Analysis of the Structural Organization and Expression of the Vrn-D1 Gene Controlling Growth Habit (Spring vs. Winter) in Aegilops tauschii Coss.

The duration of the vegetative period is an important agronomic characteristic of cereal crops. It is mainly influenced by the Vrn (response to vernalization) and Ppd (response to photoperiod) genes. In this work, we searched for alleles of several known genes of these two systems of response to external conditions in 15 accessions of Aegilops tauschii Coss. (syn. Ae. squarrosa L.), with the aim of studying the impact these alleles have on the vegetative period duration and growth habit. As a result, three allelic variants have been found for the Vrn-D1 gene: (i) one intact (winter type), (ii) one with a 5437 bp deletion in the first intron and (iii) one previously undescribed allele with a 3273 bp deletion in the first intron. It has been shown that the spring growth habit of Ae. tauschii can be developed due to the presence of a new allele of the Vrn-D1 gene. Significant differences in expression levels between the new allelic variant of the Vrn-D1 gene and the intact allele vrn-D1 were confirmed by qPCR. The new allele can be introgressed into common wheat to enhance the biodiversity of the spring growth habit and vegetative period duration of plants.


Introduction
The duration of the vegetative period is one of the most important adaptive and agronomic traits of cereal crops, which has a substantial impact on plant productivity and minimizes the impact of biotic and abiotic stresses [1].Cereal crops have three signal systems of response to external conditions that affect the duration of plants' vegetative period (earliness): (i) response to vernalization (Vrn genes), (ii) response to photoperiod (day length) (Ppd genes) and (iii) earliness per se (Eps genes) [2][3][4].
Vernalization is an impact of low positive temperatures, which is necessary to initiate spike formation [5].This is necessary to protect the floral meristems of winter plants from damage by negative temperatures in winter.Spring plants (S), which do not require exposure to low temperatures to initiate flowering, turned out to be more suitable for cultivation in the continental regions of the northern and southern latitudes from 40 • to 60 • [6].Genetic control of the response to vernalization in Ae. tauschii has been poorly studied.Most studies of the genetic regulation of these systems of response to external conditions have been carried out in the hexaploid wheat Triticum aestivum L., and so most of the current studies in this area are based on the loci identified in this species.In common wheat, four Vrn gene loci have been identified (VRN-1, VRN-2, VRN-3 and VRN-D4) and are noted for the following: changes to their structure cause differences in growth habit and response to vernalization [7][8][9][10][11][12][13]. The VRN-1 genes (Vrn-A1, Vrn-B1, Vrn-D1) and their paralog Vrn-D4 (the VRN-D4 locus) are MADS-box transcription factors homologous to the APETALA-1 (AP-1) gene in Arabidopsis thaliana (L.) Heynh.[7,[13][14][15].Expression of these genes in the leaves initiates the heading of wheat plants [16]; however, without exposure to low temperatures, their pre-mRNA binds to the RNA-binding protein TaGRP2, homologous to GRP7 in Arabidopsis, so the intact alleles are expressed at a low basal level [12,13,17].When wheat plants grow at low positive temperatures, TaGRP2 modifies O-GlcNAcylation, allowing it to interact with a carbohydrate-binding protein called VER2.The interaction between VER2 and O-GlcNAc-TaGRP2 reduces the amount of TaGRP2 in the nucleus, leading to the accumulation of the mRNA of the VRN-1 genes [17].The processes described are typical of winter varieties; however, mutations in the first intron of the VRN-1 and VRN-D4 genes, which affect TaGRP2 binding sites, as well as mutations in the promoters of the VRN-1 genes allow them to be expressed without exposure to low temperatures and thus to confer a spring growth habit [8,11,[18][19][20][21][22][23][24][25][26].The interaction diagram of VRN genes is shown in Figure 1.It has been shown that the main role of proteins encoded by the VRN-1 genes in the leaves is decreasing the expression levels of the Zinc-finger/CCT domain transcription factor-1 (ZCCT-1) and Zinc-finger/CCT domain transcription factor-2 (ZCCT-2) genes (VRN-2 locus) [16].The VRN-2 genes encode proteins containing zinc finger domains and CCT domains that suppress the expression of the VRN-3 genes