Ya. V. Pirko

Generation of transgenic barley lines producing human lactoferrin using mutant alpha-tubulin gene as the selective marker

The biolistic transformation method was used for genetic improvement of three commercial cultivars of barley (Oksamytoviy, Vodogray, and Hetman). The plasmid pHLFTuBA containing target gene hLF encoding human lactoferrin under the control of the rice glutein B-1 promoter GluB-1 was used for transformation. The gene encoding mutant alfa-tubulin conferring resistance to trifluralin (dinitroaniline herbicide) was used as the selective marker. The screening of different trifluralin concentrations ranging from 0.1–30 μM was used for determination of selective concentration of the agent. Two transgenic barley lines of cultivars Oksamytoviy and Hetman’s callus line were selected after 2–3 months of cultivation on 10 μM of trifluralin. To confirm stable integration of the transformed gene, the PCR analysis of leafs from regenerated plant after their adaptation on the ground was carried out. The 734 bp fragment of the target gene was amplified from both regenerated plants.

Plant genetic transformation using carbon nanotubes for DNA delivery

The possibility of using nanocarriers based on carbon nanotubes (CNTs) to deliver genetic material into mesophyll protoplasts, callus cells, and leaf explants is discussed. Using single-walled CNTs (SWCNTs) at the concentration of 20 μg/mL and multiwalled CNTs (MWCNTs) at the concentration of 15 μg/mL, the Nicotiana tabacum L. protoplasts were genetically transformed with the plasmid construct pGreen 0029, and a transient expression of the yfp reporter gene was shown in the protoplasts. Using SWCNTs at the concentration of 40 μg/mL and MWCNTs at the concentration of 30 μg/mL, the N. tabacum callus and leaf explants were genetically transformed by the nptII gene contained in the pGreen 0029 construct and regenerant plants were obtained on a selective medium with kanamycin at the concentration of 50 mg/L. The SWCNTs-based nanocarriers demonstrated their applicability for the transformation of protoplasts and walled plant cells. At the same time, the MWCNTs-based nanocarriers demonstrated their applicability only for the transformation of protoplasts, because of a limiting role of the cellulose wall against their penetration into the cells.

Genetic Variation and Differentiation of Peat-Bog and Dry-Meadow Populations of the Dwarf Mountain Pine Pinus mugo Turra in the Highlands of the Ukrainian Carpathians

Based on electrophoretic analysis of 21 isozyme loci controlling 10 enzyme systems, the intra- and interpopulation variation was studied in two peat-bog and three dry-meadow populations of the dwarf mountain pine Pinus mugoTurra from the highlands of the Ukrainian Carpathians. In the studied samples (a total of 164 trees), on average 62% of the studied genes were polymorphic; the mean heterozygosity was 21.3%. The dry-meadow populations differed from the peat-bog populations by allele and genotype diversity and by heterozygosity although the indices characterizing population heterogeneity (Fst and Gst) were small (0.027 and 0.032, respectively). Neis genetic distances between the populations ranged of 0.011 to 0.032 with the mean of 0.018.

Bioinformatic search for cellulose synthase genes in flax (Linum usitatissimum) and their phylogenetic analysis

In silico search for the nucleotide sequences encoding cellulose synthases in the flax genome was carried out, together with the comparison of the identified sequences with the orthologous genes in dicotyledonous plants. The analysis resulted in the identification of 32 flax candidate genes, 16 of which encoded cellulose synthases with high possibility rate, while the remaining 16 encoded cellulose synthase-like proteins (Csl). The phylogenetic analysis of the protein products of the cellulose synthase genes allowed dividing them into six groups comprising cellulose synthases of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7, and CesA8. Paralogous sequences belonging to the classes CesA1/10 and CesA5/6/2/9, associated with the primary cell wall formation, were characterized by the higher intra-class similarity rate, than the orthologous sequences. At the same time, the genes that control cellulose biosynthesis for the secondary cell wall formation constituted distinct clades, CesA4, CesA7, and CesA8. The analysis of the 16 selected flax candidate cellulose synthase genes demonstrated that the flax genome contains at least 12 different variants of cellulose synthase genes, which belong to all six cellulose synthase clades. In such a way, at this stage, the cellulose synthase genes from all ten known CesA classes have been identified in the flax genome; however, their correct attribution to each of these classes requires some additional study.

