European Accessions Research Articles

The use of microsatellite polymorphisms to characterise and compare genetic variability in Avena strigosa and A. barbata

Microsatellite (SSR) polymorphism was assessed across 90 diploid Avena strigosa Schreb. and tetraploid Avena barbata Pott ex Link accessions obtained from the USDA-ARS National Small Grains Collection using 105 genomic SSRs. Eleven polymorphic SSRs that detected 69 different alleles were identified and used to genotype the 90 accessions, which were chosen from a larger set of 385 accessions based on geographical source-diversity and variable reaction responses to five Australian pathotypes of the crown rust pathogen Puccinia coronata Corda f. sp. avenae Eriks. Eight diploid and eight tetraploid clades were identified among the 90 accessions. Diploid accessions displayed the lowest genetic diversity, with all accessions being at least 86 % similar, and included accessions from countries in the Americas such as Canada, USA, Argentina, Uruguay and Brazil, and European accessions from France, Romania and Poland. Although both species formed distinct clusters in the dendrogram, a few instances of diploids showing high similarity with tetraploids and vice versa were observed. An AMOVA analysis revealed 86 % of the total genetic variation to be distributed within the two oat species, while between-species differences accounted for only 14 %. Heterozygosity (H) index values of 0.32 and 0.40 were obtained for diploids and tetraploids respectively. Our study effectively differentiated A. strigosa and A. barbata, and identified 11 SSRs suitable for future characterisation of accessions of the two species.

Characterization of 169 diverse pea germplasm accessions for agronomic performance, Mycosphaerella blight resistance and nutritional profile

Improved agronomic performance and nutritional profile are among the major objectives in pea breeding, but the narrow gene pool available in pea germplasm has slowed progress. Therefore, the objective of this work was to evaluate 169 diverse pea accessions from Eastern Europe, Western Europe, Australia and Canada for agronomic performance, Mycosphaerella blight resistance and nutritional profile under western Canadian growing conditions. In general, eastern European accessions required less days to flower, had higher protein, acid detergent fibre and neutral detergent fibre concentrations in the seeds, compared to western European and Canadian pea accessions, which had better lodging resistance, less days to maturity, higher grain yield and seed weight, greater resistance to Mycosphaerella blight and powdery mildew and higher starch concentration in the seeds. Significant correlations were observed between different traits. Principal component analysis revealed significant variation among traits with the first four principal components explaining 68 % of the total variation. The wide range of variation in agronomic performance, Mycosphaerella blight resistance and nutritional profile detected will provide useful parents for pea breeding.

Identification of a Simple Sequence Repeat molecular-marker set for large-scale analyses of pear germplasm

Simple Sequence Repeats (SSR) are molecular markers suitable to assess the genetic variation of germplasm resources; however, large-scale SSR use requires protocol optimization. The present work aimed to identify SSR markers, developed for pear and other fruit species that are effective in characterizing pear germplasm collections and in demonstrating their use in providing support for genetic breeding programs. From a total of 62 SSR markers investigated, 23 yielding reproducible and polymorphic patterns were used to genotype a sample of 42 pear accessions of the Brazilian Pear Germplasm Bank (PGB). When compared to these 23 SSR markers, a subset of eleven markers, selected based on He, PIC and PId, was used to distinguish individual accessions and perform cluster analysis with similar efficacy. Genetic diversity analysis clustered the European, Japanese and Chinese accessions in distinct groups. This markers subset constitutes a valuable tool for several applications related to pear genetic resources management and breeding.

Phenotypic diversity of autochthonous European (Prunus domestica L.) and Damson (Prunus insititia L.) plum accessions based on multivariate analysis

Forty-three European (Prunus domestica L.) and twelve Damson (P. insititia L.) plum accessions originating from different and important growing regions in former Yugoslavia were studied to assess the overall degree of polymorphism, detect similarities among accessions and assess important agronomic, fruit quality and sensorial traits. Twenty variables were scored and subjected to multivariate analysis. Results showed a considerable phenotypic diversity among plum germplasm. A high correlation was found among some evaluated variables. Principal component analysis (PCA) revealed that traits related to fruit weight, yield and soluble solids content accounted for a large proportion of the observed variability. Accessions Bela Požegača, Crvena Ranka Bardaklija, Mudara, Požegača, Car Dušan, Julka, Turgonja and Crna Petrovka cvs are recommended for fresh consumption, while Požegača, Korajka, Bosanka and Bilska Rana cvs are recommended for drying. Most of accessions can be used for processing, particularly into plum brandy, whereas some can be used as rootstocks.