in wheat and barley [18,27].The VRN-3 locus contains genes homologous to the FLOWERING LOCUS T (FT) gene in Arabidopsis thaliana (named TaFT in wheat) known as florigene [10].In wheat, these genes encode polypeptides that can bind to FDL2 proteins [28] and proteins in the 14-3-3 family [29] and are transported as a single complex from leaves to the apical meristem, where they interact with the promoter region of the VRN-1 genes [7,14].This causes the expression of the VRN-1 genes in the growth cone, which leads to the transition from the vegetative to the generative phase of development [15,28].It has been demonstrated that a spring growth habit can be conferred by mutations in the VRN-1, VRN-D4 and VRN-3 genes, which govern their expression without exposure to low positive temperatures (vernalization) [10,13,16], or due to mutations reducing the number of proteins encoded by the VRN-2 genes or changing their amino acid composition [30][31][32].
The interaction between VER2 and O-GlcNAc-TaGRP2 reduces the amount of TaGRP2 in the nucleus, leading to the accumulation of the mRNA of the VRN-1 genes [17].The processes described are typical of winter varieties; however, mutations in the first intron of the VRN-1 and VRN-D4 genes, which affect TaGRP2 binding sites, as well as mutations in the promoters of the VRN-1 genes allow them to be expressed without exposure to low temperatures and thus to confer a spring growth habit [8,11,[18][19][20][21][22][23][24][25][26].The interaction diagram of VRN genes is shown in Figure 1.It has been shown that the main role of proteins encoded by the VRN-1 genes in the leaves is decreasing the expression levels of the Zincfinger/CCT domain transcription factor-1 (ZCCT-1) and Zinc-finger/CCT domain transcription factor-2 (ZCCT-2) genes (VRN-2 locus) [16].The VRN-2 genes encode proteins containing zinc finger domains and CCT domains that suppress the expression of the VRN-3 genes in wheat and barley [18,27].The VRN-3 locus contains genes homologous to the FLOW-ERING LOCUS T (FT) gene in Arabidopsis thaliana (named TaFT in wheat) known as florigene [10].In wheat, these genes encode polypeptides that can bind to FDL2 proteins [28] and proteins in the 14-3-3 family [29] and are transported as a single complex from leaves to the apical meristem, where they interact with the promoter region of the VRN-1 genes [7,14].This causes the expression of the VRN-1 genes in the growth cone, which leads to the transition from the vegetative to the generative phase of development [15,28].It has been demonstrated that a spring growth habit can be conferred by mutations in the VRN-1, VRN-D4 and VRN-3 genes, which govern their expression without exposure to low positive temperatures (vernalization) [10,13,16], or due to mutations reducing the number of proteins encoded by the VRN-2 genes or changing their amino acid composition [30][31][32].Not only the Vrn genes but also the Ppd and Eps genes contribute to changes in the duration of the vegetative period.Of the Ppd genes, the ones that have the greatest influence on the trait are the homologous genes at the PPD-1 locus [33][34][35][36][37][38], which belong to the pseudoresponse regulator (PRR) family [37,[39][40][41][42]. Plants can develop earliness due Not only the Vrn genes but also the Ppd and Eps genes contribute to changes in the duration of the vegetative period.Of the Ppd genes, the ones that have the greatest influence on the trait are the homologous genes at the PPD-1 locus [33][34][35][36][37][38], which belong to the pseudoresponse regulator (PRR) family [37,[39][40][41][42]. Plants can develop earliness due to large deletions or insertions of transposons in the promoter region of the PPD-1 genes [37,38,43,44] as well as due to changes in the copy number of these genes (CNVs) [35].Earliness per se (Eps) is the difference in the heading time of the varieties that do not show differences in response to vernalization or photoperiod changes [45].Earliness per se is controlled by a number of minor genes, the effects of which can be determined only in the absence of vernalization and response to photoperiod effects [46].Although the genes and QTLs (quantitative trait loci) for earliness per se have been little studied [46], they are known to increase developmental rates in any growth phase [47].
