Genetic polymorphism of white mistletoe (Viscum album L.) in Ukraine

Aim. The aim of the study was to establish genetic differences between V. album growing in different parts of Ukraine. Methods. White mistletoe samples collected in different regions of Ukraine were used in the study. The method of estimating the intron length polymorphism of β-tubulin genes was used. Amplified DNA fragments were fractionated by non-denaturing polyacrylamide gel electrophoresis and visualized by silver nitrate staining. Results. The genotypes of 91 white mistletoe plants were analyzed. DNA profiles of white mistletoe with a specific amplicons of β-tubulin gene introns were obtained, which allowed to differentiate the samples from each other. Fingerprinting data were used for cluster analysis and dendrogram construction. Conclusions. It was found that the analyzed mistletoe samples did not differ by geographical factor and were characterized by a low level of genetic diversity in the studied samples. Keywords: Viscum album L., intron length polymorphism, β-tubulin, genetic variability, Ukraine.

Transcriptome-wide Development and Utilization of Novel Intron-length Polymorphic (ILP) Markers in Common Vetch (Vicia Sativa Subsp. Sativa)

Common vetch (Vicia sativa subsp. sativa) is one of the most economically important forage legumes with rich nutritional value and multiple uses. Though the large-scale development and application of microsatellite markers have been conducted in common vetch germplasm evaluation, the investigation and exploitation of intron-length polymorphic (ILP) markers have not been systematically analyzed. In this study, the intron positions of common vetch genome were located by aligning the RNA-Seq sequences of common vetch with Medicago truncatula, soybean (Glycine max), and Arabidopsis thaliana genomic sequences, and used for VsILP marker development. A total of 10,400 markers were generated from 44,582 common vetch unigenes. Out of 300 randomly selected VsILP markers, 283 resulted in successful amplification in common vetch. Among these markers, 40 produced length variation in 30 common vetch accessions, collectively yielding 166 alleles with an average of 4.0 alleles per locus. The polymorphic information content (PIC) values extended from 0.06 to 0.81 with a mean of 0.49. Of the 283 VsILP markers, 84.8% exhibited transferability to leguminous and non-leguminous species. We presented here the first large-scale development of ILP markers in common vetch and their utility in germplasm evaluation and transferability, which will be valuable for further comparative genomic studies, genetic relationship assessments, and marker-assisted breeding of leguminous and non-leguminous species.

β-tubulin intron length polymorphism among forms var. glabra and var. laxa of napa cabbage

Aim. Main aim of this research was identification of genetic distances between different genotypes of napa cabbage (B. rapa ssp. pekinensis) and diversity identification in var. glabra and var. laxa forms. Methods. Molecular genetic analysis of napa cabbage genotypes was conducted out using method of β-tubulin intron length polymorphism (TBP). Results. Molecular profiles of different napa cabbage (B. rapa ssp. pekinensis) genotypes were identified. Number of amplified β-tubulin intron fragments was significantly varying – from 12 to 24 for each genotype. Basing on obtained results a dendrogram was built, which shows genetic distances among studied accessions. Conclusions. In present study 7 genotypes of B. rapa ssp. pekinensis were analyzed, received from IPK (Gatersleben) and Crop Research Institute (Prague) gene banks. Basing on obtained results it was established that systematic diversification of two forms var. glabra and var. laxa is not being confirmed by molecular genetic methods, such as TBP, and in this case, genetic difference between populations and cultivars was more significant. Keywords: Brassicaceae, Brassica rapa ssp. pekinensis, ILP, TBP, napa cabbage, β-tubulin intron length polymorphism.

γ-tubulin gene intron length polymorphism of Arabidopsis thaliana

Aims. Verification of the possibility of using the γ-tubulin gene intron length polymorphism method in genetic studies of plants on the example of Arabidopsis thaliana. Methods. The γ-tubulin gene intron length polymorphism evaluating method was used. Amplified fragments DNA were fractionated by electrophoresis in non-denaturing polyacrylamide gel. DNA bands were detected using silver nitrate staining. Results. Arabidopsis was first time analyzed using the γ-tubulin gene intron length polymorphism method. During amplification with degenerate primers 2 amplicons (520 bp and 555 bp) were formed in all samples. However, using selected arabidopsis-specific primers for the second intron of the γ-tubulin genes, it was possible to find several samples that differ in their DNA profile. Conclusions. It is established that the proposed method can be used in molecular genetic studies of plants. Moreover, the developed specific primers for γ-tubulin gene introns can probably be used both for the study of Arabidopsis and related species. The use of degenerate primers can be useful in the study of plants for which there is no information about their genome. Keywords: molecular-genetic markers, intron length polymorphism, γ-tubulin, A. thaliana.

Comprehensive identification of alternative back-splicing in human tissue transcriptomes

Circular RNAs (circRNAs) are covalently closed RNAs derived from back-splicing of genes across eukaryotes. Through alternative back-splicing (ABS), a single gene produces multiple circRNAs sharing the same back-splice site. Although many ABS events have recently been discovered, to what extent ABS involves in circRNA biogenesis and how it is regulated in different human tissues still remain elusive. Here, we reported an in-depth analysis of ABS events in 90 human tissue transcriptomes. We observed that ABS occurred for about 84% circRNAs. Interestingly, alternative 5′ back-splicing occurs more prevalently than alternative 3′ back-splicing, and both of them are tissue-specific, especially enriched in brain tissues. In addition, the patterns of ABS events in different brain regions are similar to each other and are more complex than the patterns in non-brain tissues. Finally, the intron length and abundance of Alu elements positively correlated with ABS event complexity, and the predominant circRNAs had longer flanking introns and more Alu elements than other circRNAs in the same ABS event. Together, our results represent a resource for circRNA research—we expanded the repertoire of ABS events of circRNAs in human tissue transcriptomes and provided insights into the complexity of circRNA biogenesis, expression, and regulation.

Ist and IIIrd intron length polymorphism of actin genes as a tool for flax DNA-profiling

Aim. The purpose of the work was to evaluate the possibility of using the polymorphism of the Ist and IIIrd introns of actin genes for DNA plant genotyping using flax varieties as model. Methods. 16 varieties of Ukrainian flax were analyzed. PCR was conducted using self-developed species-specific primers for the Ist and IIIrd introns of flax actin genes. DNA fragments were separated by electrophoresis in a 6% polyacrylamide gel and visualized by silver stains. Results. As a result of the evaluation of the Ist and IIIrd intron length polymorphism of actin genes, the species-specific DNA profiles of 16 flax varieties containing the target amplicons were obtained. The 7 allele phenotypes (PIC = 0.62) were detected for the Ist introns of the actin genes, and 3 allelic phenotypes (PIC = 0.32) for the IIIrd intron of actin genes. The highest level of polymorphism in the flax varieties was detected by evaluating the Ist intron length polymorphism of actin genes. Conclusions. Evaluation of the polymorphism of the Ist and IIIrd introns of actin genes allows genotyping and obtaining DNA profiles of flax varieties, which demonstrates the feasibility of further using both approaches for molecular genetic analysis of plants. Keywords: gene introns, length polymorphism, actin genes, flax (Linum usitatissimum L.).