卷 53, 编号 6 (2017)

Rapid advances in sequencing technologies of second- and even third-generation made the whole genome sequencing a routine procedure. However, the methods for assembling of the obtained sequences and its results require special consideration. Modern assemblers are based on heuristic algorithms, which lead to fragmented genome assembly composed of scaffolds and contigs of different lengths, the order of which along the chromosome and belonging to a particular chromosome often remain unknown. In this regard, the resulting genome sequence can only be considered as a draft assembly. The principal improvement in the quality and reliability of a draft assembly can be achieved by targeted sequencing of the genome elements of different size, e.g., chromosomes, chromosomal regions, and DNA fragments cloned in different vectors, as well as using reference genome, optical mapping, and Hi-C technology. This approach, in addition to simplifying the assembly of the genome draft, will more accurately identify numerical and structural chromosomal variations and abnormalities of the genomes of the studied species. In this review, we discuss the key technologies for the genome sequencing and the de novo assembly, as well as different approaches to improve the quality of existing drafts of genome sequences.

The genes of α-expansins of woody plants are of great interest for genetic engineering, since they can potentially be used to improve the tree growth parameters. In the flora of Russia, model woody plants for plant biotechnology are aspen (Populus tremula L.) and black poplar (Populus nigra L.). The objective of this study was to determine the role of α-expansin-encoding genes, aspen PtrEXPA3 and black poplar PnEXPA3, in the regulation and maintenance of woody plant growth. To achieve this goal, the PtrEXPA3 expression level were determined upon exogenous phytohormone treatment, the action of stress factors, and constitutive expression of the PnARGOS-LIKE gene. In addition, transgenic aspen plants with constitutive expression of the black poplar PnEXPA3 gene were generated, and their morphological analysis was carried out. The highest PtrEXPA3 mRNA level was detected in young intensely growing aspen leaves, and furthermore, expression of the gene was induced by exogenous cytokinins and auxins. In response to NaCl and constitutive expression of the PnARGOS-LIKE gene, the PtrEXPA3 mRNA level decreased. Transgenic aspen plants with constitutive PnEXPA3 expression were characterized by the decreased size of leaves, petioles, and internodes, as well as the increased size of leaf epidermal cells, while the stem size remained unchanged. Taken together, the data obtained enable the suggestion that the PtrEXPA3 and PnEXPA3 genes encode cytokinin- and auxin-regulated, leaf-specific expansins that are involved in the cell expansion.

FRUITFULL (FUL) transcription factors, SlFUL1 and SlFUL2, are directly involved in the regulation of tomato fruit development. In the present study, complete sequences of novel SlFUL2 orthologous genes were isolated and structurally characterized in five accessions of domestic tomato Solanum lycopersicum and closely related wild species S. pimpinellifolium, having different morphological characteristics. In the studied genes, overall level of nucleotide sequence variation was 1.94%. Nine out of 11 exon-specific SNPs led to amino acid substitutions in the functionally important MADS, K, and C domains of the SlFUL2 orthologues. Considerable differences in the level and spatiotemporal dynamics of the expression patterns in different tomato organs at the species and intraspecific levels were observed.

The intraspecific structure of the European anchovy (Engraulis encrasicolus) was studied on the basis of mitochondrial cytochrome b gene (cytb) fragment variability in 84 individuals from seven localities of the Black Sea and the Sea of Azov. The data on haplotype and nucleotide diversity and the values of neutrality tests suggested expansive growth of anchovy populations in the Azov-Black Sea basin. All samples from anchovy populations demonstrated a high level of haplotype diversity (Hd = 0.962). Two dominant haplotypes were identified, the frequencies of which were not directional, and they were present in all localities. Sequence analysis of the mitochondrial cytb gene fragment showed no differentiation between the Sea of Azov and the Black Sea subspecies.

Тhe DNA testing of autosomal recessive deafness type 1A (DFNB1A, MIM 220290) is complicated when deaf patients have only monoallelic (heterozygous) recessive mutations in the GJB2 (Сх26) gene that is uninformative for establishment of diagnosis. Such patients may be “random” heterozygous carriers of GJB2 mutations as well as have the mutant allele in a cis-regulatory region of GJB2 gene, in element genes encoding other connexins: GJB6 (Сх30) or GJB3 (Сх31). Previous studies of genetic causes of hearing loss in patients from Yakutia were directed to search for only mutations in the GJB2 gene, and the DNA diagnostics was uninformative for 9.7% (38/393) of the patients with monoallelic GJB2 mutations. In this work the search for mutations in genes GJB3 and GJB6 and two deletions с.del(GJB6-D13S1830) and с.del(GJB6-D13S1854) to the cis-regulatory region of GJB2 gene was conducted in 35 patients with GJB2 monoallelic mutations and in 104 normal hearing individuals. The genes studied are two synonymous substitution c.489G>A (р.Leu163Leu) (GJB6) and c.357C>T (р.Asn119Asn) (GJB3) have been found, probably do not have clinical significance, and two nonsynonymous substitution c.301G>A (p.Glu101Lys) (GJB6) and с.580G>A (p.Ala194Thr) (GJB3). Additional experimental evidences are needed for confirmation of pathogenic significance of detected nonsynonymous substitutions in development of hearing loss in studied patients. Diagnosis of the DFNB1A was confirmed in only one patient, who was discovered by the deletion с.del(GJB6-D13S1830) (GJB2) in combination with a recessive mutation с.35delG (GJB2). In general, our results indicate low contribution of mutations in genes GJB6 and GJB3 in hearing loss etiology in Yakutia.