Proanthocyanidin diversity in the EU ‘HealthyHay’ sainfoin (Onobrychis viciifolia) germplasm collection

This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80g/100g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P<0.001; R2=0.49). A negative correlation existed between PA content (thiolysis) and mDP (P<0.05; R2=−0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics.

Formalizing morphologically cryptic biological entities: New insights from DNA taxonomy, hybridization, and biogeography in the leafy liverwort Porella platyphylla (Jungermanniopsida, Porellales)

Recognition and formalization of morphologically cryptic species is a major challenge to modern taxonomy. An extreme example in this regard is the Holarctic Porella platyphylla s.l. (P. platyphylla plus P. platyphylloidea). Earlier studies demonstrated the presence of three isozyme groups and two molecular lineages. The present investigation was carried out to elucidate the molecular diversity of P. platyphylla s.l. and the distribution of its main clades, and to evaluate evidence for the presence of one vs. several species. We obtained chloroplast (atpB-rbcL, trnL-trnF) and nuclear ribosomal (ITS) DNA sequences from 101 Porella accessions (P. platyphylla s.l., P. × baueri, P. cordaeana, P. bolanderi, plus outgroup species) to estimate the phylogeny using parsimony and likelihood analyses. To facilitate the adoption of Linnean nomenclature for molecular lineages, we chose a DNA voucher as epitype. Phylogenies derived from chloroplast vs. nuclear data were congruent except for P. platyphylla s.l., including a North American lineage that was placed sister to P. cordaeana in the chloroplast DNA phylogeny but sister to the Holarctic P. platyphylla s.str. in the nuclear DNA phylogeny. European and North American accessions of P. cordaeana and P. platyphylla form sister clades. The genetic structure of P. platyphylla s.l. reflects morphologically cryptic or near cryptic speciation into Holarctic P. platyphylla s.str. and North American P. platyphylloidea. The latter species is possibly an ancient hybrid resulting from crossings of P. cordaeana and P. platyphylla s.str. and comprises several distinct molecular entities.

Towards better tasting and more nutritious carrots: Carotenoid and sugar content variation in carrot genetic resources

The genetic diversity present in crop species is crucial for crop improvement and consequently enables development of new, nutritious agricultural products. Seed collections held in genebanks facilitate access to this diversity and in the case of carrot (Daucus carota), they consist of material collected from across the world, permitting access to the diversity present in the whole genepool. However to date, no systematic evaluation of chemical composition has been made on this material. This study aimed to determine the available variation in carotenoid and sugar content in the roots of 118 carrot accessions in order to understand how the levels of these nutritious compounds vary across the global carrot genepool. The carotenoid and sugar content in root tissue was assessed. The total carotenoid content was related to root colour and ranged from 0 to 40mg per 100g fresh weight. Orange rooted European accessions were more carotenoid-rich than Asian accessions, and advanced cultivars contained on average 20% more carotenoids than older cultivated material (landraces). Sugar content varied from 5.1% to 13.6% and European and American materials contained on average 18% more total sugars than Asian accessions. Orange rooted advanced cultivars had a lower ratio of non-reducing to reducing sugars than landraces. The results obtained indicate that several accessions with elevated content of carotenoids and/or sugars exist in genebank collections. The results are of significance as they support more efficient utilisation of genebank material in breeding programmes which deliver novel cultivars with enhanced nutritional value. The occurrence of such a range of sugar and carotenoid contents supports the role of genebank collections as a source of diversity useful in the creation of novel and nutritionally improved cultivars.

Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium

In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids. Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL-accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network. Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine. Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.