It has been shown that the Vrn and Ppd genes are the main contributors to changes in the duration of the vegetative period of cereal crops [3,4,46].Furthermore, they can shorten the vegetative period in the presence of the dominant alleles of the Vrn and Ppd genes in the plant genome, either alone [37,48,49] or in combination [48,49].The genes at these loci found in T. aestivum L. have been poorly studied in the donor species of the elementary genomes of common wheat; however, they may contain valuable alleles of the Vrn and Ppd genes.Because information about their alleles is scarce, so is the pool of those from which plant breeding can benefit.Ae. tauschii is the donor of the D genome of hexaploid wheats [50][51][52][53][54][55][56][57].Most accessions of this species represent plants with a winter growth habit [58][59][60]; however, some of the spring accessions that were found there, too, had rather short vegetative periods [58][59][60].
Only a few mutations in the structure of the Vrn-D1 gene (VRN-1 locus) and the ZCCT-D1 and ZCCT-D2 genes (VRN-2 locus) of Ae. tauschii have been described [30,61,62].Detection of mutant alleles of the Vrn-D3 gene (VRN-3 locus) has not yet been reported.The sequence of the Vrn-D4 gene of Ae. tauschii is not yet known [13].It has been shown that the Vrn-D4 sequence is present only in the D genome of common wheat, where it developed due to a translocation between the long arm of chromosome 5A and a proximal region of 5DS [13].As was shown in wheat, spring varieties contain mutant alleles of the VRN-1 genes with various deletions and insertions in the promoter region and deletions in the first intron [8,11,[18][19][20][21][22][23][24][25][26].No changes in the promoter region of the Vrn-D1 gene in spring Ae. tauschii accessions have been found [26].At the same time, two mutations in the first intron have been described: a 5437 bp deletion and a 109 bp tandem duplication.The spring growth habit is induced only by this deletion, since the deleted sequence contains the binding sites for a repressor protein similar to TaGRP2 in wheat, which reduces the expression level of the wild allele Vrn-D1 in the absence of exposure to low temperatures [17,62].As was shown previously, deletions or mutations leading to the appearance of positively charged amino acids in the CCT domain are responsible for the recessive of the ZCCT-1 and ZCCT-2 alleles, which account for the spring growth habit of wheat and barley [9,31,63].Nonfunctional alleles of these genes in Ae. tauschii were found in a population originating from Iran [30,61].These accessions contained an allelic variant of the ZCCT-D1 gene with a 24 bp deletion in the first intron, located 6 bp upstream of the splicing acceptor site.Such a mutation alters splicing, resulting in the absence of translation of 35 amino acids in front of the CCT domain or in the translation of part of the intron [30].For the dominant gene ZCCT-D2, a shift of the reading frame was shown, which was due to a 1 bp deletion in the second exon, and for that reason the CCT domain in the translated protein was not observed [30].These mutations in the structure of the ZCCT-D1 and ZCCT-D2 genes cause dysfunction of the proteins they encode.
No mutant alleles of the Ppd genes with a confirmed effect on photoperiod sensitivity have been detected in Ae. tauschii [37,[64][65][66].In common wheat, the dominant allele Ppd-D1a with a 2089 bp deletion in the promotor region has been described in the D genome, due to which plants were able to complete ontogenesis under short-day conditions [37]; however, this allele appeared in the T. aestivum genome independently of Ae. tauschii [37,[64][65][66].There is only one report about the discovery of a dominant allele of the Ppd-D1 gene in Ae. tauschii [67]; however, nothing is known about the molecular characteristics of this allele.
As research into the genetics of Ae. tauschii is limited compared to common wheat, the alleles of its genes that affect vegetative period duration remain unknown [26,61,62], although they could be introgressed into commercial cultivars.
The aim of this work was to study the alleles of the Vrn and Ppd genes that affect the duration of the vegetative period in spring accessions of Ae. tauschii.As a result, two allelic variants of the Vrn-D1 gene have been found that differ from their wild-type counterpart.One variant of this gene had a known deletion (5437 bp) in the first intron.The other allele had a previously undescribed 3273 bp deletion in the first intron.Using real-time PCR (qPCR), we showed significant differences in expression level between the new allelic variants of the Vrn-D1 gene and the intact allele.

