Klaus J. Dehmer

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.

Development and application of SINE-based markers for genotyping of potato varieties

Potato variety discrimination based on morphological traits is laborious and influenced by the environment, while currently applied molecular markers are either expensive or time-consuming in development or application. SINEs, short interspersed nuclear elements, are retrotransposons with a high copy number in plant genomes representing a potential source for new markers. We developed a marker system for potato genotyping, designated inter-SINE amplified polymorphism (ISAP). Based on nine potato SINE families recently characterized (Wenke et al. in Plant Cell 23:3117–3128, 2011), we designed species-specific SINE primers. From the resulting 153 primer combinations, highly informative primer sets were selected for potato variety analysis regarding number of bands, quality of the banding pattern, and the degree of polymorphism. Fragments representing ISAPs can be separated by conventional agarose gel electrophoresis; however, automation with a capillary sequencer is feasible. Two selected SINE families, SolS-IIIa and SolS-IV, were shown to be highly but differently amplified in Solanaceae, Solaneae tribe, including wild and cultivated potatoes, tomato, and eggplant. Fluorescent in situ hybridization demonstrated the genome-wide distribution of SolS-IIIa and SolS-IV along potato chromosomes, which is the basis for genotype discrimination and differentiation of somaclonal variants by ISAP markers.

Metabolite profiling of red and blue potatoes revealed cultivar and tissue specific patterns for anthocyanins and other polyphenols

Main conclusionMetabolite profiling of tuber flesh and peel for selected colored potato varieties revealed cultivar and tissue specific profiles of anthocyanins and other polyphenols with variations in composition and concentration.Starchy tubers of Solanum tuberosum are a staple crop and food in many countries. Among cultivated potato varieties a huge biodiversity exists, including an increasing number of red and purple colored cultivars. This coloration relates to the accumulation of anthocyanins and is supposed to offer nutritional benefits possibly associated with the antioxidative capacity of anthocyanins. However, the anthocyanin composition and its relation to the overall polyphenol constitution in colored potato tubers have not been investigated closely. This study focuses on the phytochemical characterization of the phenolic composition of a variety of colored potato tubers, both for peel and flesh tissues. First, liquid chromatography (LC) separation coupled to UV and mass spectrometry (MS) detection of polyphenolic compounds of potato tubers from 57 cultivars was used to assign groups of potato cultivars differing in their anthocyanin and polyphenol profiles. Tissues from 19 selected cultivars were then analyzed by LC separation coupled to multiple reaction monitoring (MRM) to detect quantitative differences in anthocyanin and polyphenol composition. The measured intensities of 21 anthocyanins present in the analyzed potato cultivars and tissues could be correlated with the specific tuber coloration. Besides secondary metabolites well-known for potato tubers, the metabolic profiling led to the detection of two anthocyanins not described for potato tuber previously, which we tentatively annotated as pelargonidin feruloyl-xylosyl-glucosyl-galactoside and cyanidin 3-p-coumaroylrutinoside-5-glucoside. We detected significant correlations between some of the measured metabolites, as for example the negative correlation between the main anthocyanins of red and blue potato cultivars. Mainly hydroxylation and methylation patterns of the B-ring of dihydroflavonols, leading to the formation of specific anthocyanidin backbones, can be assigned to a distinct coloring of the potato cultivars and tuber tissues. However, basically the same glycosylation and acylation reactions occur regardless of the main anthocyanidin precursor present in the respective red and blue/purple tissue. Thus, the different anthocyanin profiles in red and blue potato cultivars likely relate to superior regulation of the expression and activities of hydroxylases and methyltransferases rather than to differences for downstream glycosyl- and acyltransferases. In this regard, the characterized potato cultivars represent a valuable resource for the molecular analysis of the genetic background and the regulation of anthocyanin side chain modification.

Analysis of wild Lactuca accessions: conservation and identification of redundancy

Germplasm accessions of wild Lactuca species are maintained worldwide in ex situ collections as gene reservoirs for quality and disease resistance traits for cultivated lettuce. Accessions of 12 Lactuca species from 6 genebanks were compared via morphological characterization and AFLP (Amplified Fragment Length Polymorphism)-based profiling to estimate the extent of duplication. A method of assessing redundancy within very similar, but not identical accessions, is proposed, based on 352 polymorphic AFLP products. Seven duplication groups showed a high level of AFLP similarity, and one pair of Lactuca saligna accessions displayed identical AFLP profiles. In several cases, the morphological assessment indicated that a taxonomic reclassification of accessions was necessary. Candidate duplicates were identified using population parameters and inter- and intra-accession variability. The implications of these findings on the conservation of wild species are discussed

Towards an Enhanced Utilization of Plant Genetic Resources in Grass Breeding by Characterization and Evaluation Trials

Characterizations and evaluations of the most important grass species maintained there belong to the fundamental tasks of the Malchow Satellite Collection of the IPK Genebank. In recent years and in cooperation with universities, breeding companies and research institutions, several trials were performed in this respect.

