Allelic variants for Waxy genes in common wheat lines bred at the Lukyanenko National Grain Center

This article presents the results of a molecular marker-assisted study of allelic variants of Wx genes in common wheat ( Triticum aestivum L.) lines. The study was carried out as part of the work on the transfer of null alleles of the genes Wx-A1 , Wx-B1 , and Wx-D1 to the varieties of soft wheat and creation of breeding material with modified activities of the main enzymes involved in amylose biosynthesis. The lines were obtained at the Department of Breeding and Seed Production of Wheat and Triticale, National Center of Grain named after P.P. Lukyanenko, by crossing mutant forms carrying inactive (null) alleles of genes Wx-A1 , Wx-B1 , and Wx-D1 with bread wheat cultivars. The molecular markers selected for the study allowed identification of valuable breeding material carrying both single null alleles of Wx genes and their combinations in its genome. A combination of two null alleles ( Wx-A1b + Wx-D1b ) was detected in 30 lines. The presence of three null alleles ( Wx-A1b + Wx-B1b + Wx-D1b ), which corresponded to fully Wx wheat, was found in one line. We selected 37 lines that combined the presence of the Wx-B1e allele with the Wx-A1b and Wx-D1b null alleles. The Wx-A1b + Wx-B1e combination was identified in 26 lines, and 24 lines carried the combination of alleles Wx-B1e + Wx-D1b . The mutant forms PI619381, PI619384, and PI619386 were identified as carriers of the functional Wx-B1e allele. The Wx-A1b and Wx-B1e alleles could have been transferred to the studied lines from the donors used or from the Starshina and Korotyshka varieties, respec-tively. The mutant forms used in the crosses are donors of the Wx-B1b and Wx-D1b alleles. The use of molecular markers chosen by us for identification of the allelic state of the Wx-A1 , Wx-B1 , and Wx-D1 genes can provide grounds for marker-assisted selection for this trait. Selected lines found to possess null alleles of the Wx genes are applicable in breeding programs aimed at the improvement of technological qualities of grain and raise of bread wheat varieties with modified starch properties.


Introduction
An urgent task of common wheat breeding programs is the creation of varieties with improved technological qualities of grain.
A starch fraction of about 70 % of the total dry matter in wheat grain can significantly affect the quality of the final use of common wheat flour (Zeng et al., 1997). The broad use of wheat starch in the chemical and food industries is due to its properties. These include: hygroscopicity, neutrality of taste, good tolerance of heat treatment, moderate viscosity, and emulsion stabilization (Maningat, Seib, 1997). One of the interesting properties is the ability of grains to swell in warm liquid. Another distinctive feature is the ability to form pastes stable under thermal stress or long-term storage (Maningat et al., 2009).
Starch quality is closely related to the ratio between amylose and amylopectin, the two major constituents of starch. Granule-bound starch synthase (GBSSI) is the enzyme responsible for amylose synthesis in wheat grain. As some important technological properties of starch, such as gelatinization, bonding, and gelation, depend on the amylose/amylopectin ratio (Zeng et al., 1997). The GBSSI enzyme, or Waxy protein, has been the subject of many studies in recent years. In common wheat, this enzyme is encoded by three homologous Waxy (Wx) genes located on chromosomes 7A (locus Wx-A1), 7D (Wx-D1), and 4A (Wx-B1), (Shure et al., 1983;Chao et al., 1989;Yamamori et al., 1994). Each of the Wx genes has several allelic variants. The most common wild-type alleles are called Wx-A1a, Wx-B1a, and Wx-D1a (Yamamori et al., 1994;Nakamura et al., 1995). These alleles carry no mutations, and they intensely express GBSSI protein. Another type of allele (null allele) is nonfunctional and less common. It leads to a decrease in amylose content in starch. It is known that the Wx-B1 gene has the greatest effect on amylose content in common wheat starch, followed by Wx-D1 and Wx-A1 (Yamamori et al., 1994). Functional alleles different from those of the wild type were also isolated, but their effects remain poorly understood. The presence of three null alleles for the Wx-A1, Wx-B1, and Wx-D1 genes leads to complete elimination of GBSS1, absence of amylose synthesis, and the formation of amylopectin-type starch (Nakamura et al., 2002).
At present, molecular marking methods are in broad use to identify the allelic state of Wx genes. They make it possible to detect various alleles of Wx genes, including null alleles, and can be used as the basis for breeding programs aimed at producing common wheat with a modified amylose/amylopectin ratio (Nakamura et al., 1995;Kiribuchi-Otobe et al., 1997).
Earlier, M.V. Klimushina et al. (2012) applied molecular markers to a study of the allelic composition of Wx genes in 99 varieties and lines of common wheat bred at the Lukyanenko National Grain Center (NGC). They found that most accessions had wild-type alleles (which do not reduce amylose content in starch). The data obtained motivated the work on the transfer of null alleles of the Wx-A1, Wx-B1, and Wx-D1 genes to common wheat varieties of NGC and the creation of breeding material with altered activities of the main enzymes involved in amylose biosynthesis.
This article presents the results of molecular markerassisted analysis of F 6 lines of common wheat for allelic variants of Wx genes. The purpose of the work was to select valuable genotypes carrying both individual null alleles and their combinations for their subsequent involvement in the breeding process aimed at obtaining varieties with improved technological qualities of grain.