Identification of the allelic state of the Lr34 leaf rust resistance gene in soft winter wheat cultivars developed in Ukraine

The distribution of alleles at the Lr34 locus associated with leaf rust resistance has been studied in soft winter wheat (Triticum aestivum L.) cultivars developed in Ukraine. To determine the allelic state of the Lr34 locus, codominant molecular marker cssfr5 has been used. Cultivars with the revealed Lr34(+) and Lr34(−) alleles have been identified as potentially resistant or susceptible, respectively. A collection of 81 cultivars from the main breeding centers of Ukraine has been examined; the Lr34(+) allele has been revealed in 44% of the tested cultivars. The obtained results have been compared with general data on the leaf rust resistance of wheat cultivars from different countries.

Genetic variation and differentiation of Abies alba Mill. populations from Ukrainian Carpathians

Using electrophoretic analysis of 11 enzyme systems, we studied the genetic structure and differentiation of eight natural populations of silver fir Abies alba Mill. in the Ukrainian Carpathians. Of 24 isozyme loci identified, 66.8% proved to be polymorphic. The mean numbers of alleles and genotypes per locus in the populations were respectively 3.1 and 4.5. Each A. alba tree was on average heterozygous at 15.9% of genes. In six populations, the genotypic distribution for all of the loci examined corresponded to Hardy-Weinberg proportions. The populations studied had low levels of subdivision (FST = 0.018; GST = 0.019 ) and differentiation. Nei’s genetic distances between the A. alba populations in the region ranged from 0.002 to 0.009, being on average 0.006.

[Population-genetic variation in Scots pine Pinus sylvestris L. from the main forest regions of Ukraine].

Using electrophoretic analysis of 22 isozyme loci controlling ten enzyme systems, we studied intrapopulation and interpopulation variation of Scots pine Pinus sylvestris L. in the main forest regions of Ukraine. In 15 of the populations examined, 76.5% of genes were polymorphic, and an average plant was shown to be heterozygous at 23.2% of the genes. The lowest and highest values of major polymorphism parameters were characteristic of respectively the relic populations of Ukrainian Carpathians and the populations from the steppe and forest-steppe zones. Nei’s genetic distances between the populations varied from 0.006 to 0.031 (on average 0.016). Cluster analysis failed to show clear trends in the population distribution relative to their geographical position.

The homologous identification of the stem rust resistance genes Rpg5, Adf3 and RGA1 in the relatives of barley

The barley genes Rpg5, RGA1 and Adf3, which provide a strong resistance to many pathotypes of stem rust, were cloned a few years ago, but it was still unclear whether their homologues were represented in wheat and in related species. The paper describes the results of a bioinformatic research to determine the homologues of Rpg5, RGA1 and Adf3 in the genomes of Triticum aestivum and several wild grasses, which breeders usually use as sources of stem rust resistance, and which are available in the genome databases. It was found that the Th. elongatum sequence Q9FEC6 and T. aestivum sequence Q43655 were the highly identical homologues of the Adf3 sequence. T. urartu M8A999 sequence and T. aestivum W5FCU1 sequence were found to be the closest homologues of Rpg5 complete protein sequence, but the identity of their kinase domains was not as clear as that of the other domains. The separate Rpg5 kinase part analysis did not provide the strong evidences that its orthologs were present in our corn species. T. urartu M7ZZX9 sequence and T. aestivum W5FFP0 and W5FI33 sequences were shown to be the homologues of RGA1. The analysis of the predicted active sites allowed finding out the difference between sequences of Rpg5, RGA1, Adf3 protein and their homologues.

Comparative analysis of the efficiency of intron-length polymorphism of β-tubulin genes and microsatellite loci for flax varieties genotyping

Efficacy of the β-tubulin introns lenght polymorphism method (TBP) was used for Ukrainian bredeed flax cultivars genotyping. For this purpose, TBP data were compared with data produced using the two most effective species-specific SSR markers. Both methods were used to evaluate intra- and intercultivar flax polymorphism. For each cultivar, PIC data (Polymorphism Information Content) and the range of allele lenghts, as well as the number of allele phenotypes, were calculated using TBP and SSR markers. The dendrograms, built using Nei and Li’s similarity coefficient, differ for SSR and TBP markers. Most flax cultivars of Ukrainian selection were genetically heterogeneous. The TBP method was highly efficient for differentiation of flax genotypes versus SSR analysis.

Analysis of cellulose synthase gene expression strategies in higher plants using RNA-sequencing data

The transcriptomes from different organs and tissues of western poplar, eucalyptus, soybean, and common bean were studied. The expression level of cellulose synthase genes was notably different in different types of tissues and organs in studied plants. For common bean and eucalyptus transcriptome, the domination of certain cellulose synthase genes was typical. These prevailing genes made up more than 50% of the total expression pull of cellulose synthases. On the contrary, cellulose synthase expression pulls of western poplar and soybean were distributed between multiple genes. The different expression strategies of CesA-genes may reflect phylogenetic processes that occurred in genomes studied.