Growth Habit of the Ae. tauschii Accessions
The growth habit (spring/winter) was determined for 15 accessions of Ae. tauschii, eleven of which had spring habit, and four had winter habit (Table 1).The spring growth habit has been previously studied for four accessions: KU-2009 (Pakistan), K-608 (Georgia), K-992 (Afghanistan) and K-864 (origin unknown) [26], and the same was confirmed in the present study.In addition to these four, all other spring accessions developed spikes without preliminary vernalization no later than 20 days after the heading of the spring control accession, so they were classified as spring growth-habit accessions.The Palestinian, Chinese and Iranian accessions and the winter control K-1740 did not develop spikes 80 days after the heading of the spring accession KU-20-6.The significance of differences in vegetative period duration was confirmed using the LSD 0.05 criterion calculated with a one-way analysis of variance (ANOVA).It was found that accessions K-608, C21-51-43, C21-5118, C21-4030, C21-5112 were significantly superior to the spring control KU-20-6 in days from planting to heading (Table 1).No significant differences in the timing of heading were found between the winter accessions and the winter control K-1740 (Table 1).

Determination of the Structure of the Vrn Genes
In each of all 15 Ae.tauschii accessions studied, the structure of the first intron of the Vrn-D1 gene was analyzed.To determine the presence of an intact intron in the winter accessions, the PCR analysis was performed using the primers AetVrn-D1_Intr1F2 and AetVrn-D1_Intr1R2 (Figure 2C).For each winter accession, a 998 bp PCR product was obtained, indicating the presence of an intact intron (Figure 2A).In the spring accessions, the PCR analysis was performed to determine the presence of a 5437 bp deletion in the first intron (with the primers AetVrn-D1_Intr1F1 and AetVrn-D1_Intr1R1) (Figure 2C).For all accessions except the K-992 from Afghanistan, a 747 bp PCR product similar to that for the control KU-20-6 was obtained, which indicates the presence of this deletion in the first intron of these accessions (Figure 2B).No PCR product was found in K-992 (Figure 2B).LSD0.05 6.73 * Significant differences between accessions and controls are indicated.

Determination of the Structure of the Vrn Genes
In each of all 15 Ae.tauschii accessions studied, the structure of the first intron of the Vrn-D1 gene was analyzed.To determine the presence of an intact intron in the winter accessions, the PCR analysis was performed using the primers AetVrn-D1_Intr1F2 and AetVrn-D1_Intr1R2 (Figure 2C).For each winter accession, a 998 bp PCR product was obtained, indicating the presence of an intact intron (Figure 2A).In the spring accessions, the PCR analysis was performed to determine the presence of a 5437 bp deletion in the first intron (with the primers AetVrn-D1_Intr1F1 and AetVrn-D1_Intr1R1) (Figure 2C).For all accessions except the K-992 from Afghanistan, a 747 bp PCR product similar to that for the control KU-20-6 was obtained, which indicates the presence of this deletion in the first intron of these accessions (Figure 2B).No PCR product was found in K-992 (Figure 2B).It has been previously discovered that the spring growth habit can be formed due to small deletions in the region of the first intron of the ZCCT-D1 gene and the region of the second exon of the ZCCT-D2 gene.Therefore, we examined these critical gene regions in our accessions [30].PCR with the primer pairs AetZCCT-D1_Intr1F1, AetZCCT-D1_Intr1R1, and AetZCCT-D2_Ex2F1, AetZCCT-D2_Ex2R1 primers gave 302 bp and 539 bp products, respectively.Sequencing of the PCR products and alignment of the ZCCT-D1 and ZCCT-D2 sequences presented in GenBank showed the absence of any other mutation in these regions (information on the sequences obtained is given in the Data Availability Statement section).

Determination of the Structure of the Ppd Genes
In the spring accessions, the PCR analysis was performed with the primers Ppd-D1_F and Ppd-D1_R2 to determine the presence of a 2089 bp long deletion in front of the coding region of the Ppd-D1 gene.No PCR product was found in any of the accessions.PCR amplification using the primers Ppd-D1_F and Ppd-D1_R1 gave a 454 bp product, which indicates the presence of an intact region of this gene (Figure 3).It has been previously discovered that the spring growth habit can be formed due to small deletions in the region of the first intron of the ZCCT-D1 gene and the region of the second exon of the ZCCT-D2 gene.Therefore, we examined these critical gene regions in our accessions [30].PCR with the primer pairs AetZCCT-D1_Intr1F1, AetZCCT-D1_Intr1R1, and AetZCCT-D2_Ex2F1, AetZCCT-D2_Ex2R1 primers gave 302 bp and 539 bp products, respectively.Sequencing of the PCR products and alignment of the ZCCT-D1 and ZCCT-D2 sequences presented in GenBank showed the absence of any other mutation in these regions (information on the sequences obtained is given in the Data Availability Statement section).

