Th. J. L. van Hintum

Analysis of the wild potato germplasm of the series Acaulia with AFLPs: implications for ex situ conservation.

Abstract The wild potato germplasm of the series Acaulia maintained at the Centre for Genetic Resources, The Netherlands, currently consists of 314 accessions. This collection comprises seed samples of the species Solanum acaule (ssp. acaule, ssp. aemulans, ssp. palmirense and ssp. punae) and Solanum albicans collected from South America. In order to validate taxonomic classification, to investigate the extent of redundancy and to study the distribution of genetic diversity across the collection area, the entire collection was analysed with two AFLP primer pairs on two plants per accession. Within the entire sample a total number of 130 polymorphic bands were scored for the two primer pairs. An UPGMA cluster analysis grouped the majority of plants according to the species and subspecies. A total number of 16 misclassifications were identified, including four cases that did not seem to belong to the series Acaulia. Two accessions were found to consist of plants of different AFLP clusters. AFLP data also allowed the taxonomic classification of the subspecies of 97 accessions that previously were described as S. acaule only. For 126 accessions the two individuals studied displayed identical AFLP profiles. Forty six of these 126 accessions shared their profiles with both or single plants of other accessions. These were all tested for identical profiles for a third primer pair, resulting in 15 duplication groups consisting of a total number of 22 accessions and 14 single plants. Analyses of molecular variance (AMOVA) were performed to examine the distribution of genetic variation. Comparison of geographic distances between the collection site of plants and the number of AFLP polymorphisms revealed no consistent relationship between geographic distance and genetic diversity. AFLP analysis appeared to be an efficient method to verify taxonomic classification and to identify redundancies in the wild germplasm of the series Acaulia. Implications of the results for the ex situ conservation of wild potato germplasm are discussed.

Genetic distance sampling: a novel sampling method for obtaining core collections using genetic distances with an application to cultivated lettuce

This paper introduces a novel sampling method for obtaining core collections, entitled genetic distance sampling. The method incorporates information about distances between individual accessions into a random sampling procedure. A basic feature of the method is that automatically larger samples are obtained if accessions are further apart and smaller samples if accessions are closer together. Genetic distance sampling can be used in conjunction with predefined stratifications of the accessions. Sample sizes are determined automatically; they depend on the distances between accessions within strata. The method is applied to the collection of cultivated lettuce of the Centre for Genetic Resources, the Netherlands. In this paper, genetic distances between accessions are obtained using AFLP marker data. However, genetic distance sampling can be applied using any measure of genetic distance between accessions. Some properties of genetic distance sampling are discussed.

Marker-assisted rationalisation of genetic resource collections: a case study in flax using AFLPs

