Screening of the genotypes of bread wheat (Triticum aestivum L.) by the allelic variants of Waxy genes and HMW glutenin subunits

Screening genotypes of wheat by allelic variants of Waxy and HMW-GS genes is a constitutional unit of marker-assisted selection of varieties with high values of desirable properties for flour-baking and technological attributes of the grain. The aim of the study was to produce a molecular screening of samples of spring soft wheat of local selection lines for the detection of genotypes of Triticum aestivum L. with valuable alleles of Waxy and HMW-GS genes. Seventy samples of wheat were subjected to DNA testing for the identification of genotypes with alleles of Waxy and HMW-GS genes. Molecular screening of wheat samples with the selected systems of molecular marking of the allelic variants of the analyzed genes made it possible to detect three partially Waxy lines with a combination of two null-alleles (Wx-A1b and Wx-B1b) in the study collection, as well as 33 plants with an economicallyvaluable combination of Ax2*/5+10 subunits of HMW-GS, the genotypes of which are considered as starting material for further selection work on the creation of spring soft wheat cultivars with high quality indicators of grain. The approaches applied in the study to identify Waxy and HMW-GS alleles are effective methods of evaluating their allelic polymorphism.


Introduction
Screening wheat genotypes for allelic variants of Waxy [1][2][3] and HMW glutenin subunits (HMW-GS) [4][5][6] is an integral part for marker-assisted selection of varieties with high value as indicators for flour-baking and technological properties of the grain. It is known that combinations of nonfunctional null alleles of Waxy genes in the genomes of wheat have a direct influence on the development of amylopectin-type starch with a reduced or complete absence of amylose, which, in turn, considerably affects the technological properties of starch and wheat flour [7][8][9].Taking into account the positive influence of HMW glutenin subunits Dx5, Dy10 (5+10), and Ax2* on the increase of flour-baking qualities, as well as the negative influence of subunits Dx2, Dy12 (2+12), and Axnull causing a reduction in baking properties of wheat, the combination of Ax2*/5+10 subunits is one of the most economically-valuable and desirable for the selection associated with high quality properties of the grain [4,5,10].

Digital signature
This PDF has been certified using digital signature with a trusted timestamp to assure its origin and integrity. A verification trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certificate properties provide further details such as certification time and a signing reason in case any alterations made to the final content. If the certificate is missing or invalid it is recommended to verify the article on the journal website. Currently, molecular genetic methods are the most accurate tools for assessing allelic polymorphism of Waxy [1,8,11] and HMW-GS [5,6,10] genes. The systems of molecular marking of allelic variants of Waxy [1,12,13] and HMW-GS [6,10,14] of wheat using DNA technologies selected for allelic polymorphism of genes analyzed make it possible to identify Triticum aestivum L. genotypes and so expand the range of donors and sources of economically-valuable alleles. In general, DNA technologies are integrated into fundamental and applied research aimed at the search, mobilization, and preservation of genetic resources of cultivated plants and their wild types [15][16][17]. The purpose here is to study, preserve, and apply to the biodiversity of cultivated plant forms, as well as managing selection of new genotypes with high economical productivity and resistance against bio-and abiostressors.
The main aim of the present study was a molecular screening of spring soft wheat samples from the collection of the Tatar Scientific Research Institute of Agriculture in order to detect potentially useful T. aestivum genotypes bearing allelic variants of Waxy and HMW-GS genes, valuable for selection and associated with high values of indicators for flour-baking and technological properties of the grain.

