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Information on genetic diversity and germplasm characterization is essential for successful crop improvement. Diverse data sets (pedigree, morphological, biochemical, DNA based-markers) are employed in various aspects of plant analysis. The objective of this study was to determine the efficiency of phenotypic and RAPD markers in diversity assessment often alfalfa (Medicago spp.) accessions from Europe, North America and Australia. Field experiment was designed as a randomised complete block with three replications over two consecutive years (2004, 2005) at one location. Twelve morpho-agronomic traits were recorded on 50 plants per each accession. Genomic DNA's from 16-20 randomly selected individual plants per accession were used for RAPD analysis. Six primers selected in this study generated a total of 93 polymorphic RAPD bands. The number of polymorphic bands detected per primer ranged from 11 to 20. Genetic distances (GD) among investigated accessions and two-dimensional principal coordinate analysis (2D PCoA) based on phenotypic and molecular data were obtained. The average GD between (0.283-0.416) and within (0.247-0.332) accessions based on RAPD data was higher than GD values obtained by morpho-agronomic traits (0.171-0.354 and 0.157-0.261, respectively). 2D PCoA based on GD from RAPD data grouped most of the studied individual plants to four clusters according to their geographical or taxonomy origin. 2D PCoA based only on morpho-agronomic data did not group plants congruently to their origin, probably due to a strong environmental influence on studied traits. Our results indicated that the RAPD markers were effective in assessing genetic diversity within and between studied alfalfa accessions. In addition, the obtained results suggested that the RAPD markers might be useful for grouping of germplasm with similar genetic background and for prescreening of potential heterotic groups in our breeding programme.

In order to comply with the European Union regulatory threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, it is important to trace GMOs from the field. Appropriate sampling methods are needed to accurately predict the presence of GMOs at the field level. A 2-year field experiment with two maize varieties differing in kernel colour was conducted in Slovenia. Based on the results of data mining analyses and modelling, it was concluded that spatial relations between the donor and receptor field were the most important factors influencing the distribution of outcrossing rate (OCR) in the field. The approach for estimation fitting function parameters in the receptor (non-GM) field at two distances from the donor (GM) field (10 and 25 m) for estimation of the OCR (GMO content) in the whole receptor field was developed. Different sampling schemes were tested; a systematic random scheme in rows was proposed to be applied for sampling at the two distances for the estimation of fitting function parameters for determination of OCR. The sampling approach had already been validated with some other OCR data and was practically applied in the 2009 harvest in Poland. The developed approach can be used for determination of the GMO presence at the field level and for making appropriate labelling decisions. The importance of this approach lies in its possibility to also address other threshold levels beside the currently prescribed labelling threshold of 0.9% for food and feed.

A zymography technique based on native polyacrylamide gel electrophoresis (PAGE) has been devised, which enables the substrate specificities, content and pH profiles of proteolytic enzymes to be determined in an unfractionated tissue extract. Enzymes were visualized by exogenous application of small molecule substrates that fluoresce when hydrolyzed. The linearity of response, treatment of background fluorescence, and effects of diffusion of substrate and enzyme were taken into account. Based on these studies, successive application of different substrates on the same gel has enabled the presence and specificity of individual enzymes to be determined. Differences in the concentrations and profiles of enzymes, resulting from environmental factors or ontogeny of the organism, can be assessed from crude extracts on a single gel. The technique was applied to aminopeptidases and peptidases in crude Phaseolus vulgaris leaf extracts. One enzyme active against Ala-AMC (7-amino-4-methylcoumarin), one enzyme active against Z-Arg-AMC, several enzymes active against Leu-AMC, and (for the first time in plants) several enzymes active against Phe-AMC were identified. The technique is very sensitive, and microgram quantities of total protein led to picomoles of liberated AMC, with a linear response over a 32-fold range of concentration. The experimental procedure, including electrophoresis, is rapid, taking approximately 1 h.

Hbr markers are based on location presence/absence of the Heartbreaker family of miniature inverted repeat transposable elements (MITEs). Together with the cost-effective technique--Hbr display were developed in 2000. We chose 15 populations from the Slovenian maize germplasm bank and described ten individual samples per each population by 268 Hbr markers and 35 morphological traits (IPGRI descriptors). Samples from the same population had highly similar DNA fingerprints, while the between populations differences were very high. Therefore, only a minor part of the total genetic variance existed within populations (23.3%), and the major part among populations (76.7%). Beli zob (the only dent type population) and stajerski dvanajsterec were the most divergent populations, others were closely related. They shared the majority of bands in the way that each band was shared by different set of populations. This is suggesting the origin from the common gene pool and the high extent of migrations.