Abstract Removing redundant germplasm from collections is one of the options for genebanks to increase the efficiency of their genetic resource management. Molecular characterisation of germplasm is thereby becoming more and more important to verify suspected duplication. AFLPs were used to characterise 29 flax accessions of material derived from research activities (hereafter termed ’’breeder’s line”). Based on similar accession names, the breeder’s lines could be classified into three series (’M 25’, ’Ru’ and ’Rm’) that were expected to contain redundancies. In addition, 12 reference cultivars were analysed. A total number of 144 polymorphic bands (59.8%) were scored among the 164 individuals investigated. In general, relatively high levels of intra-accession variation were found, even for the cultivars examined. This finding was not in line with the low outcrossing rates reported for flax. A cluster analysis grouped the ’Ru’ and ’Rm’ series together, indicating their close genetic relationship. An analysis of molecular variance (AMOVA) showed a significant group effect (fibre/oil flax) only for ’M 25’, explaining 34% of the variation observed within this series. For the cultivars 40.5% of the variation was distributed among accessions within groups and all pairwise comparisons were significantly different, except for one case. Both for the series of breeder’s lines and the cultivars the major part of the variation was distributed among individuals within accessions. This component constituted 80.7% and 83.6% of the total variation for the ’Ru’ and ’Rm’ series, respectively. Pairwise comparisons of accessions were performed by AMOVA in order to identify redundant germplasm. Stepwise bulking of accessions until all remaining accessions were significantly different showed that the 29 accessions of breeder’s lines could be reduced to 14. Only a small negative effect of this bulking approach on the among-population component of variance was observed, showing a reduction of 2.6%. Results are discussed in relation to improving the efficiency of collection management.Removing redundant germplasm from collections is one of the options for genebanks to increase the efficiency of their genetic resource management. Molecular characterisation of germplasm is thereby becoming more and more important to verify suspected duplication. AFLPs were used to characterise 29 flax accessions of material derived from research activities (hereafter termed ’’breeder’s line”). Based on similar accession names, the breeder’s lines could be classified into three series (’M 25’, ’Ru’ and ’Rm’) that were expected to contain redundancies. In addition, 12 reference cultivars were analysed. A total number of 144 polymorphic bands (59.8%) were scored among the 164 individuals investigated. In general, relatively high levels of intra-accession variation were found, even for the cultivars examined. This finding was not in line with the low outcrossing rates reported for flax. A cluster analysis grouped the ’Ru’ and ’Rm’ series together, indicating their close genetic relationship. An analysis of molecular variance (AMOVA) showed a significant group effect (fibre/oil flax) only for ’M 25’, explaining 34% of the variation observed within this series. For the cultivars 40.5% of the variation was distributed among accessions within groups and all pairwise comparisons were significantly different, except for one case. Both for the series of breeder’s lines and the cultivars the major part of the variation was distributed among individuals within accessions. This component constituted 80.7% and 83.6% of the total variation for the ’Ru’ and ’Rm’ series, respectively. Pairwise comparisons of accessions were performed by AMOVA in order to identify redundant germplasm. Stepwise bulking of accessions until all remaining accessions were significantly different showed that the 29 accessions of breeder’s lines could be reduced to 14. Only a small negative effect of this bulking approach on the among-population component of variance was observed, showing a reduction of 2.6%. Results are discussed in relation to improving the efficiency of collection management.

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.

Comparison of anonymous and targeted molecular markers for the estimation of genetic diversity in ex situ conserved Lactuca.

The anonymous marker systems microsatellites (simple sequence repeats), amplified fragment length polymorphisms and sequence-specific amplified polymorphisms were compared with the targeted marker systems sequence-related amplified polymorphisms, target region amplification polymorphisms and nucleotide binding site profiling for their ability to describe the genetic diversity in a selected set of 80 Lactuca accessions. The accessions were also described morphologically, and all characterisation methods were evaluated against the genetic diversity assessed by a panel of three crop experts. The morphological data showed a low level of association with the molecular data, and did not display a consistently better relationship with the experts’ assessments in comparison with the molecular data. In general, the diversity described by the targeted molecular markers did not differ markedly from that of the anonymous markers, resulting in only slight differences in performance when related to the expert-based assessments. It was argued that markers targeted to specific gene sequences may still behave as anonymous markers and that the type of marker system used is irrelevant when at low taxonomic levels a clear genetic structure is absent due to intensive breeding activities.

Marker-Assisted Acquisition and Core Collection Formation: A Case Study in Barley using AFLPs and Pedigree Data

A problem that often occurs in deciding which germplasm should be acquired to expand the diversity of a plant genetic resources collection, and which accessions should be included in a core collection, is the lack of proper data. The usefulness of an AFLP-based protocol to assist in acquisition decisions and in core collection formation was examined by using 52 barley cultivars. For validation purposes, pedigree data of the cultivars were used to calculate the ‘effective number of origin lines’ (no1), a parameter introduced in earlier research that was defined as the number of alleles per locus, not identical by descent, in a set of lines. Two AFLP primer combinations were able to distinguish all 52 cultivars from each other, and to discriminate between spring and winter crop types. Using the year of origin of the cultivars, the historical development of no1 showed a stepwise pattern, indicating the periodical release of genetically similar cultivars, alternated by the incorporation of new material. Comparison of AFLP data between cultivars and both their parents was possible in five cases. These comparisons revealed a high likelihood that the correct parents were involved but a rather skewed contribution of parents to offspring, suggesting that backcrossing had been applied. Treating the 25 cultivars that were released before 1980 and played an important role in barley cultivation as a basic collection, and the 27 more recent cultivars as potential candidates for acquisition, no1 values generated by a marker-based approach largely followed those using a random approach. Given this poor performance, a marker-based protocol to assist in acquisition decisions was not considered useful for the analysed material. If the 52 cultivars were considered to be the collection from which a core collection had to be selected, the marker-based selection showed much better results compared to a random selection. About half of the total number of origin lines could be captured with a quarter of the collection, indicating the potential utility of AFLPs in core collection formation.

