensive to conserve ex situ. A genebank collection of 47 perennial kales and 34 reference samples from the main Brassica oleracea crop types were characterized with seven microsatellite markers in order to verify potential redundancies and to obtain more insight in the position of perennial kales within B. oleracea. Based on the obtained results and on data from previous studies, the collection was reduced with 49% to 24 perennial kales. Considering this level of reduction, it was estimated that the investments made for the final verification by microsatellite analysis are returned after only 4-year time. A principal coordinate plot clearly separated the perennial kales from the other crop types of B. oleracea, except in one case. This deviating accession of vegetatively preserved perennial kale clustered closely together with the single seed-preserved accession of perennial kale included in the study. These two accessions occupied an intermediate position between the group of vegetatively propagated perennial kales and the group of seed-propagated Brassica accessions, suggesting a hybridization background with another B. oleracea crop type. The microsatellite study demonstrated a close genetic relationship among the investigated perennial kales and their unique position within B. oleracea.

Morphological and chemical variations of sweet flag (Acorus calamus L.) in the Czech and Finnish gene bank collection

The aim of this study was to analyze both the morphological and chemical characteristics of <i>Acorus calamus</i> plants collected from the natural sites in the Czech Republic and to compare them to the sweet flags growing in Finland, Slovenia and Canada, which are concentrated in the Gene Bank collections in Finland. The sweet flags of Finnish, Slovenian and Czech origin were found to be phenotypically and chemically very similar. They all represent the European triploid type of <i>Acorus calamus</i> var. <i>calamus</i>. The average essential oil content was established at the value of 1.50% of dry material in Finland and 1.91% in the Czech Republic. This amount is not sufficient according to the Czech norm for <i>Radix calami</i> quality (minimal amount is 2%) but the content of carcinogen β-asarone, which was established at 9−21% in the essential oil in all the analyzed sweet flags, did not exceed the maximal recommended value 0.5% of dry drug mass.

Comparison of different planting methods in relation to grain yield of wheat

The objective of this paper was to evaluate the adequacy of different methods of planting in small plots and compared with the grain yield rank of wheat genotypes in large plots under different planting densities. Significant differences were found among cultivars, same cultivars under different planting densities, years of testing and planting densities. The estimated values of heritability in narrow sense for grain yield were high and varied from 68 to 99 %, depending on the method of planting and planting density. Comparison of correlation among cultivar’s rank between Method 1 (planting with drilling machine) and other Methods (manual planting) revealed that these relationships were dependant upon the year of testing and varied from low to high, positive or negative and from significant to non-significant. The highest correlation was found between Method 1 and Method 3 (20 kernels/hill) in both years of testing. If we wish to test a larger number of genotypes from gene bank collections or advanced lines in early phase of breeding program for grain yield on small plots we can recommend the use of hill plots with planting density as close as possible to those in large plots.

Ex situ collections of cultivated flax (Linum usitatissimum L.) and other species of the genus Linum L.

An overview of ex situ collections of cultivated flax (Linum usitatissimum L.) and 53 other species of the genus Linum L. is presented. The names of 33 genebanks in 23 countries preserving Linum germplasm and the species preserved at these places are provided. World genebanks engage in ex situ preservation of about 48,000 accessions of cultivated flax (Linum usitatissimum). However, possibly only 10,000 accessions of these are unique. Of the ca. 200 species of the genus Linum, other than L. usitatissimum, 53 are reported to be represented in the world’s ex situ collections with a total of about 900 accessions. Of these, 279 accessions alone refer to the wild progenitor of cultivated flax, Linum bienne Mill. However, L. bienne from the region of origin of cultivated flax is rarely represented in genebank collections. References to publications describing the techniques for ex situ conservation and the infraspecific variation of cultivated flax are made. It is suggested that taxonomic species delimitation within the genus Linum is in need of clarification and that the variation in Linum species for ornamental use or use in breeding is investigated.

The distribution of genetic diversity in a Brassica oleracea gene bank collection related to the effects on diversity of regeneration, as measured with AFLPs.

The ex situ conservation of plant genetic resources in gene banks involves the selection of accessions to be conserved and the maintenance of these accessions for current and future users. Decisions concerning both these issues require knowledge about the distribution of genetic diversity within and between accessions sampled from the gene pool, but also about the changes in variation of these samples as a result of regenerations. These issues were studied in an existing gene bank collection of a cross-pollinating crop using a selection of groups of very similar Dutch white cabbage accessions, and additional groups of reference material representing the Dutch, and the global white cabbage gene pool. Six accessions were sampled both before and after a standard regeneration. 30 plants of each of 50 accessions plus 6 regeneration populations included in the study were characterised with AFLPs, using scores for 103 polymorphic bands. It was shown that the genetic changes as a result of standard gene bank regenerations, as measured by AFLPs, are of a comparable magnitude as the differences between some of the more similar accessions. The observed changes are mainly due to highly significant changes in allele frequencies for a few fragments, whereas for the majority of fragments the alleles occur in similar frequencies before and after regeneration. It is argued that, given the changes of accessions over generations, accessions that display similar levels of differentiation may be combined safely.

126. Preliminary results of the cryopreservation of Malus germplasm from the fruit gene bank collection at the Institute of Fruit Breeding, Dresden

126. Preliminary results of the cryopreservation of Malus germplasm from the fruit gene bank collection at the Institute of Fruit Breeding, Dresden

Preservation of plant genetic resources in the biotechnology era

Thousands of years ago humans began domesticating crops as a food source. Among the wild germplasm available, they selected those that were best adapted for cultivation and utilization. Although wild ancestors have continued to persist in regions where domestication took place, there is a permanent risk of loss of the genetic variability of cultivated plants and their wild relatives in response to changing environmental conditions and cultural practices. Recognizing this danger, plant ex situ genebank collections were created since the beginning of the last century. World-wide, more than 6 million accessions have been accumulated including the German ex situ genebank in Gatersleben, one of the four largest global collections, housing 150,000 accessions belonging to 890 genera and 3032 species. This review summarizes the ex situ plant genetic resources conservation behavior with a special emphasis on German activities. Strategies for maintenance and management of germplasm collections are reviewed, considering modern biotechnologies (in vitro and cryo preservation). General aspects on genetic diversity and integrity are discussed.