Fourteen microsatellite markers were used to describe genetic diversity in a sample of 128 common bean (Phaseolus vulgaris L.) accessions cultivated within the territory of Slovenia and its nearby regions between 1800 and 2000. The accessions were grouped into three periods, Period I comprising accessions from the beginning of the 19th century, while the other two periods included accessions from the middle (Period II) and the end of the 20th century (Period III). Seven control accessions of known Mesoamerican and Andean origin were also included in the study. A total of 130 alleles were generated. Allelic richness, in terms of number of alleles per locus, was 6.07 for Period I, 6.71 for Period II and 6.07 for Period III. In the UPGMA dendrogram, all studied accessions were intermixed in three main clusters, indicating that the diversity in the time periods overlapped. Two clusters consisted of accessions of Andean and Mesoamerican origin, while the third represents additional variation, which existed in this area already 200 years ago. The analysis of molecular variance showed that a great part of genetic diversity has been preserved till today, confirming the results of cluster analysis. The calculation of number of alleles per locus revealed no significant quantitative change in genetic diversity over the last 200 years of common bean cultivation. However, the calculation of genetic distances indicated slight qualitative shifts in genetic diversity of common bean germplasm over time, while the calculations of allelic frequency variation and polymorphic information content revealed recent decline of some alleles' frequencies. These findings should stress the need for establishing an appropriate strategy of genetic resources management.

Genetic variation in the southern green stink bug Nezara viridula (Linnaeus) from 11 geographically separated sampling locations (Slovenia, France, Greece, Italy, Madeira, Japan, Guadeloupe, Galapagos, California, Brazil and Botswana) was studied by sequencing 16S and 28S rDNA, cytochrome b and cytochrome c oxidase subunit I gene fragments and random amplified polymorphic DNA (RAPD) analysis. Sequencing revealed 11 distinct haplotypes clustering into lineages A, B and C. Lineage C was characteristic for a single analysed specimen from Botswana. Lineage B was detected in Japan, and it probably arose in Asia. Haplotypes of European and American specimens belonged to lineage A; specimens from France, Slovenia, Madeira and Brazil shared highly similar haplotypes (>99%) from subgroup A1, while all the specimens from Greece, California, Galapagos and Guadeloupe shared a haplotype from subgroup A2. RAPD data were more variable but consistent with mtDNA sequences, revealing the same clustering. They separated the Botswanian specimen from Japanese specimens and from a group of more closely related specimens from Europe and America. Sequence and RAPD results both support the African origin of N. viridula, followed by dispersal to Asia (lineage B) and, more recently, by expansion to Europe and America (lineage A). RAPD analysis revealed two highly supported subgroups in Japan, congruent with mtDNA lineages A2 and B, suggesting multiple colonization of Japan. Invariant sequences at the 28S rDNA combined with other results do not support the hypothesis that cryptic (sibling) species exist within the populations investigated in this study.

A set of introgression lines (ILs), containing individual chromosome segments from the wild nightshade Solanum lycopersicoides bred into the genetic background of cultivated tomato (Lycopersicon esculentum), has been developed. A primary group of 56 lines was selected for maximum representation of the S. lycopersicoides genome (approximately 96% of the total map units), homozygosity, and a minimum number of introgressed segments per line. A secondary set of 34 lines provides increased map resolution in certain regions. Approximately 34% of the lines were sterile in the homozygous condition, but could be maintained by heterozygotes. To facilitate identification of segregating ILs, restriction fragment length polymorphism probes were converted to higher throughput cleaved amplified polymorphic sequence markers, which supplement allozyme and morphological loci. Strong segregation distortion was observed in F2 progeny of heterozygous ILs, with an excess of L. esculentum alleles in most regions. For introgressions on distal chromosome 1L, a preferential transmission of S. lycopersicoides alleles was observed in the male germ line. Homozygous ILs generally yielded less seed from self pollination than corresponding heterozygotes, indicating that sterility effects were recessive. This IL library provides a novel resource for genetic studies of traits found in S. lycopersicoides.

F(1) hybrids between the cultivated tomato (Lycopersicon esculentum) and the wild nightshade Solanum lycopersicoides are male sterile and unilaterally incompatible, breeding barriers that impede further crosses to tomato. Meiosis is disrupted in 2x hybrids, with reduced chiasma formation and frequent univalents, but is normal in allotetraploid hybrids, indicating the genomes are homeologous. In this study, a partially male-fertile F(1) was backcrossed to tomato, producing the first BC(1) population suitable for genetic mapping from this cross. BC(1) plants were genotyped at marker loci to study the transmission of wild alleles and to measure rates of homeologous recombination. The pattern of segregation distortion, in favor of homozygotes on chromosomes 2 and 5 and heterozygotes on chromosomes 6 and 9, suggested linkage to a small number of loci under selection on each chromosome. Genome ratios nonetheless fit Mendelian expectations. Resulting genetic maps were essentially colinear with existing tomato maps but showed an overall reduction in recombination of approximately 27%. Recombination suppression was observed for all chromosomes except 9 and 12, affected both proximal and distal regions, and was most severe on chromosome 10 (70% reduction). Recombination between markers on the long arm of this chromosome was completely eliminated, suggesting a lack of colinearity between S. lycopersicoides and L. esculentum homeologues in this region. Results are discussed with respect to phylogenetic relationships between the species and their potential use for studies of homeologous pairing and recombination in a diploid plant genome.