Reduction of duplication in a Brassica oleracea germplasm collection

SummaryTo reduce the number of accessions in the Brassica oleracea collection of the Centre for Genetic Resources The Netherlands (CGN) groups of accessions were bulked. Accessions in a group were selections from the same landrace or old variety, and were chosen, with the help of crop experts, on the basis of their history and morphology. Since this approach was received rather sceptically in the genebank community, a genetical analysis of some of the groups and their components (named varieties) was made to justify the procedures. A selection of white cabbage and Brussels sprouts, representing two crop types of B. oleracea, was made: eleven white cabbage groups comprising 43 components, and nine Brussels sprouts groups comprising 24 components. Each group and all of its components were described on the basis of electrophoresis of nine isoenzymes on 30 plants. Most components were rightly classified; 16 percent were misclassified. All these misclassifications were within similar groups. It could be concluded that pooling of the components included in this study was justified. In two cases the isozymic patterns suggested that the groups could even have been larger. Only in the case of the two groups of Brussels sprouts ‘Bedfordshire’ this was a real option. In the other case involving the white cabbage groups ‘Langedijker Bewaar Gewoon’, ‘Langedijker Bewaar Graag’ and ‘Late Herfstdeen’ this was not an option since these landraces, although having a common genetic background, clearly have a distinct identity as defined by morphology and history.

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.

A note on the measurement of genetic diversity within genebank accessions of lettuce (Lactuca sativa L.) using AFLP markers

This paper discusses a statistical approach for measuring genetic diversity within genebank accessions of a self-fertilising species. This approach is applied to lettuce (Lactuca sativa L.), using AFLP marker data on a set of 1,390 accessions, representing six different lettuce types. Knowledge of the within-accession genetic diversity is important for decisions about the way accessions have to be maintained by genebanks. It is argued that if the within-accession diversity is small, as can be expected for a self-fertilising species like L. sativa, the best approach is to sample as many accessions as possible with only two plants per accession and estimate the within-accession diversity by the proportion of accessions of which the individuals are different.

Distribution of genetic diversity in wild European populations of prickly lettuce (Lactuca serriola): implications for plant genetic resources management

Genetic variation in Lactuca serriola, the closest wild relative of cultivated lettuce, was studied across Europe from the Czech Republic to the United Kingdom, using three molecular marker systems, simple sequence repeat (SSR, microsatellites), AFLP and nucleotide-binding site (NBS) profiling. The ‘functional’ marker system NBS profiling, targeting disease resistance genes of the NBS/LRR family, did not show marked differences in genetic diversity parameters to the other systems. The autogamy of the species resulted in low observed heterozygosity and high population differentiation. Intra-population variation ranged from complete homogeneity to nearly complete heterogeneity. The highest genetic diversity was found in central Europe. The SSR results were compared to SSR variation screened earlier in the lettuce collection of the Centre for Genetic Resources, the Netherlands (CGN). In the UK, practically only a single SSR genotype was found. This genotype together with a few other common SSR genotypes comprised a large part of the plants sampled on the continent. Among the ten most frequent SSR genotypes observed, eight were already present in the CGN collection. Overall, the CGN collection appears to already have a fair representation of genetic variation from NW Europe. The results are discussed in relation to sampling strategies for improving genebank collections of crop wild relatives.