Narrow species concepts in the Frullania dilatata–appalachiana–eboracensis complex (Porellales, Jungermanniopsida): evidence from nuclear and chloroplast DNA markers

We investigated the phylogeny of a Holarctic-Asian group of Frullania species, the Frullania dilatata–F. appalachiana–F. eboracensis complex, using multiple accessions of morphologically circumscribed taxa and three molecular markers (nrITS region, cp DNA trnL-F and atpB-rbcL regions). Maximum parsimony and likelihood analyses indicated monophyly of morphologically defined taxa. Our phylogenies support a species rather than a subspecies concept within the complex, with four species in North America (F. appalachiana, F. eboracensis, F. parvistipula and F. virginica), and two species in Europe (F. dilatata and F. parvistipula). Accessions of F. dilatata from Southeast Europe and Asia are separated from other European accessions, indicating a former disjunct range of the species.

Genetic diversity and linkage disequilibrium studies on a 3.1-Mb genomic region of chromosome 3B in European and Asian bread wheat (Triticum aestivum L.) populations

Genetic diversity and linkage disequilibrium (LD) were investigated in 376 Asian and European accessions of bread wheat (Triticum aestivum L.). After a first and rapid screening about diversity and genetic structure at the whole genome scale using 70 simple sequence repeats (SSRs), we focused on a sequenced contig (ctg954) of 3.1 Mb located on the short arm of chromosome 3B of cv. Chinese Spring, using 32 SSRs and 10 single nucleotide polymorphisms. This contig is part of a multiple fungal resistance region. Mean polymorphism information content value on the 32 SSRs was slightly higher in the Asian genepool (0.396) than that for the European (0.329) pool. Compared with results at the whole genome scale, data from this 3.1-Mb region indicated similar trends in genetic diversity indices between both genepools. Population structure and molecular variance analyses demonstrated significant genetic differentiation and geographical subdivision in both groups of accessions. Concerning LD at the contig level, the European population had a significantly higher mean r(2) value (0.23) than the Asian population (0.18), indicating a stronger LD in the European material. With a mean of 1 marker every 74 kb, the resolution reached here allowed to perform a detailed comparative analysis of the LD and genetic diversity along the complete 3.1-Mb region in both genepools. A sliding-window approach revealed some interesting regions of the contig where LD is increasing when genetic diversity is decreasing. This study provides an in-depth understanding of molecular population genetics in European and Asian wheat gene pools, and prospects for association mapping of important sources of fungal disease resistance.

Evolution of self-compatibility in Arabidopsis by a mutation in the male specificity gene

Ever since Darwin's pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components.

Morphological characteristics of some accessions of grass pea (Lathyrus sativus L.) grown in Europe and nutritional traits of their seeds

Thirty-one European accessions of grass pea (Lathyrus sativus L.) originating from Italy, Spain, Northern France, Germany and Poland grown in the same soil-climatic conditions in Poland were investigated. The morphological traits as well as the seed morphology and nutrient contents were studied. Substantial differentiation in some morphological characteristics was noticed. The plants could be assigned to two groups: one group comprising the lines from the Mediterranean basin (Italy and Spain) and the other group containing the accessions from West-central Europe (Northern France, Germany and Poland). The grass pea plants produced from the seeds obtained from West-central Europe appeared to be about 10 cm taller than the Mediterranean accessions and had predominantly white flowers. Their seeds were more than two times smaller than seeds of the accessions of Italian or Spanish origin. There were not noted any considerable differences in nutrient contents or antinutritional factors in seeds between these two groups of Lathyrus accessions. A negative correlation between β-ODAP and tannin contents (r = −0.379, P ≤ 0.05) and a positive correlation (r = 0.843, P ≤ 0.01) between a tannin content and flower color intensity were found. A white or creme colour was associated with a low tannin level in the seeds, while the colourful blooming accessions had higher tannin contents.

Evidence for genetic differentiation and divergent selection in an autotetraploid forage grass (Arrhenatherum elatius)

