Temporal changes in genetic diversity of common bean (Phaseolus vulgaris L.) accessions cultivated between 1800 and 2000.

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 differentiation of geographically separated populations of the southern green stink bug Nezara viridula (Hemiptera: Pentatomidae).

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.

Genetic introgression between wild and stocked salmonids and the prospects for using molecular markers in population rehabilitation: the case of the Adriatic grayling (Thymallus thymallus L. 1785).

In the north Adriatic basin, a morphologically and genetically distinct lineage of grayling is found, designated as the Adriatic grayling. In Slovenia, the Adriatic grayling is restricted to the Soca river system, where it is critically endangered. The most pertinent threat is stocking with non-native, highly divergent Sava (Danubian) drainage stock, and this activity has been going on for more than four decades. The present study was designed to characterise the genetic structure of the Adriatic grayling in Slovenia, with particular emphasis on estimating the degree of introgression with non-indigenous stocked grayling. We analysed polymorphism at 154 microsatellite loci in samples representing grayling from the Adriatic and Danubian drainage stock. A relatively high number (12) of alleles, diagnostic for the Adriatic grayling, were identified. However, a correspondence analysis based on individual multilocus genotypes also revealed that there is no distinctive Adriatic group but rather a dispersed multitude of individuals that cannot be unambiguously distinguished from the more homogenous Danubian population. A Bayesian analysis of individual admixture coefficients confirmed this pattern and revealed extensive introgression between the Adriatic grayling and stocked grayling of Danubian origin. Average individual admixture coefficients showed that only between 50 and 60% of the original gene pools remained, and only few non-introgressed indigenous individuals could be identified. Microsatellite-based individual admixture analysis appear to be an important tool for identifying remaining non-introgressed indigenous individuals that could be used for restoring the original populations.

Allelic differences in bovine kappa-CN gene which may regulate gene expression.

The most common kappa casein (kappa-CN) variants, kappa-CN A and kappa-CN B, are synthesised differentially in the lactating mammary gland of heterozygous animals (kappa-CN AB). In this study we evaluated several approaches for quantification of allele specific mRNA transcripts. The most consistent results were obtained using allele specific RT-PCR and capillary electrophoresis. On average, 13.4% more allele B specific than A specific transcripts were found. DNA sequencing of the proximal promoter region in several homozygous animals (kappa-CN AA, BB, EE) did not reveal any allele specific polymorphisms. Using the EMSA and DNase I footprinting we confirmed functional binding sites for three transcription factors (AP-2, NF1 and MGF) within the kappa-CN proximal promoter region. Sequence analysis of the 3'-UTR of the kappa-CN gene revealed seven allele specific sites. Two of these allelic differences were close to previously identified 3'-end regulatory sequences. In addition, allele specific differences in length between mRNAs of both variants were found. The two later findings suggest a possible post translational control determining content differences of kappa-CN in milk.

Allelic differences in bovine kappa - CN gene which may regulate gene expression.

The most common kappa casein (κ-CN) variants, κ-CN A and κ-CN B, are synthesised differentially in the lactating mammary gland of heterozygous animals (κ-CN AB). In this study we evaluated several approaches for quantification of allele specific mRNA transcripts. The most consistent results were obtained using allele specific RT-PCR and capillary electrophoresis. On average, 13.4% more allele B specific than A specific transcripts were found. DNA sequencing of the proximal promoter region in several homozygous animals (κ-CN AA, BB, EE) did not reveal any allele specific polymorphisms. Using the EMSA and DNase I footprinting we confirmed functional binding sites for three transcription factors (AP-2, NF1 and MGF) within the κ-CN proximal promoter region. Sequence analysis of the 3'-UTR of the κ-CN gene revealed seven allele specific sites. Two of these allelic differences were close to previously identified 3'-end regulatory sequences. In addition, allele specific differences in length between mRNAs of both variants were found. The two later findings suggest a possible post translational control determining content differences of κ-CN in milk.