Materials and methods
The study was conducted with 502 lines of common wheat generation F 6 . The lines were obtained at NGC by crossing carriers of inactive (null) alleles Wx-A1, Wx-B1, Wx-D1 to commercial varieties of common wheat. The mutant forms PI619381, PI619384, PI619376, PI619386, PI619377, and PI619378, in which the functional Waxy protein was not synthesized, were used as null alleles donors. These wheat forms were obtained from CIMMYT Turkey as part of a collaboration on the exchange of breeding material. The mutant forms had been created at the National Center for the Study of Small-Grain Germplasm, USDA-ARS, United States, by crossbreeding of Bai Huo common wheat varieties from China, a null allele carrier of the Wx-D1 gene, to Kanto 107 and Ike varieties from the USA, carrying null alleles of the Wx-A1 and Wx-B1 genes. Varieties Starshina, Vassa, Utrish, Tabor, Esaul, Kuma, Grom, and Sila, raised at NGC, and variety Korotyshka, bred at the Belgorod Research Institute of Agriculture, were used as recipients. The crossing combinations of the lines under study are shown in Table 1. DNA was isolated from 5 to 7-day-old etiolated wheat seedlings according to the method (Plaschke et al., 1995). The lines were genotyped for the allelic state of Wx genes by PCR. Primers were selected on the basis of literature data; their names and amplification conditions are presented in Table 2. The reaction mixture of the volume 25 μl contained 1× buffer for TaqDNA polymerase (50 mM KCl, 20 mM TrisHCl pH 8.4, 2-5 mM MgCl 2 , and 0.01 % tween-20), 2 mM MgCl 2 , 0.2 mM each dNTP, 12.5 pM each primer, 50 ng of DNA and 1 U of Taq polymerase.
Amplification was carried out according to the conditions recommended by the authors with minor modifications (see Table 2). PCR products were resolved by agarose gel elec-Allelic variants for Waxy genes in common wheat lines bred at the Lukyanenko National Grain Center trophoresis in 0.5 × TBE buffer. The gel concentration ranged from 1.5 to 2.0 % depending on the size of the amplified fragment. The gels were stained with ethidium bromide and photographed under ultraviolet light using an Infiniti 1000 photo box. The 100 bp M 24 DNA marker SibEnzyme was used as a molecular weight ladder.