The use of local provenances in restoration, agriculture and forestry has been identified as measure to sustain biological diversity and to improve local productivity. However, the delineation of regional provenances is challenging because it requires the identification of well-defined groups based on spatiogenetic differentiation and/or the evidence of local adaptation. In this study, we investigate genetic variation at 186 AFLP loci in 46 European accessions of the important grassland species Arrhenatherum elatius and ask (1) whether genetic variation within accessions differs between European geographical regions; (2) at which spatial scale populations are structured across Europe and (3) whether putatively adaptive markers contribute to this pattern and whether these markers can be related to climatic site conditions. Basic expectations of population genetics are likely to be altered in autotetraploid species, thus, we adopted a band-based approach to estimate genetic diversity and structuring. Compared to other grasses A. elatius showed high genetic diversity and considerable differentiation among accessions (Phi(ST) = 0.24). Accessions separated in a Western European and a Central/Eastern European group, without further structure within groups. A genome scan approach identified four potentially adaptive loci, whose band frequencies correlated significantly with climatic parameters, suggesting that genetic differentiation in A. elatius is also the result of adaptive processes. Knowledge on adaptive loci might in the long run also help to adapt ecosystems to adverse climate change effects through assisted migration of ecotypes rather than introduction of new species.

Genetic Relationships of Chinese Grape Accessions to European and American Cultivars Assessed by Microsatellite Markers

ABSTRACTA total of 45 grape accessions were used to investigate the genetic polymorphism and relationships among Chinese and other European or America grape accessions by microsatellite markers. Eighty-six alleles were detected in 9 simple sequence repeat (SSR) loci with an average of 9.6 alleles per locus. Genetic similarity ranged from 0.38 to 0.83 with an average value of 0.58. This indicated sufficient diversity among the accessions. Based on cluster analysis and principal coordinate analysis, the results showed that all accessions could be divided into three major groups and the clustering pattern was related to the classical ecogeographical grouping: occidentalis, pontica and orientalis. Those from the same ecological group could cluster preferentially in most cases into one group, suggesting the positive correlation between the cluster results and the geographical origin of the grape accessions. Wine grapes were significantly differentiated from table grapes no matter whether the accessions were from China or other countries.

The influence of European and American wild germplasm in hop (Humulus lupulus L.) cultivars

Microsatellite variation at the nuclear and chloroplast genomes was evaluated for wild European and wild American hops, in order to assess the genetic diversity and origin of cultivated hops. Seven nuclear loci and 32 chloroplast loci were used in the analysis of 182 hop accessions including wild European (68), wild American (48), and cultivars (66). A total of 116 alleles were identified using 7 nuclear microsatellites showing different averages of polymorphism and distribution in the wild American and European accessions and cultivars. Two main groups were established as revealed by several statistical analyses; one including European wild accessions and cultivars and a second group consisting of American wild accessions. Three polymorphic chloroplast microsatellite loci were detected, six alleles were scored which defined a total of five haplotypes that were exclusive or presented different distribution between American and European wild accessions. A major influence of the wild European haplotypes was detected among hop cultivars. To the best of our knowledge, this is the first work reporting the use of chloroplast microsatellites in hops.

Identification of a resistance gene Rpi-dlc1 to Phytophthora infestans in European accessions of Solanum dulcamara.

Initial screening of 14 Solanum dulcamara accessions enabled the identification of individuals resistant and susceptible to Phytophthora infestans. Crosses between contrasting genotypes resulted in three F2–BC1 populations segregating for resistance to late blight in a laboratory assay and under field conditions. Genetic profiling of one of these populations using 128 AFLP primers generated three markers linked to the resistant phenotype. Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers. Thus, we describe a first resistant gene, named Rpi-dlc1, from S. dulcamara, a Solanum species native to Europe. In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence. This indicated the possible presence of additional Rpi genes.

Diversity and the origin of the European population of Triticum dicoccum (Schrank) Schuebl. As revealed by chromosome analysis

Cluster analysis of the Triticum dicoccum chromosome passports by artificial neural networks and UPGMA divided the European T. dicoccum population into two groups, West European and Volga-Balkan. The West European T. dicoccum accessions displayed a predominance of the marker translocation T7A:5B (67% of the accessions), which was also found in a few accessions from other countries (Turkey, Iran, and northern Africa), and were similar in chromosome C-banding patterns. The Volga-Balkan T. dicoccum accessions differed in the C-banding patterns of some chromosomes from the West European accessions, thus probably originating from another founder population. It was assumed that the T. dicoccum accessions carrying the T7A:5B translocation had a common origin and that the wild T. dicoccum population of the Middle East (Syria and Lebanon) contributed to the origin of West European T. dicoccum.

