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.

Identification of Candidate Gene-Based Markers for Girth Growth in Rubber Trees

Girth growth is an important factor in both latex and timber production of the rubber tree. In this study, we performed candidate gene association mapping for girth growth in rubber trees using intron length polymorphism markers (ILP) in identifying the candidate genes responsible for girth growth. The COBL064_1 marker developed from the candidate gene (COBL4) regulating cellulose deposition and oriented cell expansion in the plant cell wall showed the strongest association with girth growth across two seasons in the Amazonian population and was validated in the breeding lines. We then applied single molecule real-time (SMRT) circular consensus sequencing (CCS) to analyze a wider gene region of the COBL4 to pinpoint the single nucleotide polymorphism (SNP) that best explains the association with the traits. A SNP in the 3’ UTR showing linkage disequilibrium with the COBL064_1 most associated with girth growth. This study showed that the cost-effective method of ILP gene-based markers can assist in identification of SNPs in the candidate gene associated with girth growth. The SNP markers identified in this study added useful markers for the improvement of girth growth in rubber tree breeding programs.

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.

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.

Development of a multiparent advanced generation intercross (MAGIC) population for genetic exploitation of complex traits in Brassica juncea: glucosinolate content as an example

Multiparent advanced generation intercross (MAGIC) populations have recently been developed to allow the high-resolution mapping of complex quantitative traits. This article describes the development of one MAGIC population and verifies its potential application for mapping quantitative trait loci (QTLs) in B. juncea. The population was developed from eight founders with diverse traits and composed of 408 F6 recombinant inbred lines (RILs). To develop one rapid and simplified way for using the MAGIC population, a subset of 133 RILs as the primary mapping population were genotyped using 346 intron-length polymorphism (ILP) polymorphic markers. The population lacks significant signatures of population structure that are suitable for the analysis of complex traits. Genome-wide association mapping (GWAS) identified three major glucosinolate (GSL) QTLs of QGsl.ig01.1 on J01 for indole GSL (IG), QGsl.atg09.1 on J09 and QGsl.atg11.1 on J11 for aliphatic GSL (AG) and total GSL (TG). The candidate genes for QGsl.ig01.1, QGsl.atg09.1 and QGsl.atg11.1 are GSH1, GSL-ALK and MYB28, which are involved in converting glutamate and cysteine to γ–EC, the accumulation of glucoraphanin, and the whole process of AG metabolism, respectively. One effective method for association mapping of quantitative traits in the B. juncea MAGIC population is also suggested by utilization of the remaining 275 RILs and incorporation of the novel kompetitive allele specific PCR (KASP) technique. In addition to its QTL mapping purpose, the MAGIC population could also be potentially utilized in variety development by breeders.

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.).

Distribution of mistletoe (Viscum album L.), which parasitizes different woody plants species, in Kyiv and its genetic characteristics

Aim. The aim of the work was to study the species diversity of mistletoe host trees with the establishment of damage degrees and the study of the mistletoe genetic characteristics in Kiev. Methods. The β-tubulin first intron length polymorphism evaluating method (TBP) was used. Amplified fragments DNA were fractionated by electrophoresis in non-denaturing polyacrylamide gel. DNA bands were detected using silver nitrate staining. Results. The species diversity of Viscum album L. host-trees was studied in Kiev. Among them, plants of the genus Pinus and angiosperms that belong to 8 genera (Acer, Fraxinus, Acacia, Populus, Tilia, Salix, Malus, Sorbus), wherein 47% belong to the genus Acer. It was revealed that among infected trees, 30% had severe damage to mistletoe, 28% moderate and 42% weak. Based on the analysis of DNA marker polymorphisms, the molecular-genetic profiles of V. album, which grows on various species of host trees, were obtained and analyzed. Conclusions. The species diversity of V. album host-trees was studied and the degree of their infection was assessed in Kiev. Mistletoe, which grows on angiosperms, is characterized by a greater degree of genetic polymorphism than that which grows on gymnosperms. Keywords: Viscum album L., host-plant, introns of genes, length polymorphism, β-tubulin.

Exploring the basis of 2-propenyl and 3-butenyl glucosinolate synthesis by QTL mapping and RNA-sequencing in Brassica juncea

Brassica juncea is used as a condiment, as vegetables and as an oilseed crop, especially in semiarid areas. In the present study, we constructed a genetic map using one recombinant inbred line (RIL) of B. juncea. A total of 304 ILP (intron length polymorphism) markers were mapped to 18 linkage groups designated LG01-LG18 in B. juncea. The constructed map covered a total genetic length of 1671.13 cM with an average marker interval of 5.50 cM. The QTLs for 2-propenyl glucosinolates (GSLs) colocalized with the QTLs for 3-butenyl GSLs between At1g26180 and BnapPIP1580 on LG08 in the field experiments of 2016 and 2017. These QTLs accounted for an average of 42.3% and 42.6% phenotypic variation for 2-propenyl and 3-butenyl GSLs, respectively. Furthermore, the Illumina RNA-sequencing technique was used to excavate the genes responsible for the synthesis of GSLs in the siliques of the parental lines of the RIL mapping population, because the bulk of the seed GSLs might originate from the siliques. Comparative analysis and annotation by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) revealed that 324 genes were involved in GSL metabolism, among which only 24 transcripts were differentially expressed genes (DEGs). Among those DEGs, 15 genes were involved in the biosynthesis and transport of aliphatic GSLs, and their expression patterns were further validated by qRT-PCR analysis. These RNA-Seq results will be helpful for further fine mapping, gene cloning and genetic mechanisms of 2-propenyl and 3-butenyl GSLs in B. juncea.