Identification of new nucleotide sequences of the Glu-B1-1 gene encoding x-type glutenins in bread wheat.

Studies of the genetic base and polymorphism of bread wheat cultivars aimed at identifying alleles of genes associated with high baking and other economically valuable traits seem to be relevant, since bread wheat, along with all representatives of the Triticeae tribe, has a huge genetic potential for creating cultivars with high technological and rheological properties of grain flour. The aim of this study was sequencing and analysis of the nucleotide sequences of the Glu-B1-1 gene, and analysis of the predicted amino acid sequences of its protein product in three cultivars of bread wheat. Thus, in the course of genotyping cultivars and lines of bread wheat for the Glu-B1-1 gene, in the cultivars 'Avesta', 'Leningradka krupnozernaya' and line C-75094, previously undescribed changes in the size of amplifiable regions of the Glu-B1-1 gene for high-molecular weight glutenins were found. Comparative analysis of the nucleotide sequences of these genes with known sequences showed the presence of two deletions in 'Avesta' and C-75094 and the presence of seven single-nucleotide substitutions in 'Leningradka krupnozernaya'. Alignment of the predicted Glu-B1 amino acid sequences of the studied accessions and the standard cultivar carrying the Glu-B1-a allele showed that deletions in the amino acid sequences of 'Avesta' and C-75094 accessions are localized in the central domain of the protein and affect the amount of tri-, hexa-, and nonapeptides, and in 'Leningradka krupnozernaya', a decrease in GQQ and PGQGQQ by one unit was revealed. In addition, substitutions of five amino acids were found in 'Leningradka krupnozernaya'. Thus, we have found previously undescribed deletions and substitutions in the nucleotide sequences of the Glu-B1-1 gene for high-molecular-weight glutenins, which lead to changes in amino acid sequences in functionally important regions, namely, in the central domains of protein molecules. The identified mutations can be used for genotyping bread wheat cultivars.

Genetic polymorphism of high-molecular-weight glutenin subunit loci in bread wheat varieties in the Pre-Ural steppe zone.

High-molecular-weight glutenins play an important role in providing high baking qualities of bread wheat grain. However, breeding bread wheat for this trait is very laborious and, therefore, the genotyping of variety samples according to the allelic composition of high-molecular-weight glutenin genes is of great interest. The aim of the study was to determine the composition of high-molecular-weight glutenin subunits based on the identification of the allelic composition of the Glu-1 genes, as well as to identify the frequency of the Glu-1 alleles in bread wheat cultivars that are in breeding work under the conditions of the Pre-Ural steppe zone (PSZ). We analyzed 26 winter and 22 spring bread wheat varieties from the PSZ and 27 winter and 20 spring varieties from the VIR collection. Genotyping at the Glu-A1 locus showed that the Ax1 subunits are most common in winter varieties, while the predominance of the Ax2* subunits was typical of spring varieties and lines. In the Glu-B1 locus, the predominance of alleles associated with the production of the Bx7 and By9 subunits was revealed for both winter and spring varieties. In the case of the Glu-D1 gene, for all the wheat groups studied, the composition of the Dx5+Dy10 subunits was the most common: in 92.3 % of winter and 68.2 % of spring PSZ accessions and in 80 % of winter and 55 % of spring VIR accessions. The analysis of genotypes showed the presence of 13 different allelic combinations of the Glu-A1, Glu-B1, Glu-D1 genes in the PSZ varieties, and 19 combinations in the VIR varieties. The b b/al/с d allelic combination (Ax2* Вх7+Ву8/8*/9 Dx5+Dy10) turned out to be the most common for the PSZ spring varieties and lines, while for the PSZ winter accessions it was a с d (Ax1 Вх7+By9 Dx5+Dy10); the b с a and b с d genotypes (Ax2* Вх7+Ву9 Dx2+Dy12 and Ax2* Вх7+Ву9 Dx5+Dy10, respectively) occur with equal frequency among the VIR spring accessions; in the group of VIR winter varieties, the combination of the a b/ al d alleles (Ax1 Вх7+Ву8/8* Dx5+Dy10) prevails. The most preferred combination of alleles for baking qualities was found in the spring variety 'Ekaterina' and winter varieties 'Tarasovskaya 97', 'Volzhskaya S3', as well as in lines k-58164, L43510, L43709, L-67, L-83, which are recommended for further breeding programs to improve and preserve baking qualities in the conditions of the Pre-Ural steppe zone.

[Genome Editing in Species of the Tribe Triticeae with the CRISPR/Cas System].

The tribe Triticeae includes important agricultural crops, such as bread wheat, durum wheat, barley, rye, and triticale. Research in the field of reverse genetics and genetic engineering of Triticeae received a new impetus as the CRISPR/Cas genome editing system came into broad use. The review describes and analyzes the data on recent advances in genomic editing of cultivated plants of the tribe Triticeae and tools used in the field. The tools most commonly used for genome editing in Triticeae include the codon-optimized Cas9 gene under the control of the maize ubiquitin gene promoter and guide RNAs under the control of Pol III promoters U6 and U3 in one or more binary vectors. Phosphinothricin and hygromycin resistance genes are used as selectable genes. Agrobacterium-mediated transformation and biolistics are performed to obtain genome-edited plants, and immature embryos are used as explants. Approaches developed to overcome the problem of low regenerative capacity of Triticeae include in planta transformation of shoot apical meristems, transformation of microspores and pollen grains, and the use of haploinductors. Bread wheat and barley were subject to genomic editing in the majority of studies published to date, and durum wheat and triticale were recently used in CRISPR/Cas knockout studies of target genes. Further progress in the development of genome editing of cultivated plants of the tribe Triticeae should be aimed at expanding the range of species and varieties involved and overcoming the problems of low regenerative capacity. This will allow genetic modification of elite varieties, which will be in demand in agricultural production.

[PCR-based evaluation of sequence specificity of DNA fragmentation by ultrasound].

Ultrasonic fragmentation, which is a simple and convenient method for the mechanical degradation of DNA, is widely used in modern genome studies as one of the sample preparation steps. It has been recently found that the DNA breaks occur more often in the regions containing 5'-CG-3' dinucleotides. We studied the influence of the 5'-CG-3' dinucleotides on the efficiency of the 28S rRNA gene amplification during PCR with sonicated DNA of Mantis religiosa. It was shown that the amplification rate depends on the template length and the number of 5'-CG-3' dinucleotides. Amplification of the DNA regions with a higher 5'-CG-3' density is less efficient because of their higher sensitivity to ultrasound. The amount of the amplified DNA templates is inversely proportional to the 5'-CG-3'number.

[DNA amplification using PCR with abutting primers].

DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.