Results
A total of 502 soft wheat generation F 6 lines were analyzed for the allelic state of the Wx-A1, Wx-B1, and Wx-D1 genes with the use of molecular markers. The numbers of samples studied for each crossing combination, as well as the numbers of identified alleles, are presented in Table 3.
The study of the allelic states of the Wx-A1 gene in the lines was carried out using the codominant marker designed by T. Nakamura et al. (2002). The null allele of the Wx-A1 gene was detected in all crossing combinations except for No. 59 and 60. Altogether, 122 lines carrying Wx-A1b were identi-fied; in the rest, the Wx-A1a allele (wild type) was present. The presence of the Wx-A1b null allele was confirmed in all donors used (mutant for the Wx-A1, Wx-B1, and Wx-D1 genes).
To identify the allelic state of the Wx-B1 gene, a marker developed by A. McLauchlan et al. (2001) was used at the first step. Samples with identified Wx-B1b null alleles were then rescreened with the marker proposed by L.S. Vanzetti et al. (2009) (Figure). L.S. Vanzetti et al. (2009) have shown that the use of a molecular marker developed by A. McLauchlan et al. (2001) does not discriminate Wx-B1e and the Wx-B1b null allele, whereas no amplification occurs in samples with null allele with the use of the other marker. This can cause errors stemming from poor DNA isolation and PCR inhibition in some samples (Klimushina et al., 2012).
The null allele Wx-B1b was detected in one line obtained from cross combination No. 56, in four lines of combination No. 62, and in five lines of combination No. 71. It was also found that out of six mutant forms used as donors, the Wx-B1b allele was present in three: PI619376, PI619377, and PI619378. A functional allele of Wx-B1e, other than Wx-B1a, was detected in 108 lines. It was absent from combinations No. 56, 59, 62, and 71.
The nonfunctional allele Wx-D1b was detected in 100 lines, in all crossing combinations except for No. 56 and 59. The largest number of lines with this allele were identified in the combination of crossing No. 70. As a result of the analysis, lines carrying combinations of null alleles of the Wx genes were selected (Table 4).
The combination of two null alleles (Wx-A1b + Wx-D1b) was detected in 19 lines from cross combination No. 58, in three lines in combinations No. 61 and 62, in two lines in combination No. 70, and in one line in combination No. 71. The presence of three null alleles (Wx-A1b + Wx-B1b + Wx-D1b), which corresponds to fully Wx wheat, was found in line 56-12Mc4, obtained from crossing the mutant form PI 619376 to Vassa variety (crossing combination No. 56). Thirty-seven lines were selected combining the presence of the Wx-B1e allele with the null alleles Wx-A1b and Wx-D1b. The combination (Wx-A1b + Wx-B1e) was identified in 26 lines, and 24 lines carried the combination of alleles (Wx-B1e + Wx-D1b).

Discussion
The chosen molecular markers revealed common wheat lines bearing single null alleles of Wx genes or their combinations.
The use of molecular markers to identify the allelic states of the Wx-A1, Wx-B1, and Wx-D1 genes can provide grounds for marker-assisted selection for this trait. At the initial stages of selection to study the allelic state of the Wx-B1 gene, screening with the codominant marker designed by M. Saito et al. (2009)  The selected lines with the Wx gene alleles identified therein are of interest for breeding programs aimed at improving the technological qualities of grain and obtaining common wheat varieties with new starch properties. Wx-B1b null allele lines are promising for the production of special types of noodles, such as udon or ramen. This is due to their high swelling volumes and high paste viscosity peak, which are observed in wheats with low amylose contents. For example, the suitability of Australian common wheat varieties for the production of Japanese udon noodles is partly due to the low level of amylose in these varieties (Oda et al., 1980;Toyokawa et al., 1989). It has also been found that most of them lack Wx-B1 protein (Yamamori et al., 1994). The properties of starch from fully Wx wheats (bearing the null alleles Wx-A1b, Wx-B1b, and Wx-D1b) are not suitable for use in the production of noodles, but may be useful for industrial purposes. The use of fully Wx-wheats in regular flour mixtures increases the weight yield of the product and the volume of baked bread, and pure flour obtained from Wx wheat varieties has a low specific volume and a sticky crumb structure and is not suitable for bakery products (Hayakawa et al., 2004). The maximum content of Wx wheat flour without significant negative changes in the quality of bakery products is 30 %. However, Wx wheat flour can serve as improver, and it contributes to the long-term storage of finished products (Hayakawa et al., 2004).

Conclusion
The study of allelic variants of Wx genes is an important step in the breeding of common wheat varieties with starch composition modified without chemical modification. As a result of the work, valuable source material was selected for breeding common wheat with improved technological qualities of grain.