Single Nucleotide Polymorphism (SNP) Markers for Allele Quantification in Lolium (Poaceae): Development and First Applications

Lolium grasses are widely distributed and used, and their economic importance is significant. We report the development of Lolium single nucleotide polymorphism (SNP) markers that were derived from barley EST data and that were suitable for allele quantification (AQ) via pyrosequencing (PSQ). Polymorphism information content (PIC) values for 22 developed markers ranged between 0.23 and 0.93 (average PIC value 0.80). The markers were used to characterize a collection of 2829 Lolium accessions. Eleven SNP markers could be mapped in the Lolium perenne VrnA mapping population. This is the first report on fingerprinting a comprehensive genebank collection by quantification of SNP alleles. The markers clearly distinguish between L. perenne and L. multiflorum and will prove as very useful for the management of germplasm collections.

Pleistocene climate changes explain large-scale genetic variation in a dominant grassland species, Lolium perenne L.

Aim Grasslands have been pivotal in the development of herbivore breeding since the Neolithic and are still nowadays the most widespread agricultural land-use across Europe. However, it remains unclear whether the current large-scale genetic variation of plant species found in natural grasslands of Europe is the result of human activities or natural processes. Location Europe. Taxon Lolium perenne L (perennial ryegrass). Methods We reconstructed the phylogeographic history of L. perenne, a dominant grassland species, using 481 natural populations including 11 populations from closely related taxa. We combined the Genotyping-by-Sequencing (GBS) and Pool-sequencing (Pool-seq) methods to obtain high-quality allele frequency calls of ~ 500 k SNP loci. We performed genetic structure analyses and demographic reconstructions based on the site frequency spectrum (SFS). We additionally used the same genotyping protocol to assess the genomic diversity of a set of 32 cultivars representative of the L. perenne cultivars widely used for forage purposes. Results Expansion across Europe took place during the Würm glaciation (12-110 kya), a cooling period that decreased the dominance of trees in favour of grasses. Splits and admixtures in L. perenne fit historical sea level changes in the Mediterranean basin. The development of agriculture in Europe (7-3.5 kya), that caused an increase in the abundance of grasslands, did not have an effect on the demographic patterns of L. perenne. We found little differentiation between modern cultivars and certain natural variants. However, modern cultivars do not represent the wide genetic variation found in natural populations. Main conclusions Demographic events in L. perenne can be explained by the changing climatic conditions during the Pleistocene. Natural populations maintain a wide genomic variability at continental scale that has been underused by recent breeding activities. This variability constitutes valuable standing genetic variation for future adaptation of grasslands to climate change, safeguarding the agricultural services they provide.

Patterns of Spring Growth and Phenology in Natural Populations of Lolium perenne Under Contrasting Field Conditions

The ecotypic diversity of perennial ryegrass (Lolium perenne L.) is a major genetic resource for breeding programs. In three replicated micro-sward trials in France, Belgium and Germany, we measured spring growth and recorded heading date of round 400 genebank accessions from the natural diversity of L. perenne that were selected as to represent the wide range of variability in this species. We observed marked differences between trial locations as well as interaction between accessions and locations in the timing of spring growth rates along growing-degree-days (GDDs). These preliminary results are part of a wider project aiming to investigate the natural adaptation of perennial ryegrass to various regional climates across its spontaneous area of presence in Europe.

Enhancing the Sustainable Use of Lolium perenne Genetic Resources from Genebanks in Plant Breeding and Research

Lolium perenne is one of the most important grass species in Europe and subject of intensive breeding and research. Hence, there is a substantial need for well described, diverse and easily available genetic resources. The Satellite Collections North of the IPK Genebank continuously characterize and evaluate genebank accessions of oil and fodder crops. As an example, results from characterization and evaluation trials of 336 L. perenne accessions collected in Bulgaria, Croatia, Ireland and Spain are presented. Traits considered were, amongst others, phenological development, ear emergence, biomass production, disease susceptibility and winter hardiness. Results from the three-year evaluation trials showed a high variability regarding these traits. Generally, the Spanish ecotypes revealed a high biomass production level, while the Bulgarian ecotypes were characterized by a low rust susceptibility. The Irish ecotypes showed a better growth after winter than the ecotypes from the other countries in the third scoring year. These results have been transferred to the in-house developed Genebank Information System (GBIS) and to the international EURISCO information system. Their access via EURISCO is demonstrated.

Ex-situ evaluations of Lolium perenne L. ecotypes collected in Bulgaria, Croatia, Spain and Ireland reveal valuable breeding material

Between 2002 and 2010, a total of 366 Lolium perenne L. genebank accessions originating from collection trips to Bulgaria, Croatia, Spain and Ireland were evaluated in field experiments together with commercial check varieties at the island of Poel in northern Germany. Ten plant parameters were visually scored, including the development before and after winter, spring growth, heading, plant biomass and the incidence of disease symptoms. The ecotypes collected in Croatia and Spain achieved high scores for all the growth parameters evaluated, particularly in the first and second experimental year, and were well comparable to the check varieties. The Bulgarian ecotypes exhibited a low plant biomass development after the first cut and before winter but a high productivity in spring and a low susceptibility to rust infection. The ecotypes from Ireland revealed a low aftermath heading in the second experimental year and a superior growth after winter in the third year. In conclusion, the L. perenne ecotypes studied showed a high variability regarding important agronomic attributes and several accessions were identified that exceeded the average performance of the check varieties.