Genetic variability analysis of Russian cultivars of ryegrass (Lolium) based on SCoT markers.

BACKGROUND: Ryegrass is a promising crop for the development of meadow farming in the world. More than 1000 cultivated varieties widely used in feed production have been developed, based on the main species - perennial ryegrass (Lolium perenne L.) and annual one (Lolium multiflorum Lam.). Development and implementation of the modern methods of plant varietal and species identification are of great importance. In recent years, molecular markers have been successfully used for these purposes, which increase the accuracy of the breeding material evaluation at a significant reduction of time and labor costs. The aim of this study was to assess the discriminatory potential of the new SCoT marking technique for the identification of Russian perennial (Lolium perenne L.) and annual (Lolium multiflorum Lam.) ryegrass varieties. RESULTS: Out of the total number of the tested SCoT-primers, 8 polymorphic ones were selected, which demonstrates the high stability and reproducibly amplified DNA fragments. These primers generated 107 PCR products, where 37 were found to be polymorphic. The average number of amplicons per primer was 13. The size of the PCR products varied from 349 to 2718 bp (see Table 3). The polymorphic ratio of the tested markers was 30.8%. The marker SCoT-06 was characterized by the maximum number of PCR products and the highest level of polymorphism (50%). The effective number of alleles (ne) ranged from 1.35 to 1.58 with a mean of 1.48 per locus. The average value of the PIC and Shannon index (I) were 0.35 and 0.46, respectively. The unique PCR fragments were revealed for the identification of tested varieties. Analysis of molecular variance (AMOVA) showed that the level of genetic diversity between ryegrass species (59%) was more than between varieties within a species (41%). Based on binary matrix data, clustering and PCoA analysis (see Figs. 1 and 2) of the samples were carried out that divided them into two groups according to species. CONCLUSIONS: We found a set of markers that can be useful tools for ryegrass varieties identification. The level of intravarietal polymorphism turned out to be higher than the differences between varieties because of the possible significant heterogeneity of the varietal material. The information obtained can be used in breeding programs to create improved ryegrass varieties adapted to Russian climatic conditions.

Mapping candidate QTLs related to plant persistency in red clover.

Red clover (Trifolium pratense L.) is a diploid (2n = 14), self-incompatible legume that is widely cultivated as a forage legume in cold geographical regions. Because it is a short-lived perennial species, improvement of plant persistency is the most important objective for red clover breeding. To develop a marker-assisted selection (MAS) approach for red clover, we identified candidate QTLs related to plant persistency. Two full-sib mapping populations, 272 x WF1680 and HR x R130, were used for QTL identification. Resistance to Sclerotinia trifoliorum and Fusarium species, as well as to winter hardiness, was investigated in the laboratory and in field experiments in Moscow region (Russia), and Sapporo (Japan). With the genotype data derived from microsatellite and other DNA markers, candidate QTLs were identified by simple interval mapping (SIM), Kruskal-Wallis analysis (KW analysis) and genotype matrix mapping (GMM). A total of 10 and 23 candidate QTL regions for plant persistency were identified in the 272 x WF1680 and the HR x R130 mapping populations, respectively. The QTLs identified by multiple mapping approaches were mapped on linkage group (LG) 3 and LG6. The significant QTL interactions identified by GMM explained the higher phenotypic variation than single effect QTLs. Identification of haplotypes having positive effect QTLs in each parent were first demonstrated in this study for pseudo-testcross mapping populations in plant species using experimental data.

Construction of a consensus linkage map for red clover (Trifolium pratense L.).

BACKGROUND: Red clover (Trifolium pratense L.) is a major forage legume that has a strong self-incompatibility system and exhibits high genetic diversity within populations. For several crop species, integrated consensus linkage maps that combine information from multiple mapping populations have been developed. For red clover, three genetic linkage maps have been published, but the information in these existing maps has not been integrated. RESULTS: A consensus linkage map was constructed using six mapping populations originating from eight parental accessions. Three of the six mapping populations were established for this study. The integrated red clover map was composed of 1804 loci, including 1414 microsatellite loci, 181 amplified fragment length polymorphism (AFLP) loci and 204 restriction fragment length polymorphism (RFLP) loci, in seven linkage groups. The average distance between loci and the total length of the consensus map were 0.46 cM and 836.6 cM, respectively. The locus order on the consensus map correlated highly with that of accession-specific maps. Segregation distortion was observed across linkage groups. We investigated genome-wide allele frequency in 1144 red clover individuals using 462 microsatellite loci randomly chosen from the consensus map. The average number of alleles and polymorphism information content (PIC) were 9.17 and 0.69, respectively. CONCLUSION: A consensus genetic linkage map for red clover was constructed for the first time based on six mapping populations. The locus order on the consensus map was highly conserved among linkage maps and was sufficiently reliable for use as a reference for genetic analysis of random red clover germplasms.

Comprehensive structural analysis of the genome of red clover (Trifolium pratense L.).

With the aim of establishing the basic knowledge and resources needed for applied genetics, we investigated the genome structure of red clover Trifolium pratense L. by a combination of cytological, genomic and genetic approaches. The deduced genome size was approximately 440 Mb, as estimated by measuring the nuclear DNA content by flow cytometry. Seven chromosomes could be distinguished by microscopic observation of DAPI stained prometaphase chromosomes and fluorescence in situ hybridization using 28S and 5S rDNA probes and bacterial artificial chromosome probes containing microsatellite markers with known positions on a genetic linkage map. The average GC content of the genomes of chloroplast, mitochondrion and nucleus were shown to be 33.8, 42.9 and 34.2%, respectively, by the analysis of 1.4 Mb of random genomic sequences. A total of 26,356 expressed sequence tags (ESTs) that were grouped into 9339 non-redundant sequences were collected, and 78% of the ESTs showed sequence similarity to registered genes, mainly of Arabidopsis thaliana and rice. To facilitate basic and applied genetics in red clover, we generated a high-density genetic linkage map with gene-associated microsatellite markers. A total of 7159 primer pairs were designed to amplify simple sequence repeats (SSRs) identified in four different types of libraries. Based on sequence similarity, 82% of the SSRs were likely to be associated with genes. Polymorphism was examined using two parent plants, HR and R130, and 10 F(1) progeny by agarose gel electrophoresis, followed by genotyping for the primer pairs showing polymorphisms using 188 F(1) plants from the mapping population. The selected 1305 microsatellite markers as well as the previously developed 167 restriction fragment length polymorphism markers were subjected to linkage analysis. A total of 1434 loci detected by 1399 markers were successfully mapped onto seven linkage groups totaling 868.7 cM in length; 405 loci (28%) were bi-parental, 611 (43%) were specific to HR and 418 (29%) were specific to R130. Each genetic linkage group was linked to a corresponding chromosome by FISH analysis using seven microsatellite markers specific to each of the linkage groups as probes. Transferability of the developed microsatellite markers to other germplasms was confirmed by testing 268 selected markers on 88 red clover germplasms. Macrosynteny at the segmental level was observed between the genomes of red clover and two model legumes, Lotus japonicus and Medicago truncatula, strongly suggesting that the genome information for the model legumes is transferable to red clover for genetic investigations and experimental breeding.