The genomic dosage of active (transcription-capable) ribosomal genes (AcRG) was determined in the genomes of 172 individuals, carriers of various chromosomal abnormalities: 116 individuals with numerical autosomal abnormalities (Down syndrome and Robertson translocations), 36 individuals with numerical abnormalities of gonosomes (Klinefelter, Turner, trisomy X, and disomy Y syndromes), and 20 carriers of various structural abnormalities of chromosomes. In the control sample of healthy individuals with a normal karyotype (N = 318), the AcRG number varied from 120 to 190 copies with mean of 150 ± 1 copies per diploid genome. In all the studied samples of carriers of chromosomal abnormalities, the genomic dosage (GD) of AcRG does not exceed the limits of variation of this feature in the control sample. However, in all the samples, the characteristic differences in the GD of AcRG were revealed. In accordance with the expectation, in patients with Down syndrome, the mean GD of AcRG was 10% higher, and in carriers of Robertson translocations, the maximum AcRG dose was 20% less than in the control. It can be concluded that about 10% of patients with Down syndrome and up to 50% of carriers of Robertsonian translocations die in the prenatal or early postnatal period because of excess or deficiency of AcRG in their genomes. A significant narrowing of the limits of the variation of the GD of AcRG in a sample of age-specific (over 10 years) patients with Down syndrome in comparison with the newborns was revealed. Obviously, Down syndrome carriers with low and high doses of AcRG predominantly die during the first years of life. The GD of AcRG of carriers of numerical anomalies of gonosomes predominantly fall into the region of medium, adaptive doses, and the carriers of structural chromosomal abnormalities predominantly survive when the dose of AcRG in the genome is greater than the mean in the control sample. For the first time, data on the association of the viability of carriers of different variants of chromosomal abnormalities with the number of active copies of rRNA genes in their genomes are presented.

A diploid population is analyzed with respect to the set of L autosomal loci. The attention is focused on the description of possible gametic disequilibria defined as deviations of the current population state from the reference state with the same allelic concentrations but with independent combination of alleles in gametes. The disequilibrum description is not based on any assumptions about microevolution factor pressures, generation overlapping, mating systems, etc. It is simply used to represent the current genetic structure of the population in terms of gametic disequilibria and allele concentrations. As the indicators of linkage disequilibrium, the basis vectors of subspaces of admissible deviations are chosen that were originally suggested as eigenvectors of a linearization matrix for the model of generalized nonepistatic selection in a series of papers by S. Karlin and U. Liberman. In the present paper, these characteristics are considered from a completely different, new point of view, namely, as the basis gametic disequilibria, and admissible deviations are decomposed into the basis ones. The disequilibrium hierarchical organization with respect to inducing marginal deviations from appropriate reference states on the locus subsets of different sizes is revealed. Each of the basis disequilibria is characterized by a set of loci K, the so-called deviation support. For a basis disequilibrium of a certain level of hierarchy, there is a threshold m (equal to the quantity of loci in K) for the number of loci which is necessary for that disequilibrium detection. Thus, it is required to analyze no less than m loci simultaneously to reveal the existing basis deviation from linkage equilibrium. The justification of the results is based on the use of linear algebra machinery, Kronecker multiplication, and linear subspace of admissible deviations. Properties of marginal deviations on various subsets of loci are considered in terms of gametic disequilibria.

The association of polymorphism T-786C of the endothelial NO-synthase gene NOS3 with the functional state of the left ventricle (LV) was studied among residents of the West Siberian region with ischemic heart disease (IHD), including that combined with diabetes mellitus type 2 (DMT2). Carriers of genotype–786TT had the greatest ejection fraction of the left ventricle in the group of patients without DMT2 (p = 0.012). This dependence was not revealed in the group of patients with IHD combined with DMT2. In the group with genotype–786TC, the frequency of left ventricular hypertrophy was higher among patients with DMT2 than patients without it (p = 0.025). There was no association between NOS3T–786C polymorphism and the severity of functional class of heart failure in both the groups.