Determination of the Structure of the Ppd Genes
In the spring accessions, the PCR analysis was performed with the primers Ppd-D1_F and Ppd-D1_R2 to determine the presence of a 2089 bp long deletion in front of the coding region of the Ppd-D1 gene.No PCR product was found in any of the accessions.PCR amplification using the primers Ppd-D1_F and Ppd-D1_R1 gave a 454 bp product, which indicates the presence of an intact region of this gene (Figure 3).

Analysis of the Nucleotide Sequence of the Intron of the Vrn-D1 Gene
Because the previously described mutations in the Vrn-D1, ZCCT-D1 and ZCCT-D2 genes were not found in spring accession K-992 and also because mutations in the VRN-1 genes are most often found in spring-type cereals [8,11,[18][19][20][21][22][23][24][25][26], we explored the structure of the Vrn-D1 gene in K-992.Mutations responsible for the spring growth habit are localized in the promoter region or in the first intron of the VRN-1 genes.It was previously shown that the promoter region of the Vrn-D1 gene in K-992 does not contain any disorders [26].For that reason, we carried out a PCR analysis with the primers AetVrn-D1_Intr1F2 and AetVrn-D1_Intr1R2 (Figure 2C) to confirm the presence of an intact allele of vrn-D1, but no PCR product was found.Presumably, the first intron of the Vrn-D1 gene in K-992 had a different deletion, which was shorter than the 5437 bp deletion in KU-20-6, but covered the binding region for the repressor protein identified in the spring varieties of wheat and barley [11,68,69].We developed a direct primer AetVrn-D1_Intr1F3 lying outside the critical region while being within the KU-20-6 deletion (Figure 2C).PCR analysis with the primers AetVrn-D1_Intr1F3 and AetVrn-D1_Intr1R1 gave a 275 bp product, which indicated the presence of a previously undescribed deletion in the first intron of the Vrn-D1 gene in accession K-992.
To determine the exact location of the deletion, we performed PCR amplification with the direct primer Ex1/C/F and the reverse primer AetVrn-D1_Intr1R1 (Figure 2C).The resulting 3500 bp PCR product was sequenced by the Sanger method using a forward and a reverse primer.The nucleotide sequence determined using the primer AetVrn-D1_Intr1R1 did not differ from its matching region in the Vrn-D1 gene, so we came to a conclusion that the deletion is located at the 5' end of the first intron.Sequencing using the primer Ex1/C/F and alignment against the nucleotide sequence of the intact allele of the Vrn-D1 gene in Ae. tauschi showed that K-992 contained a 3273 bp deletion in the first intron of the Vrn-D1 gene, which does not fully include the critical region that is responsible for the spring growth habit in wheat and barley [11,68,69] (Figure 4).It was also found that this deletion includes the RIP-3 sequence, an important region of the gene, affecting the degree of binding of the pre-mRNA gene to the protein TaGRP2 [13] (Figure 4).

Analysis of the Nucleotide Sequence of the Intron of the Vrn-D1 Gene
Because the previously described mutations in the Vrn-D1, ZCCT-D1 and ZCCT-D2 genes were not found in spring accession K-992 and also because mutations in the VRN-1 genes are most often found in spring-type cereals [8,11,[18][19][20][21][22][23][24][25][26], we explored the structure of the Vrn-D1 gene in K-992.Mutations responsible for the spring growth habit are localized in the promoter region or in the first intron of the VRN-1 genes.It was previously shown that the promoter region of the Vrn-D1 gene in K-992 does not contain any disorders [26].For that reason, we carried out a PCR analysis with the primers AetVrn-D1_Intr1F2 and AetVrn-D1_Intr1R2 (Figure 2C) to confirm the presence of an intact allele of vrn-D1, but no PCR product was found.Presumably, the first intron of the Vrn-D1 gene in K-992 had a different deletion, which was shorter than the 5437 bp deletion in KU-20-6, but covered the binding region for the repressor protein identified in the spring varieties of wheat and barley [11,68,69].We developed a direct primer AetVrn-D1_Intr1F3 lying outside the critical region while being within the KU-20-6 deletion (Figure 2C).PCR analysis with the primers AetVrn-D1_Intr1F3 and AetVrn-D1_Intr1R1 gave a 275 bp product, which indicated the presence of a previously undescribed deletion in the first intron of the Vrn-D1 gene in accession K-992.
To determine the exact location of the deletion, we performed PCR amplification with the direct primer Ex1/C/F and the reverse primer AetVrn-D1_Intr1R1 (Figure 2C).The resulting 3500 bp PCR product was sequenced by the Sanger method using a forward and a reverse primer.The nucleotide sequence determined using the primer AetVrn-D1_Intr1R1 did not differ from its matching region in the Vrn-D1 gene, so we came to a conclusion that the deletion is located at the 5' end of the first intron.Sequencing using the primer Ex1/C/F and alignment against the nucleotide sequence of the intact allele of the Vrn-D1 gene in Ae. tauschi showed that K-992 contained a 3273 bp deletion in the first intron of the Vrn-D1 gene, which does not fully include the critical region that is responsible for the spring growth habit in wheat and barley [11,68,69] (Figure 4).It was also found that this deletion includes the RIP-3 sequence, an important region of the gene, affecting the degree of binding of the pre-mRNA gene to the protein TaGRP2 [13] (Figure 4).The results of the study of the first intron structure of the Vrn-D1 gene, the study of the structure of the promoter region of the Ppd-D1 gene, and the determination of the nucleotide sequences of the first intron of the ZCCT-D1 gene and the second exon of the ZCCT-D2 gene are presented in Table 2.