Evaluation of phenotypic variation in a worldwide germplasm collection of safflower (Carthamus tinctorius L.) grown under organic farming conditions in Germany

An investigation was conducted to determine the extent of diversity and relationships among a worldwide safflower (Carthamus tinctorius L.) germplasm collection and to find out adapted accessions that can be used in an organic safflower breeding program in Germany. A total of 468 accessions was studied under organic farming conditions at Kleinhohenheim experimental station during the seasons of 2004 and 2005. All the accessions were evaluated for 12 phenotypic traits and three rated diseases. Multivariate analyses have been used to measure the diversity in a subset of 200 accessions and 11 geographical regions. Generally, the study showed that there was a large genetic variation within accessions. A coefficient of variation (CV%) for investigated traits and diseases ranged from 2.9 to 91.0% with the highest CV was recorded for yield/m², yield/plant and seeds/plant. The most accessions that originated in Europe revealed relatively better performance compared to non-Europeans. High yielding, early maturing, and disease tolerant accessions were identified. However, the low oil content (8.7–22.8%) is the primary concern in this germplasm collection. The degree of heritability varied between 10% for lodging to 86% for plant height. Genotypic coefficient of correlation (r g) was slightly higher for many traits than the respective phenotypic coefficient. Oil content and seed yield/m² were highly significantly correlated (r g = 0.78). The genotypic coefficient of correlation showed that selection for seeds/plant and thousand kernel weight was effective for improvement of seed yield and oil content. The results of the principal component analysis and the clustering pattern of accessions were consistent with the results of analysis of variance. About 78% of the total phenotypic diversity in the germplasm was explained on the basis of four principal components and 88% of the total variation among geographical regions was contributed by the first three principal components. The distribution of the accessions within clusters has no apparent relationship with the geographical origin. However, many of the European accessions have a tendency to stay together.

Global chloroplast phylogeny and biogeography of bracken (Pteridium; Dennstaedtiaceae)

Bracken ferns (genus Pteridium) represent an ancient species complex with a natural worldwide distribution. Pteridium has historically been treated as comprising a single species, but recent treatments have recognized several related species. Phenotypic plasticity, geographically structured morphological variation, and geographically biased sampling have all contributed to taxonomic confusion in the genus. We sampled bracken specimens worldwide and used variable regions of the chloroplast genome to investigate phylogeography and reticulate evolution within the genus. Our results distinguish two major clades within Pteridium, a primarily northern hemisphere Laurasian/African clade, which includes all taxa currently assigned to P. aquilinum, and a primarily southern hemisphere Austral/South American clade, which includes P. esculentum and P. arachnoideum. All European accessions of P. aquilinum subsp. aquilinum appear in a monophyletic group and are nested within a clade containing the African P. aquilinum taxa (P. aquilinum subsp. capense and P. aquilinum subsp. centrali-africanum). Our results allow us to hypothesize the maternal progenitors of two allotetraploid bracken species, P. caudatum and P. semihastatum. We also discuss the biogeography of bracken in the context of the chloroplast phylogeny. Our study is one of the first to take a worldwide perspective in addressing variation in a broadly distributed species complex.

Haplotypes of Fallopia introduced into the US

In the US, clonal growth of Fallopia japonica, Fallopia sachalinensis and their hybrid Fallopia x bohemica (Polygonaceae) is prominent, yet sexual reproduction and hybridization contribute to the genetic complexity of swarms. The contribution to this diversity from multiple introductions is unknown. Using 800 bp of the non-coding chloroplast marker accD–rbcL, we compared 21 Japanese haplotypes with 46 US samples from 11 states, 2 Canadian samples, and 6 European samples from 4 countries, in order to investigate if there were repeated introductions from Asia. While most North American and all European haplotypes accessions in our collection matched a single widespread haplotype, we identified 8 other haplotypes. Three haplotypes of F. japonica (including the widespread haplotype) and one F. sachalinensis matched previously identified Japanese haplotypes, supporting the hypothesis of multiple introductions in the US. Five additional US haplotypes were detected once. Four of these differed from Japanese haplotypes by one single nucleotide polymorphism (SNP), possibly indicating a recent in situ change. The fifth haplotype represents a garden cultivar, which differed from all F. japonica haplotypes. It therefore appears that the US genetic diversity of these taxa has three sources: intra-specific reproduction, inter-specific reproduction, and multiple sources of introduction.