Genetic variation of GLI-B1 locus in Ukrainian bread wheat varieties and lines

Aim. To investigate polymorphism of Gli-B1 locus in modern Ukrainian bread wheat cultivars, to analyze the distribution of alleles and to compare the received data with the “core-collection of wheat cultivars” presented by Dr. E. Metakovsky. Methods. Eighty one bread wheat cultivars and lines from different plant breeding institutions and stations of Ukraine were tested using allele-specific primers to Gli-B1 locus developed by Zhang et al. [2003]. PCR products were fractionated in polyacrylamide gel (PAG) and then were stained by silver nitrate. Allelic variants of gliadins were analyzed by electrophoresis in acid polyacrylamide gel (APAGE). Results. Nine allelic variants of gliadins were revealed by APAGE and six alleles of Gli-B1 locus were detected by PCR-analysis. In 52 % of modern Ukrainian bread wheat cultivars we revealed Gli-B1b allelic variant, according to PCR - Gli-B1.1 allele with a 369 bp amplification fragment. In the genotypes of Ukrainian wheat cultivars, the 1RS.1BL translocation, which carries resistance genes, is frequent, as was detected by the absence of any amplification fragments with Gli-B1 primers. The correspondence between allelic variants of gliadins and alleles of Gli-B1 locus is discussed. Conclusions. DNA polymorphism of Gli-B1 locus examined in our research coincides with the diversity of allelic variants of gliadins, which were detected by APAGE method for Ukrainian bread wheat cultivar. However, PCR-analysis with applied primers carried out in this study does not distinguish the alleles that correspond to the Gli-B1c, Gli-B1g and Gli-B1e allelic variants of gliadins. The most common allele (52 %) for the in-vestigated Ukrainian wheat varieties is Gli-B1.1 allele, which was characterized by the amplification fragment of 369 bp, and the presence of 1RS.1BL translocation, which corresponds to Gli-B1b and Gli-B1l allelic variants of gliadins obtained by APAGE, respectively.


Introduction
Gliadins are large families of wheat storage proteins with similar amino acid sequences. Monomeric gliadin peptides are held together with glutenin polymers via intramolecular disulfide bonds and form gluten complex during mixing wheat grain flour and water [1]. It is considered that gliadins contribute to dough properties such as viscosity and extensibility, whereas glutenins affect strength and elasticity [2,3]. Moreover, gliadins contain CD epitopes, that can cause coeliac disease in 1-3 % of population. Thus, studying the gliadins gene sequences will advance the development of low gluten wheat by using RNA interference or Crispr/Cas 9 technologies in order to create safe food for people with this disease [4][5][6][7][8].
Each Gli locus encodes a group of gliadin polypeptides (allelic variant) inherited together as a simple Mendelian trait. There is a range of 40 to 150 copies of α-genes in Gli-2 loci, 15-40 and 15-18 copies of γ-and ω-gliadin genes at Gli-1 loci respectively [14][15][16], de-pending on the wheat cultivar. A large number of pseudogenes have also been reported for each gene family, and some of them had very low expression level.
Multiple allelism of gliadins has been revealed by the acid polyacrylamide gel electrophoresis method [17,18]. Allelic variants differing from each other in a number of bands and their electrophoretic mobility are encoded by one Gli locus. 182 allelic variants for six main gliadin loci in more than 1000 common wheat cultivars have been described by Metakovsky et al. [18]. The most frequent allelic variants are Gli-B1b (28.2 %) and Gli-D1b (39.4 %) [18].
Contribution of Gli-B1 locus to breadmaking quality is higher than that of Gli-A1 and Gli-D1 loci [19]. Better quality is observed in cultivars with GLD 1B1 and GLD 1B2 (Gli-B1b and Gli-B1d) allelic variants of gliadins. To date, 26 allelic variants of gliadins of Gli-B1 locus were described and cataloged [18].
Only one consensus sequence (MG560141.1) of Gli-B1 locus with length 6 535 908 bp was generated. Based on PacBio platform sequencing data, six γ-, one ω-and one δ-gliadin active genes and eight pseudogenes were reported for Gli-B1 locus of Chinese Spring cultivar, which have Gli-B1a allelic variant [18,20]. To date, there are no full nucleotide sequences of Gli-B1 locus of other wheat cultivars with different allelic variants of Gli-B1 locus. Based on AF234646 and M13712 sequences, allelespecific SNP markers (Gli-B1.1 and Gli-B1.2) for γ-gliadin pseudogene of Gli-B1 locus were developed by Zhang et al. [21]. Amplification fragments lengths variation obtained with marker Gli-B1.2 in wheat varieties was observed due to the presence of microsatellite in the sequence, as reported by Devos et al. [22]. Polischuk et al. [23] applied PCR with the allele-specific primers for the analysis of six near-isogenic lines and 12 Ukrainian bread wheat cultivars and showed the correspondence between allelic variants of gliadins and Gli-B1 alleles. Also, the correspondence between PCR fragments length polymorphism (detected using primers developed by Zhang [21]) and allelic variants of gliadins at Gli-B1 locus had been reported for 46 common wheat cultivars in our previous studies [24]. In this research we aimed to explore polymorphism of Gli-B1 locus in modern Ukrainian bread wheat cultivars (most of which have been registered in The State Register of Plant Varieties Suitable for Dissemination in Ukraine after 2000 year), to analyze the distribution of alleles and to compare received data with "core-collection of wheat cultivars" presented by Dr. E. Metakovsky.  [25] and 13 reference wheat cultivars -Miro novskaya 808, Mironovskaya 61, Donskaya Polukar likovaya, Albatros, Strumok, Suneca, Federation, Bezostaya 1, Escualo, Ardec, Caia, Prinqual, Krasnodonka (the reference cultivars from "core collection of common wheat", which have been collected by Dr. E. Meta kovsky with the aim to represent the widest polymorphism of allelic variants of Gli-B1 locus), were used for comparison and verification of amplification fragments length.

Materials and Methods
For research we used five grains per variety. Each grain was divided into two parts. One half of the grain was used for analysis of allelic variants of gliadins by using electrophoresis according to F. O. Poperelya's method in acid polyacrylamide gel (APAGE) [26]. Gliadin allelic variants were identified using the catalogue of Е. Metakovsky [18].
Allele frequency was calculated considering heterogeneous cultivars [29]. Nei's genetic variation index [30] was calculated according to the formula where p i is the frequency of the certain allele at the locus.
Based on PCR analysis all cultivars were divided into six groups. The first and the second groups included wheat cultivars with Gli-B1.1 allele, the third group combined cultivars that did not have any amplification fragments, and other three groups were characterized by Gli-B1.2 allele.
The first group was the largest and included cultivars Vyshyvanka, MIP Assol, Oberih Myronivsky, Mariia, Sonata and other with the amplification fragment of 369 bp in Gli-B1.1 locus. The same size amplification products were identified in cv. Bezostaya 1, Mironovskaya 808 and Albatros (Fig. 1). This group included 36 wheat cultivars and lines with Gli-B1b allelic variant of gliadins (Table 1).
Only one wheat line Gli-B1-12 belonged to the second group. In PCR with allele-specific primers line Gli-B1-12 produced a 400 bp amplification fragment of Gli-B1.1 allele as in variety Federation (Fig. 1). Both cultivar and line were characterized by Gli-B1o allelic variant.
The third group included 21 wheat cultivars that did not produce in PCR any amplification fragments (null allele) due to the 1RS.1BL wheat-rye translocation (cultivar Ledia, Fig. 1,  Fig. 2). All cultivars in the group had Gli-B1l allelic variant as expected (Table 1).
Seven wheat cultivars and lines that produced a 397 bp amplification fragment of Gli-B1.2 allele as in reference varieties Escualo and Prinqual, were assigned to the fourth group  (Table 1).
Only one cultivar belonged to the fifth group which was distinguished by Gli-B1.2 allele with the amplification fragment of 403 bp. The reference cultivars ˗ Ardec (402 bp), Caia (403 bp) and Krasnodonka (401 bp) were used to identify amplification fragment length of cultivar Madiarka (Fig. 2). All the above varieties have Gli-B1h allelic variant ( Table 1).
The sixth group was distinguished by Gli-B1.2 allele with the amplification fragment of 409 bp. We revealed this amplification fragment for the cv. Bilotserkivska Napivkarlykova and line KS 22-04 as in reference cv. Donskaya Polykarlikovaya and Suneca (Fig. 2). Gli-B1d allelic variant of gliadins, which is predominant in Mexico, Canada and Krasnodar (Russia) [18], was detected for all cultivars in this group. Due to the controversial results with Polischuk et al. [23], who had pointed out that Gli-B1.1 allele corresponds to Gli-B1d allelic variant, cultivar Strumok was used to   Table 1). The results obtained in our research demonstrated wide distribution of two main alleles of Gli-B1 locus among modern Ukrainian wheat cultivars. The first allele is Gli-B1.1 with the amplification fragment of 369 bp, according to PCR, which corresponds to Gli-B1b allelic variant of gliadins that has positive impact on dough quality and is present in the majority of bread wheat cultivars in Ukraine and some other countries, such as Bulgaria, Romania and Russia [18]. The second allele is null allele in PCR with allele-specific primers, which corresponds to Gli-B1l allelic variant, linked to some important resistance genes. Gli-B1 loci carry not only important storage protein genes, but also are linked with the resistance and adaptation genes that provide the predominance of certain alleles in the specific environment.

Conclusions
DNA polymorphism of Gli-B1 locus examined in our research coincided with the diversity of allelic variants of gliadins, which were detected by APAGE method in Ukrainian bread wheat cultivars, and divided all cultivars into six groups at the DNA level.
Clear correspondence observed in this study allows one to distinguish some important allelic variants of gliadins such as Gli-B1b, Gli-B1l, Gli-B1d, Gli-B1h by the PCR method. However, DNA-analysis applied in this study does not distinguish the Gli-B1c, Gli-B1g and Gli-B1e allelic variants. Nevertheless, at the same time using PCR with Gli-B1.2 allelespecific primers we revealed polymorphism at Gli-B1 locus among the cultivars with Gli-B1h allelic variant of gliadins.
The correspondence between allelic variants of gliadins and alleles of Gli-B1 locus in Ukrainian bread wheat cultivars is retained as in the previously studied [24] "core collection of wheat cultivars".