Study of the Relative Expression Levels of Different Variants of the Vrn-D1 Gene in Ae. tauschii
To study the effect of the 3273 bp deletion on the expression level of the Vrn-D1 gene and to compare the expression profiles of different variants of this gene, we grew plants of three accessions: (i) winter K-1740, (ii) spring KU-2009 and (iii) spring K-992.
Days from planting to heading in the spring accessions KU-2009 and K-992 was 81 ± 9.4 and 48.5 ± 1.8 (t = 10.7),respectively, so we collected leaves for RNA isolation only until week 12 for KU-2009 and till week 7 for K-992.Winter plants K-1740 did not develop spikes during the entire vegetative period, so in this case we collected leaves for RNA isolation only till week 12, as we did for KU-2009.A quantitative comparison of the expression profiles revealed differences in the transcription levels of the Vrn-D1 gene in the leaves of different accessions at different vegetative periods (Figure 5).In K-1740, the Vrn-D1 expression level was low during all 12 weeks of development.Significant differences The results of the study of the first intron structure of the Vrn-D1 gene, the study of the structure of the promoter region of the Ppd-D1 gene, and the determination of the nucleotide sequences of the first intron of the ZCCT-D1 gene and the second exon of the ZCCT-D2 gene are presented in Table 2.
Table 2. Structural changes of the genes that control the duration of the vegetative period of the Ae.tauschii accessions studied.

Study of the Relative Expression Levels of Different Variants of the Vrn-D1 Gene in Ae. tauschii
To study the effect of the 3273 bp deletion on the expression level of the Vrn-D1 gene and to compare the expression profiles of different variants of this gene, we grew plants of three accessions: (i) winter K-1740, (ii) spring KU-2009 and (iii) spring K-992.
Days from planting to heading in the spring accessions KU-2009 and K-992 was 81 ± 9.4 and 48.5 ± 1.8 (t = 10.7),respectively, so we collected leaves for RNA isolation only until week 12 for KU-2009 and till week 7 for K-992.Winter plants K-1740 did not develop spikes during the entire vegetative period, so in this case we collected leaves for RNA isolation only till week 12, as we did for KU-2009.A quantitative comparison of the expression profiles revealed differences in the transcription levels of the Vrn-D1 gene in the leaves of different accessions at different vegetative periods (Figure 5).In K-1740, the Vrn-D1 expression level was low during all 12 weeks of development.Significant differences in Vrn-D1 expression level were revealed between the spring accession KU-2009 and the winter accession on weeks 10, 11 and 12 of development (Figure 5A); between the spring accession K-992 and each of K-1740 and KU-2009, starting from week 3 of development and throughout all subsequent weeks (Figure 5A).A comparison of Vrn-D1 expression level between K-992 and KU-2009 on week 5 before heading revealed a significantly higher number of transcripts in K-992 on week 4 before heading (Figure 5B).In the later weeks before heading, the differences in expression levels between different alleles were not significant (Figure 5B). in Vrn-D1 expression level were revealed between the spring accession KU-2009 and the winter accession on weeks 10, 11 and 12 of development (Figure 5A); between the spring accession K-992 and each of K-1740 and KU-2009, starting from week 3 of development and throughout all subsequent weeks (Figure 5A).A comparison of Vrn-D1 expression level between K-992 and KU-2009 on week 5 before heading revealed a significantly higher number of transcripts in K-992 on week 4 before heading (Figure 5B).In the later weeks before heading, the differences in expression levels between different alleles were not significant (Figure 5B).

