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

THE MOLECULAR GENETIC STUDY OF KRIM-SAGHYZ (Taraxacum hybernum Steven) USING SSR, RAPD AND ISSR MARKERS

Krim-saghyz (Taraxacum hybernum Steven) is an alternative to Hevea brasiliensis as a source of natural rubber. In Russia, krim-saghyz is common only in the Crimean Peninsula and is traditionally named after it. In spite of its potential for economical use, the genetic structure of the Crimean population of this plant is still unexplored. In this regard, the purpose of our work was a comparative molecular-genetic characterization of T. hybernum from various habitats of the Crimean Peninsula using SSR, RAPD and ISSR markers. According to the plan, we collected achenes, leaves and roots of krim-saghyz in 10 spots all over the Crimean Peninsula. We found the plants in the western part of the southern Crimean coast and the western part of the Crimean foothills, which are two general regions of the area of this species. Total DNA was extracted from dry leaves of krim-saghyz with cetyltrimethylammonium bromide (CTAB). For the first time 12 SSR, 3 RAPD and 3 ISSR markers were tested on krim-saghyz. To observe polymorphism of RAPD- and ISSR-fragments, we used analytical electrophoresis in 1.7 % agarose gel. To compare the length of SSR amplicons, we used gel-electrophoresis in 8 % polyacrylamide gel. We found that the Crimean population of krim-saghyz appears to be genetically homogeneous. This could be due to a small geographic range and apomictic reproduction of this species. However, the phenotypical diversity within the population of T. hybernum is well known from the literature. Consequently, the study of the DNA polymorphism of this species should be continued, in particular, with the help of high-resolution techniques

Analysis of nucleotide sequences of the <i>GT47</i> glycosyltransferase gene in rye cultivars differing in the content of water-soluble pentosans in grain

Rye (Secale cereale L.) is the most important crop in Russia, its grain quality depends on the content of water-soluble pentosans. The grain of rye cultivars with high content of water-soluble pentosans has good baking properties, but low fodder qualities. Methods of marker-assisted selection for this trait in rye remain undeveloped. For Triticeae, the content of pentosans in grain may be associated with the GT47 glycosyltransferase genes, but the genes of this family have not been identified in rye.  The aim of this study was amplification, sequencing, and search for single nucleotide substitutions or other mutations in the GT47 gene in various rye cultivars differing in the content of water-soluble pentosans in their grain and the viscosity of their aqueous extract. DNA from rye leaves was isolated by the standard CTAB method. Based on the nucleotide sequences of the bread wheat and barley GT47 genes, universal primers were selected, then a fragment of the open reading frames of the studied gene was amplified, and the nucleotide sequences were determined by automatic capillary sequencing.The population cultivars of rye, ‘Chulpan 7’ and ‘Podarok’, and F1 hybrids ‘KVS Aviator’, ‘KVS Magnifico’ and ‘KVS Eterno’ were analyzed. The highest content of pentosans and the highest kinematic viscosity of the aqueous extract were found in cv. ‘Chulpan 7’. The lowest values of these indicators were shown by the hybrid cv. ‘KVS Aviator’. The analysis of the nucleotide sequences of the GT47 gene revealed the presence of single-nucleotide substitutions in seven loci, in which the studied rye cultivars differed. Of these, the high-pentosan cv. ‘Chulpan 7’ and the low-pentosan cv, ‘KVS Aviator’ differed in three nucleotide substitutions: 159 (G/A), 204 (C/T), and 327 (G/A). It is suggested that these SNPs can be used for genotyping rye cultivars for the content of water-soluble pentosans in grain

Identification of new nucleotide sequences of the <i>Glu-B1-1</i> 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

Bread wheat callusogenesis and organogenesis using mature embryos as explants

Background. Bread wheat (Triticum aestivum L.) is one of the staple cereal crops, so it is of great interest to breeders and researchers and requires constant monitoring of existing cultivars, including the development of new ones through classical breeding and modern gene engineering. The key stage in these techniques is successful callusogenesis and organogenesis in target objects. With this in view, the regeneration potential of two spring (‘Saratovskaya 55’ and ‘Sigma’) and three winter (‘Tanya’, ‘Fisht’ and ‘Pamyat’) cultivars of bread wheat was assessed, and optimal conditions were identified for callus induction and organogenesis using mature embryos.   Materials and methods. Immature and mature embryos of the five bread wheat cultivars were used in the study. The in vitro morphogenetic potential was evaluated under the impact of abiotic factors: preliminary exposure of grains to cold and use of exogenous hormones (2,4-D in various concentrations). Pretreatment of wheat with cold was carried out as follows: sterilized grains were incubated on the hormonal medium at a temperature of 4 °C for 2 weeks, and then transferred to 26 °C for 4 more weeks. The efficiency of callusogenesis and rhizogenesis was assessed and the numbers of morphogenetic calluses, regenerated and acclimatized plants were calculated.   Results and conclusions. The analysis made it possible to identify the bread wheat cultivars ‘Fisht’ and ‘Sigma’ for their high morphogenetic and regenerative potential. It was also shown that exposure to cold can serve as a good stimulating factor for producing a large number of calluses, but regenerants are better induced under normal conditions. The results also depended on the concentration of hormones applied. Universal conditions for morphogenesis and regeneration were not identified

