C.C.M. van de Wiel

Use of microsatellites to evaluate genetic diversity and species relationships in the genus Lycopersicon

Abstract  In order to determine how informative a set of microsatellites from tomato is across the genus Lycopersicon, 17 microsatellite loci, derived from regions in and around genes, were tested on 31 accessions comprising the nine species of the genus. The microsatellite polymorphisms were used to estimate the distribution of diversity throughout the genus and to evaluate the efficacy of microsatellites for establishing species relationships in comparison with existing phylogeny reconstructions. Gene diversity and genetic distances were calculated. A high level of polymorphism was found, as well as a large number of alleles unique for species. The level of polymorphism detected with the microsatellite loci within and among species was highly correlated with the respective mating systems, cross-pollinating species having a significantly higher gene diversity compared to self-pollinating species. In general, microsatellite-based trees were consistent with a published RFLP-based dendrogram as well as with a published classification based on morphology and the mating system. A tree constructed with low-polymorphic loci (gene diversity <0.245) was shown to represent a more-reliable topology than a tree constructed with more-highly polymorphic loci.

DNA barcoding discriminates the noxious invasive plant species, floating pennywort (Hydrocotyle ranunculoides L.f.), from non-invasive relatives.

Floating pennywort (Hydrocotyle ranunculoides L.f.), a member of the plant family Araliaceae originating from North America, is an example of an invasive aquatic species posing serious problems to the management of waterways outside of its original distribution area in Australia and Western Europe. As a consequence, its import was banned in the Netherlands. It can be difficult to distinguish H. ranunculoides from other species of the genus on a morphological basis. In this regard, DNA barcoding may become a good alternative once this could be performed on a routine basis. In this study, we show that it is possible to distinguish H. ranunculoides from a series of closely related congeners by using a single plastid DNA sequence, trnH‐psbA.

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.

Efficient distinction of invasive aquatic plant species from non-invasive related species using DNA barcoding

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non‐invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH‐psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH‐psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.

Outcrossing and coexistence of genetically modified with (genetically) unmodified crops: a case study of the situation in the Netherlands

Abstract With the introduction of genetically modified (GM) crops the EU has demanded that individual member states enact measures to prevent inadvertent admixture — through outcrossing — of genetically modified organisms (GMOs) with products from conventional and organic farming. A literature review on out-crossing was prepared for the Coexistence Committee installed in the Netherlands in 2004. For sugar beet and potato, isolation distances do not appear to be of overriding importance, as true seeds are not part of the harvested product. The only route for admixture is through persistence of GM hybrid volunteers, and these should already be subject to strict control in good agricultural practice. Data on maize indicate that a distance larger than 25 m is needed to keep admixture below the EU labelling threshold of 0.9%, and larger than 250 m to remain below the 0.1% threshold as favoured by organic farming organizations. Oilseed rape is more complex because apart from pollen flow also persistence of volunteers in and outside arable fields, and hybridization with wild relatives play a role. At the present state of knowledge, isolation distances of 100–200 m and rotation intervals of 6–8 years might be warranted for the 0.9% threshold. It is as yet not clear whether a threshold of 0.1% is achievable in practice. The conclusions are compared with the measures recommended by the Dutch Coexistence Committee.

Spontaneous gene flow and population structure in wild and cultivated chicory, Cichorium intybus L.

Spontaneous gene flow between wild and cultivated chicory, Cichorium intybus L., may have implications for the genetic structure and evolution of populations and varieties. One aspect of this crop-wild gene flow is the dispersal of transgenes from genetically modified varieties, e.g. gene flow from GM chicory to natural chicory could have unwanted consequences. With the purpose to identify and quantify crop-wild gene flow in chicory, we analysed introgression in 19 wild chicory populations and 16 accessions of chicory varieties and landraces distributed across Northern, Central and Mediterranean Europe. The analysis used 281 AFLP markers and 75 SSAP markers giving a total of 356 polymorphic markers. Results from model based assignments with the program STRUCTURE indicated many incidents of recent gene flow. Gene flow was observed both between cultivars and wild populations, between landraces and wild populations, between different wild populations as well as between cultivars. Population structure visualized by distance-based clustering showed a North–South geographical structuring of the wild populations, and a general grouping of the cultivars corresponding to known origin. The results indicated, however, that the structuring between the two groups of wild and cultivated types was weak. As crop and wild recipients are genetically close and genes are transferred between the two types rather frequently, focus on mitigating crop-wild gene flow should be increased, before transgenic varieties are cultivated openly.

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.

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.

Pollen-mediated gene flow in maize tested for coexistence of GM and non-GM crops in the Netherlands: effect of isolation distances between fields

Abstract In 2006 and 2007, field trials were performed to study the effects of the two isolation distances indicated by the Dutch Coexistence Committee, i.e., 25 m between GM (genetically modified) and conventional maize, and 250 m between GM and deliberately non-GM (e.g., organic) maize, on pollen-mediated gene flow (PMGF) under representative agricultural conditions in the Netherlands. Each isolation distance was tested at three different locations across the Netherlands in both years. For testing PMGF with the 25 m isolation distance, GM source fields of 100 m × 100 m (1 ha) were surrounded by four equally sized non-GM receptor fields at a distance of 25 m. For testing PGMF with the 250 m isolation distance, 1-ha GM source fields were surrounded by four 50 m × 50 m (0.25 ha) receptor fields in four different directions at 250 m. For the GM source field, the maize variety DKC3421YG containing the MON810 event was used with both distances. A maize variety near-isogenic to the GM variety was grown in the receptor fields to obtain good flowering synchronicity between GM and non-GM maize and thus optimal conditions for PGMF. Levels of the transgene in grain samples from the receptor fields were measured by a validated real-time PCR (polymerase chain reaction) quantification method for the MON810 event. Analyses showed the following levels of grain admixture as a consequence of PMGF, averaged over 12 fields for each isolation distance tested: at 25 m 0.084% (individual field averages ranged from 0.009%t0 0.296%) in 2006 and 0.080% (0.002% to 0.318%) in 2007, respectively, and at 250 m 0.005% (individual field averages ranged from o to 0.040%) in 2006 and 0.007% (0 to 0.037%) in 2007, respectively. Although weather conditions clearly differed between 2006 and 2007 (a hot and dry summer in 2006 vs. a relatively wet one with about-average temperatures in 2007), outcrossing rates did not differ significantly between these years.

New traits in crops produced by genome editing techniques based on deletions

One of the most promising New Plant Breeding Techniques is genome editing (also called gene editing) with the help of a programmable site-directed nuclease (SDN). In this review, we focus on SDN-1, which is the generation of small deletions or insertions (indels) at a precisely defined location in the genome with zinc finger nucleases (ZFN), TALENs, or CRISPR-Cas9. The programmable nuclease is used to induce a double-strand break in the DNA, while the repair is left to the plant cell itself, and mistakes are introduced, while the cell is repairing the double-strand break using the relatively error-prone NHEJ pathway. From a biological point of view, it could be considered as a form of targeted mutagenesis. We first discuss improvements and new technical variants for SDN-1, in particular employing CRISPR-Cas, and subsequently explore the effectiveness of targeted deletions that eliminate the function of a gene, as an approach to generate novel traits useful for improving agricultural sustainability, including disease resistances. We compare them with examples of deletions that resulted in novel functionality as known from crop domestication and classical mutation breeding (both using radiation and chemical mutagens). Finally, we touch upon regulatory and access and benefit sharing issues regarding the plants produced.