Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70-100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.

• MeSH
• Alleles MeSH
• Bayes Theorem MeSH
• Breeding MeSH
• DNA, Plant isolation & purification   MeSH
• Phylogeny MeSH
• Genetic Variation * MeSH
• Genetic Markers * MeSH
• Genome, Plant MeSH
• Genotype MeSH
• Flax genetics   MeSH
• Molecular Sequence Data MeSH
• Multigene Family MeSH
• Polymorphism, Genetic MeSH
• Retroelements * MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Genetic Markers * MeSH
• Retroelements * MeSH

One hundred and sixty-four accessions representing Czech and Slovak pea (Pisum sativum L.) varieties bred over the last 50 years were evaluated for genetic diversity using morphological, simple sequence repeat (SSR) and retrotransposon-based insertion polymorphism (RBIP) markers. Polymorphic information content (PIC) values of 10 SSR loci and 31 RBIP markers were on average high at 0.89 and 0.73, respectively. The silhouette method after the Ward clustering produced the most probable cluster estimate, identifying nine clusters from molecular data and five to seven clusters from morphological characters. Principal component analysis of nine qualitative and eight quantitative morphological parameters explain over 90 and 93% of total variability, respectively, in the first three axes. Multidimensional scaling of molecular data revealed a continuous structure for the set. To enable integration and evaluation of all data types, a Bayesian method for clustering was applied. Three clusters identified using morphology data, with clear separation of fodder, dry seed and afila types, were resolved by DNA data into 17, 12 and five sub-clusters, respectively. A core collection of 34 samples was derived from the complete collection by BAPS Bayesian analysis. Values for average gene diversity and allelic richness for molecular marker loci and diversity indexes of phenotypic data were found to be similar between the two collections, showing that this is a useful approach for representative core selection.

• MeSH
• Alleles MeSH
• Principal Component Analysis MeSH
• Bayes Theorem MeSH
• Gene Frequency MeSH
• Genetic Variation * MeSH
• Genetic Markers genetics   MeSH
• Pisum sativum genetics   MeSH
• Microsatellite Repeats genetics   MeSH
• Minisatellite Repeats genetics   MeSH
• Population Dynamics MeSH
• Retroelements genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Genetic Markers MeSH
• Retroelements MeSH

The distinctness, uniformity and stability (DUS) requirements involve expensive, space- and time-consuming measurements of morphological traits. Moreover, for a majority of traits, interactions between genotype and environment complicate the evaluation. Molecular markers have a potential to facilitate this procedure, increase the reliability of decisions, and substantially save the time and space needed for experiments. We chose 25 varieties of pea (Pisum sativum L.) from the list of recommended varieties for cultivation in the Czech Republic, and made both a standard classification by 12 morphological descriptors and a classification by biochemical-molecular markers. Two isozyme systems, 10 microsatellite loci, 2 retrotransposons for multilocus inter-retrotransposon amplified polymorphism (IRAP), and 12 retrotransposon-based insertion polymorphism (RBIP) DNA markers were analysed. The main objective of the study was to examine the potential of each method for discrimination between pea varieties. The results demonstrate a high potential and resolving power of DNA-based methods. Superior in terms of high information content and discrimination power were SSR markers, owing to high allelic variation, which was the only biochemical-molecular method allowing clear identification of all varieties. Retrotransposon markers in RBIP format proved to be the most robust and easy to score method, while multilocus IRAP produced informative fingerprint already in a single analysis. Isozyme analysis offered a fast and less expensive alternative. The results showed that molecular identification could be used to assess distinctness and complement morphological assessment, especially in cases where the time frame plays an important role. Currently developed pea marker systems might serve also for germplasm management and genetic diversity studies.

• MeSH
• Biomarkers * MeSH
• DNA, Plant genetics   MeSH
• Species Specificity MeSH
• Pisum sativum genetics   metabolism   MeSH
• Microsatellite Repeats genetics   MeSH
• Polymorphism, Genetic MeSH
• Retroelements MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers * MeSH
• DNA, Plant MeSH
• Retroelements MeSH

In vitro clonal propagation of plants should generate identical copies of the selected genotype. However, associated stress might result in a breakdown of control mechanisms and consequent instability of the genome. We have used several molecular methods to assess the genetic stability of long-term propagated (24 years) multiple shoot in vitro culture of pea (Pisum sativum L.). We focused on assessing the stability of repetitive sequences, such as simple sequence repeats (SSR) and retrotransposons, both comprising a large part of genome. No differences were found when seedlings (Co-2004) or original seed (Co-1982) controls and long-term or newly established in vitro (one subculture cycle) samples were investigated by the SSR, inter-repeats (ISSR) or inter-retrotransposon amplified polymorphism (IRAP) method. However, the more global amplified fragment length polymorphism (AFLP) and particularly the methylation sensitive MSAP methods detected 11 and 18% polymorphism among samples, respectively. Interestingly, investigation of the global cytosine methylation status by HPCE measurement revealed no statistically significant differences. Some evidence of retrotransposon re-arrangement was observed by sequence-specific amplification polymorphism. This occurred mostly in the abundant Ty3-gypsy type Cyclop element and to a smaller extent in the Ogre element. Alternatively, no polymorphism was detected among the PDR-1 element of the Ty1-copia type retrotransposon. Based on these results, multiple shoot culture of pea maintained over a long period may be considered as a true to type multiplication method of the original genotype.

• MeSH
• Epigenesis, Genetic * MeSH
• Genetic Markers MeSH
• Pisum sativum MeSH
• Evolution, Molecular MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• Retroelements genetics   MeSH
• Tissue Culture Techniques MeSH
• Plant Shoots cytology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Genetic Markers MeSH
• Retroelements MeSH