Genetic diversity assessment in pea cultivars and lines using the SSR analysis

Background. Pea is the main leguminous crop in the Republic of Bashkortostan and widespread all over the world. The key role in the breeding of new pea cultivars is played by source material representing the phenotypic and genotypic diversity of Pisum sativum L., searched for in plant genetic resources collections. SSR markers are successfully used to study the DNA polymorphism of various genetic objects, including pea. However, the distribution of a number of microsatellite alleles in the genotypes of specific lines and cultivars of this valuable pulse crop remains practically unexplored.Materials and methods. Molecular genetic polymorphism was studied in 40 pea cultivar accessions of different ecological and geographical origin from the Vavilov Institute’s genebank of plant genetic resources or developed at regional breeding centers. Microsatellite analysis was performed using 5 SSR markers from the genomic library of microsatellites (Agrogene®, France).Results. All markers delivered good electrophoretic profiles and helped to amplify a number of alleles per locus varying from 2 (AB53) to 9 (AA355). The total number of alleles was 26, while the average number of alleles per locus was 5.2. The polymorphism information content (PIC) varied from 0.39 for locus AB53 to 0.82 for locus AA355, with the mean value of 0.60. The set of SSR markers used in the work made it possible to individualize each of the studied pea genotypes. The measured genetic distances were used to draw a dendrogram showing the distribution of genotypes according to their genetic relationship.Conclusion. Through studying the source material for pea breeding by the SSR analysis the data were obtained that provide additional information about the genetic structure of the collection and the polymorphism of the studied cultivar accessions. The results of genotyping pea cultivars and lines can be used for their genetic identification or to select parental pairs for hybridization

Development of source material for pea breeding through chemical mutagenesis and evaluation of its genetic diversity using SSR markers

Background. Pea (Pisum sativum L.) is a valuable leguminous crop of worldwide importance. The main problem of modern plant breeding is a decrease in the genetic diversity of crops, including pea. One of the ways to increase genetic polymorphism is the use of chemically induced mutagenesis. Sodium azide (NaN3) is a highly effective chemical mutagen successfully used in mutation breeding to increase the productivity of cultivated plants and enrich them with new useful traits. We used it to obtain new pea breeding material.Materials and methods. Experiments were carried out to obtain pea mutants using sodium azide at the concentrations of 1, 5 and 10 mM and the exposure time of 3 and 9 h. Molecular genetic polymorphism of the М2 plants and the original cultivar was assessed using 10 SSR markers from the microsatellite genomic library (Agrogene®, France).Results. Optimal concentrations of sodium azide and the duration of seed treatment with it were identified: 1–5 mM for 3 h. Sixteen mutant populations were obtained; in ten of them a change in the leaf type was found. An analysis of the yield structure components revealed a significant superiority (p &lt; 0.05) over the initial cultivar ‘Pamyati Khangildina’ in the mutant populations No. 1, No. 5, No. 9, No. 10, No. 15 and No. 16 in the number of seeds per pod, No. 9 and No. 16 in the weight of 1000 seeds, and No. 16 in the weight of seeds per plant. A dendrogram constructed on the basis of the SSR analysis data showed the degree of differences between the M2 populations of pea plants and the initial cultivar ‘Pamyati Khangildina’.Conclusion. The obtained mutant populations are planned to be used in pea breeding as sources of high seed numbers in pods, seed yield, seed weight per plant, and large seed size. A microsatellite analysis with 10 SSR markers revealed differences among the M2 mutant populations at the genetic level and made it possible to identify them

Changes in the content of proteins and lipids and in the state of the antioxidant system in mutant forms of <i>Amaranthus cruentus</i> L.

Background. One of the important indicators of the nutritional value of amaranth is the high content of protein and lipids in seeds. Hence, obtaining and identifying such forms of amaranth through breeding, so that they also possessed resistance to abiotic stressors, is an important task.Materials and methods. Leaves and seeds of Amaranthus cruentus L. and mutants of the second inbred generation obtained by treatment with sodium azide were analyzed. The Bradford assay was used to measure the content of total soluble protein, lipid analysis was performed by thin-layer chromatography, the state of the antioxidant system was assessed according to catalase and peroxidase activities and the rate of superoxide anion formation. Mathematical data were processed using the Statistica 10.0 software.Results. The highest concentration of total protein in seeds was 13.78 mg/g in one of the mutants obtained after treatment with 3 mM sodium azide. Fifteen fatty acids were found in amaranth seeds, and in four mutants a significant increase in the percentage of omega-6 unsaturated linoleic acid was recorded. An increase in salt tolerance compared to the control was observed in mutants No. 2 and No. 3. Mutant No. 2 under salinization demonstrated higher peroxidase activity and mutant No. 3 higher catalase activity; both mutants showed a reduced rate of superoxide anion formation compared to the control.Conclusion. Amaranth mutants identified for higher stress resistance, protein content and linoleic acid content can be recommended for further breeding to produce new cultivars of amaranth with economically valuable traits

Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof

BACKGROUND: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. METHODOLOGY: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. SIGNIFICANCE/CONCLUSION: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants