POLYMORPHISM OF SOME TRANSCRIPTION FACTOR GENES RELATED TO DROUGHT TOLERANCE IN WHEAT

T riticum spp., polymerase chain reaction, transcription factors, TaNAC2a, TaWRKY2, TaWRKY19, LEA , Td29b , drought tolerance. The aim of the research was to study polymorphism of preselected gene loci of three transcription factors ( TaNAC2a , TaWRKY2 , and TaWRKY19 ) and the Late Embryogenesis Abundant (LEA) proteins dehydrin ( Td29b ) related to wheat drought tolerance. The genes structure and chromosome location were established via bioinformatics tools. It is stated that TaWRKY2 and TaWRKY19 genes were comprised of 4 exons and 3 introns located on 2BS and 1DS chromosome arms, respectively; TaNAC2a — 2 exons and 1 intron 7AS; Td29b — single exon gene 3AS. Using polymerase chain reaction, no polymorphism was observed. Polymorphic bands were detected for TaWRKY2 locus. The screening of the distribution of the revealed polymorphic loci was carried out for a set of wheat and rye varieties, old landraces and interspecific hybrids. The polymorphism of TaWRKY2 locus indicated the presence of some other possible alleles of the gene. The obtained data are importantfor further investigations of wheat drought tolerance.

Common wheat (Triticum aestivum L.) is very important widely grown crop used for bread baking, food and animal feed. Wheat yield is the third largest cereal production in the world, after maize and rice [1]. In consequence of methods of modern plant breeding, numerous varieties with increased productivity were obtained. However, due to the recent undesired climate changes and global warming, the selection of droughttolerant germplasm donors must be constantly monitored to include them in contemporary breeding programs [2]. Marker-assisted selection (MAS) based on DNA markers can be effectively applied in the process of such selection [3][4][5][6]. While different types of DNA sequences can be employed for this purpose.
First DNA marker systems to study drought tolerance in plants were based on non-coding DNA sequences -RAPD (Random Amplified Polymorphic DNA) [7,8], SSR (Simple Sequence Repeats) [9,10] and ISSR (Inter Simple Sequence Repeats) [8] etc. Presently, attention mostly attracted to target encoding gene sequences, which play a great role in plant response to stress factors. These genes predominantly represented with transcriptional factors (TFs) and dehydrin genes [11].
In present work, our aim was to study DNA polymorphism of preselected gene loci of three transcription factors (TaNAC2a, TaWRKY2, TaWRKY19) and the LEA dehydrin (Td29b) related in their expression response to wheat drought tolerance.
WRKY transcription factors represent family of proteins that have WRKY domain (approximately 60 amino acids), involving the conserved WRKYGQK domain and a zincfinger-like motif [12,13]. These proteins are of great importance for biotic and abiotic stress responses [14,15]. Overexpression of TaWRKY2 as well as TaWRKY19 increased dehydration stress tolerance in transgenic Arabidopsis plants [12]. It was also found out that TaWRKY2 overexpressing plants had enhanced STZ and RD29B gene expressions due to temperate binding to the loci from RD29B STZ-1 and STZ-2 locus of Arabidopsis. As to TaWRKY19 transgenic plants, they had higher expression levels of DREB2A, RD29B, Cor6.6 and RD29A genes [12].
Another TF family that highly introduced in common wheat is represented with proteins containing a highly conserved NAC domain at the N-terminus and a variable transcriptional regulation domain at the C-terminus [16,17]. Overexpression of different TaNAC responded to enhanced biotic and abiotic tolerance [18,19]. It was postulated in the [16], that TaNAC2a transgenic plants of tobacco had extremely increased drought tolerance.
Special role in response to dehydration stress relates to dehydrin proteins, which help plant cell cope with osmotic changes. The number of dehydrins were described in wheat [20,21]. Late Embryogenesis Abundant (LEA) proteins belong to above mentioned group of proteins and can be candidate for wheat improvement [22]. It was reported [23] that LEA proteins accumulation enhanced stress tolerance protecting plant cells against dehydration. It was also described the importance of Td29b dehydrin in common wheat, which synthesis was highly induced by dehydration.