Material and methods
Seventy samples of wheat [68 samples of spring soft wheat and two varieties of winter soft wheat (cultivars 'Sila' and 'Starshina')] from the collection of the Tatar Scientific Research Institute of Agriculture (TatSRIA) were subjected to DNA testing to identify T. aestivum genotypes with allelic variants of Waxy and HMW-GS genes.
Nucleic acid extraction from wheat grains at milky-wax ripeness of a 2017 generation was performed using a commercial set of DNA-sorb C (Central Scientific Research Institute of Epidemiology, Russia), in accordance with the manufacturer's instructions. The principle of the method used for the isolation of nucleic acids consists of treating a sample (one ground wheat grain) with a lysis solution with proteinase K, which results in the destruction of cell membranes and the release of nucleic acids and cellular components. Dissolved nucleic acids bind to sorbed particles while other components of the lysed test material remain in the solution and are removed after the sorbent's precipitation by centrifugation and subsequent washing. When the buffer is added to the sorbent for elution, the nucleic acid transfers from the surface of the silicate into the solution, which is then separated from the sorbent particles by centrifugation. This procedure results in a highly purified nucleic acid preparation free of inhibitors of the amplification reaction, which provides a high analytical sensitivity in the PCR study. The screening of wheat lines and varieties was performed with the application of the selected systems of molecular marking of the alleles of the gene loci analyzed (Tab. 1).
An electrophoretic detection of PCR products and RFLP fragments was conducted in 2-3% agarose gels in ТBЕ buffer containing ethidium bromide followed by visualization of amplicons in UV-transilluminator (λ = 310 nm), the dimensions of which were evaluated by mobility by comparison with standard DNA markers (SibEnzyme, Russia).
Thus, there is an established predominance of the combination of subunits Ax2*/5+10 desirable for the selection on flour-baking qualities of grain over other less economicallyvaluable combinations (Fig. 3).

