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DNA markers are currently the most precise tool for forest tree species identification and can be used for comparative analyses of plant material. Molecular diagnosis of evidence and reference material is based on comparing the structure of DNA markers duplicated in the PCR reaction and estimation of the DNA profiles obtained in studied wood samples. For this purpose, the microsatellite DNA markers are the most suitable tool because of their high polymorphism and accurate detection of structural changes in the genome. The analysis of tree stump DNA profiles let avoid timely collection of data such as tree age, diameter, height and thickness, although such a piece of information may advantageous in wood identification process. For each examined tree species, i.e. Pinus sylvestris L., Picea abies (L.) Karst., Quercus robur L. and Q. petraea (Matt.) Liebl., Fagus sylvatica L., Betula pendula L., and Alnus glutinosa L., wood identification was possible via the DNA profiles established on a basis of minimum 4 microsatellite nuclear DNA loci, and at least one cytoplasmatic (mitochondrial or chloroplast) DNA marker. Determination of the DNA profiles provided fast and reliable comparison of genetic similarity between material of evidence (wood, needles, leaves, seeds) and material of reference (tree stumps) in the forest. This was done with high probability (approximately 98– 99%).
A survey of fully-sequenced chloroplast genomes revealed that in land plants there are six tRNA genes that have introns. Moreover, the length of a particular tRNA gene intron remains relatively stable across species. However, in algae, the presence of chloroplast tRNA genes containing introns is exceptional. A survey of mitochondrial plant genomes revealed intron-containing tRNA genes are rather rare features, with the exception of tRNASerGCU genes in liverworts and peat-mosses. We isolated and sequenced one mitochondrial and three chloroplast intron-containing tRNA genes and a fragment of the mitochondrial coxIII gene containing the first intron from the following liverwort species: Pellia borealis, Pellia epiphylla-species N, Pellia epiphylla-species S and Porella baueri, Porella cordaeana, Porella platyphylla. We showed that, as in the case of higher plants, the rate of nucleotide substitution is lower in the mitochondrial genome than in the chloroplast genome. Moreover, the comparison of intron nucleotide sequences enabled us to show that in the case of one allopolyploid species, Pellia borealis, organelles were transmitted from one parent species, Pellia epiphylla-species N. In the case of another allopolyploid species, Porella baueri, organelles were also inherited from one parent species, Porella cordaeana. Therefore, organellar inheritance in liverworts seems to be uniparental. It remains clear that analysis of carefully chosen chloroplast and mitochondrial DNA sequences allowed us to reconstruct historical events.
Four cpDNA regions were analyzed with the use of PCR-RFLP technique and nucleotide sequences of two mtDNA regions were characterized in order to find P. sylvestris and P. mugo species specific markers useful for studies of the species hybridization. The difference in the restriction fragment patterns of trnV-rbcL region after digestion with MvaI endonuclease was detected. The analyses of the species representatives from various geographic regions revealed that the observed polymorphism is species specific. No differences have been disclosed in the analyzed trnS-trnT, trnK1-trnK2, trnC-trnD cpDNA regions. The P. sylvestris and P.mugo mtDNA sequences of orf25 and coxI regions proved to be identical.
Genomic sequence AY661558, representing a part of the ВАС contig of the Rym4/Rym5 locus conferring resistance to the barley yellow mosaic virus complex (BaMMV/BaYMV), was exploited in order to develop SSR markers for practical barley breeding. Out of 57 SSR motifs found within this sequence, primers were designed and tested for the 5 SSRs with the highest repeat length. The polymorphic SSR marker QLB1 со-segregated with rym4 and rym5 phenotypes in respective high-resolution mapping populations developed for the construction of the original ВАС contig. The primers targeted 2 sites located 756 bp and 5173 bp downstream of the translation initiation factor 4E (Hv-eIF4E). Physical linkage of the QLB1 marker to the Rym4/Rym5 locus was confirmed experimentally on Morex ВАС 519J14, a seed ВAC of Hv-eIF4E, and ВAC 801A11, which is located proximally to Hv-eIF4E. QLB1 revealed 7 alleles in a set of 100 winter barley lines and cultivars. Five alleles were found within 673 advanced breeding lines derived from applied Polish winter barley breeding programmes, which corresponds to a PIC value of 0.684. No recombinants between Rym4/5 and QLB1 were detected, suggesting that QLB1 can be used efficiently in marker-assisted selection of the Hv-eIF4E-mediated bymovirus resistance.
The trait flowering time regulated by genes determining alisation and photoperiod sensitivity was used as an example for presenting data on comparative major gene and QTL mapping within the Triticeae. The major genes are shown to be members of homoeologous series. Furthermore it was demonstrated that in genome regions carrying major genes also QTLs for the same traits were detected.
Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici is a destructive disease of tomato crops worldwide. The use of resistant varieties is the best strategy for disease control. In the present study we analyze eight tomato lines and hybrids for Fusarium wilt disease resistance by polymerase chain reaction. Total genomic DNA was extracted from young leaves of three-week-old plants of tomato. Results of PCR of eight tomato lines and hybrids indicated that there are one dominant heterozygote, two recessive homozygotes and five dominant homozygotes. Also, results of polymerase chain reaction showed that it needs less time and is cheaper. Also by using this method, it is possible to determine genotype of plant (homozygote or heterozygote) without presence of the pathogen. Therefore, PCR technique was used in the identification gene I2 conferring resistance to F. oxysporum f. sp. lycopersici.
This work assessed genetic diversity and genetic structure using random amplified polymorphic DNA (RAPD) variation in 120 individuals of four natural populations of Taxus baccata growing in southern Poland (3 in mountains and one in lowland) to obtain basic information on this natural resource. With 9 primers, 185 highly reproducible and clear RAPD bands were obtained. Genetic diversity within populations was relatively high, with percentages of polymorphic bands ranging from 48.65% to 77.30%, averaging 69.59% (Shannon index 0.311). Global AMOVA showed that genetic variation between populations accounted for 26% of total variation, with the remainder (74%) occurring within population. Pairwise ΦPT values were not correlated with geographic distance. Two groups of populations were distinguished by ANOVA and principal coordinate analysis (PCO) based on a Euclidean metric: those growing in mountains (Nowa Wieś, Cisowa Gora, Zadni Gaj), with higher internal diversity, and those growing in lowlands (Liswarta), with lower internal diversity. The results are typical for an outcrossing, wind-pollinated and long-lived woody species
The obligatory human pathogen, Mycobacterium tuberculosis, is the most important etiological factor of tuberculosis. Unfortunately, there is little information about genetic diversity of this pathogen. The main aim of this research was the estimation of genetic diversity of M. tuberculosis on the basis of various categories of DNA markers. The genome of 32 strains were scanned by DNA markers such RAPD, IS6110 and catalase-peroxidase katG gene. All 162 identified loci were polymorphic. The genetic diversity coefficient (HT) of M. tuberculosis was 0.32 for RAPD and 0.27 for IS6110. There were 14 alleles in katG gene. All strains were characterised by the individual molecular pattern. Genetic similarity varied from 0.13 to 0.94 (RAPD markers) and from 0 to 1 for (IS6110). M. tuberculosis strains did not represent a clonal structure, single source of transmission and epidemiological relationships as well. The applied DNA markers proved to be highly efficient for analysis of genetic structure of M. tuberculosis.
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