Materials and Methods
The subject of the study was a set of wheat cultivars of Ukrainian and foreign origin (25 and 36, consequently), a set of 52 old wheat species, distant and interspecific hybrids, 4varieties of rye.
Total DNA was isolated from one kernel with the modified CTAB method [25]. Polymerase chain reaction (PCR) of 20 μl included 0.5 μM of forward and reverse primers each ( The PCR products were separated by means of electrophoresis in 2% agarose gels in lithium borate buffer, 0.1 μg/ml ethidium bromide [26]. Gels were visualized in UVlight with a photosystem Canon EOS 600D. GelAnalyzer 2010 software was applied to identify the size of amplified fragments (http://www.gelanalyzer.com). Frequencies for each combination of amplified fragments were calculated according to [27].

Results and Discussion
As it was denoted above, data on CDS only are available for those three studied transcriptional factors (TaNAC2a, TaWRKY2, and TaWRKY19) and the dehydrin (Td29b). Thus, we managed to predict the exon-intron Having carried out every CDS alignments in the database of wheat whole genome shotgun contigs, the gene structures and chromosomal location were defined for three studied transcription factors (TaNAC2a, TaWRKY2, TaWRKY19) and the dehydrin (Td29b) (Fig. 1) in accordance with [28]. Hence, TaWRKY2 and TaWRKY19 have similar structure of 4 exons and 3 introns (Fig. 1, A, B), though, they are situated in different chromosomes (TaWRKY2 -short arm of 1D chromosome; TaWRKY19 -short arm of 1B). Both primer pairs applied in the following DNA polymorphism study hybridized at the end of the fourth exon. The gene of TF TaNAC2a comprises of 2 exons and 1 intron (Fig. 1, C) and allocates at the short arm of 7A chromosome. The primer pair for this gene locus annealed at the central part of exon 2. It was established, that Td29b gene might have referred to single exon gene ( Fig. 1, D). Its location is the short arm of 3A chromosome.

Molecular genetic study
To study DNA polymorphism of the selected loci of 4 genes (three TF -TaNAC2a, TaWRKY2, TaWRKY19; and the dehydrin gene Td29b) a set of 25 Ukrainian and 37 international wheat accessions from Global Wheat Program of the International Maize and Wheat Improvement Center (CIMMYT) and the Wheat Germplasm Bank was collected. By means of applying primer pairs and PCR conditions indicated in the Table 1, we observed no polymorphism for gene loci TaNAC2a, TaWRKY19 and Td29b. There was one fragment amplified only for each sample -fragment of approximately 227 base pairs (bp) for TaNAC2a gene locus, 160 bp for TaWRKY19, 86 bp for Td29b (Fig. 2).
Following the amplification of total genomic DNA of all common wheat varieties of Ukrainian and foreign origin, there were two fragments detected for each sample. We observed three different genotypes in the studied TaWRKY2 locus. The first one   Tables 2  and 3.    According to CDS sequence of TaWRKY2 (GenBank ID EU665425.1), the primer pair for this TF locus is likely to amplify the fragment of 188 bp long. Such a fragment was observed through the study; however, not all the wheat samples possessed it. Consequently, there must be an indel mutation, which is likely to form another allele.
The old wheat landraces is the source of potential genes of interest which can be of great value for common wheat improvement in modern breeding programs. Thus, the following screening of a number of wheat landraces and interspecific hybrids was carried out. The data were indicated in the    The study of the genes, that impact greatly on drought response, is of great value for wheat improvement in present-day plant breeding programs. The current research reveals knowledge on DNA polymorphism of three transcriptional factors (TaNAC2a, TaWRKY2, TaWRKY19) and the dehydrin (Td29b) genes which can be applied for MAS. During the analysis the gene structure and chromosomal location were established. Thus, TaWRKY2 and TaWRKY19 genes comprised of 4 exons and 3 introns (2BS and 1DS, respectively); TaNAC2a -2 exons and 1 intron (7AS); Td29b -single exon gene (3AS).
In the result of this study, no polymorphism was observed for gene loci TaNAC2a, TaWRKY19 and Td29b by means of preselected primer pairs. In contrast, polymorphic bands were detected for TaWRKY2 locus that did not correspond to CDS from GenBank. This fact indicated the presence of some other possible alleles of the gene.