Discussion
Molecular genetic study identified genotypes of T. aestivum from the collection of the Tatar Scientific Research Institute of Agriculture by allelic variants of Waxy and HMW-GS genes, as well as revealing more potentially useful samples of wheat, the screening of which was performed by the use of selected PCR systems for molecular marking of the gene loci analyzed (Tab. 1, Fig. 2).
Inactive null alleles of loci Wx-A1, Wx-B1, and Wx-D1 of Waxy genes of T. aestivum have a direct influence on the formation of amylopectin-type starch (with a low content of amylose) [7,8,11]. It is noteworthy that out of the 70 samples analyzed, the great majority of plants (65.7%) were characterized by a combination of active alleles Wx-A1a/ B1a/D1a traditional for the first type of wheat and the rare combination Wx-A1g/B1a/D1a of the same type in two lines (2.9%) (Fig. 1, Tab. 2).
The principle of allelic discrimination of Wx-A1g from Wx-A1a was based on the absence of PCR product of 262 bp size in our modified method of genotyping with primers 4F-c and 4R, as well as the presence of amplified fragment Wx-A1g of 257 bp size in the setting up of PCR reactions with primers 4F and 4R [18].
The three lines developed as a result of marker-assisted selection by the crossing of winter soft wheat 'Starchina' (carrier of the null-allele Wx-A1b), courtesy of the National Center of Grain named after P. P. Lukyanenko (Russia, Krasnodar) [2], with the О-192-03-5 line of spring soft wheat (carrier of the null-allele Wx-B1b) [19] are assessed as more useful genotypes followed by the insertion of null alleles of the Wx-D1 locus (Wx-D1b, Wx-D1d, or Wx-D1e) [21,22] into their genomes in order to obtain cultivars of Waxy wheat. The allelic discrimination of Wx-B1b from Wx-B1a and Wx-B1e was achieved using the PCR method developed by us with primers 4F-c and Wx-B2R [18,19], generating in comparison to the prototype [12], PCR products reduced by 61 bp with a length of 402 bp (Wx-B1a allele) and 436 bp (Wx-B1e allele), providing a better separation of amplified fragments in agarose gel and, accordingly, increased accuracy of the interpretation of the genotyping results.
It is well known that winter soft varieties are widely used in the local selection of spring soft wheat in order to increase the genetic potential of productivity [23]. Given the positive influence of allelic variants Glu-D1d (5+10) and Glu-A1b (Ax2*) HMW-GS on the increase of flour-baking qualities, the competitive advantage of Glu-A1b over Glu-A1a (Ax1), and the negative influence of alleles Glu-D1a (2+12) and Glu-A1c (Axnull) causing a decrease in flour-baking properties of wheat, the combination Ax2*/5+10 [18] desirable for the selection is one of the most economically-valuable combinations of HMW glutenin subunits.
The well-known primers used in the present work: UMN19F+UMN19R (Ax1/Axnull and Ax2* alleles), UMN25F+UMN25R (Dx2 and Dx5 alleles) and UMN26F+UMN26R (Dy10 and Dy12 alleles) [10], were constructed for PCR identification of allelic variants of HMW-GS of T. aestivum. This was mainly by the capillary and vertical gel-electrophoresis detection methods in PAGE, which showed the effectiveness of their use in the application of PCR with the detection of the results by the method of horizontal electrophoresis in agarose gel [24].
The reliability of our results of PCR identification of the wheat genotypes studied by the use of primer sets of the series UMN [10] was further confirmed by our proposed procedure for RFLP analysis by restriction endonuclease HaeIII, providing a correct interpretation of generating PCR-RFLP fragments at their electrophoretic detection in agarose gel [18].
Over the past 5 years, a total of 223 samples of T. aestivum have been studied from the collections of the Tatar Scientific Research Institute of Agriculture, using allelic variants of Waxy and HMW-GS genes [18,19,24]. The collection includes potentially useful T. aestivum genotypes -carriers of valuable null alleles Wx-A1b and Wx-B1b, as well as rare active alleles of Wx-A1g and Wx-B1e Waxy genes including the genotypes with an economically-valuable combination Ax2*/5+10 HMW-GS, which is of potentially high interest not only to Russian, but also foreign breeders. However, unlike previous studies [18,19,24] performed in the course of marker-assisted selection, new wheat genotypes with a combination of two null alleles (Wx-A1b and Wx-B1b) of Waxy genes have been revealed by us for the first time. The new genotypes of T. aestivum presented in this study expand the circle of donors and sources of economically-valuable alleles of Waxy and HMW-GS genes which indicates the importance of the results of our research reported here.
The methods developed by us to identify allelic variants of Waxy and HMW-GS genes of wheat on the basis of PCR and RFLP analysis, which were used in the current work, are effective approaches for the assessment of their allelic polymorphism expanding the arsenal of systems of molecular marking of alleles of the genes analyzed.
In the course of any expanded screening of the world collection of T. aestivum genotypes from various geographic zones, the selection of systems of molecular marking of allelic variants of Waxy [8,21,25] and HMW-GS [6,14] should be carried out taking into the account our current knowledge of allelic polymorphism of the such genes.

Conclusion
Molecular screening of lines and cultivars of wheat in the local collection studied by selection realized with the use of the selected systems of molecular marking of allelic variants of the loci of genes analyzed made it possible to establish the presence of three partially Waxy lines (K-243-13Wx-2, K-243-13Wx-4, K-243-13Wx-6), with a simultaneous combination of two null alleles (Wx-A1b and Wx-B1b). Additionally, 33 plants with an economically-valuable combination of subunits Ax2*/5+10 of HMW-GS were identified whose genotypes are considered as a starting material for further breeding new wheat varieties with high quality indicators of grain quality. These potentially useful wheat genotypes add to the circle of donors and sources of economically-valuable alleles of Waxy and HMW-GS genes and have a high potential to meet the demands of national and foreign plant breeders.
Methods of identification of allelic variants of Waxy and HMW-GS genes of wheat used in this work, also developed by us, are effective tools for assessing their allelic polymorphism. However, given the new knowledge of allelic polymorphism of the genes analyzed, the arsenal of systems for molecular marking must be expanded further in the course of the screening of the world collection of T. aestivum genotypes in various geographic zones.