Discussion
Mutations in the Vrn and Ppd genes are of great importance for early heading [3,4,45], so the study of their various allelic variants is very important for the breeding of early maturing cultivars.Ae. tauschii populations have spring accessions, in which mutant Vrn genes appeared independently of those in T. aestivum [30,61,62].Such alleles are very important for the transfer into common wheat, as they reduce the vegetative period of plants [30].For this reason, our primary plan was to determine which growth habit Ae. tauschii has in order to be able to study the allelic composition of the Vrn and Ppd genes in the spring accessions.
In 11 spring accessions, the structure of the first intron of the Vrn-D1 gene was studied.Ten of them had a 5437 bp deletion in the first intron, which had already been detected in Ae. tauschii [62].It has been shown that this deletion accounts for a spring growth habit, which is consistent with the studies carried out on common wheat [11,68,69] and was additionally confirmed by our results (Figure 2, Tables 1 and 2).None of the accessions that we have studied had other mutations in the ZCCT-D1, ZCCT-D2 or Ppd-D1 genes.One spring accession, K-992, had an allele of the Vrn-D1 gene that differed from that described in KU-20-6.As revealed by sequencing, K-992 had a 3273 bp deletion in the first intron of the Vrn-D1 gene, which partially covered the binding region for the repressor protein identified in some spring varieties of wheat and barley [11,68,69]; this deletion also included the RIP-3 region, nucleotide substitutions that had a great impact on the degree of binding of the protein TaGRP2 to the pre-mRNA of the wheat VRN-1 genes [13].Although this deletion does not completely cover the critical region of the first intron, it can be assumed that due to the removal of the RIP-3 region from the sequence, this deletion can influence the expression of the Vrn-D1 gene in the leaves.To test this assumption, we measured the relative expression level of the Vrn-D1 gene.

Discussion
Mutations in the Vrn and Ppd genes are of great importance for early heading [3,4,45], so the study of their various allelic variants is very important for the breeding of early maturing cultivars.Ae. tauschii populations have spring accessions, in which mutant Vrn genes appeared independently of those in T. aestivum [30,61,62].Such alleles are very important for the transfer into common wheat, as they reduce the vegetative period of plants [30].For this reason, our primary plan was to determine which growth habit Ae. tauschii has in order to be able to study the allelic composition of the Vrn and Ppd genes in the spring accessions.
In 11 spring accessions, the structure of the first intron of the Vrn-D1 gene was studied.Ten of them had a 5437 bp deletion in the first intron, which had already been detected in Ae. tauschii [62].It has been shown that this deletion accounts for a spring growth habit, which is consistent with the studies carried out on common wheat [11,68,69] and was additionally confirmed by our results (Figure 2, Tables 1 and 2).None of the accessions that we have studied had other mutations in the ZCCT-D1, ZCCT-D2 or Ppd-D1 genes.One spring accession, K-992, had an allele of the Vrn-D1 gene that differed from that described in KU-20-6.As revealed by sequencing, K-992 had a 3273 bp deletion in the first intron of the Vrn-D1 gene, which partially covered the binding region for the repressor protein identified in some spring varieties of wheat and barley [11,68,69]; this deletion also included the RIP-3 region, nucleotide substitutions that had a great impact on the degree of binding of the protein TaGRP2 to the pre-mRNA of the wheat VRN-1 genes [13].Although this deletion does not completely cover the critical region of the first intron, it can be assumed that due to the removal of the RIP-3 region from the sequence, this deletion can influence the expression of the Vrn-D1 gene in the leaves.To test this assumption, we measured the relative expression level of the Vrn-D1 gene.
When grown in field conditions, the winter accession used as the control did not ear as it possessed the recessive allele vrn-D1 with a low basal level of expression.Plants of spring accessions K-992 and KU-2009 with deletions in the first intron of the Vrn-D1 gene eared without prior exposure to vernalization and had a significantly higher amount of mRNA of the Vrn-D1 gene in the leaves than the winter control had.These results support the assumption that the 3273 bp deletion in the first intron of the Vrn-D1 gene causes expression without having to expose the plants to low temperatures, leading Ae.tauschii to develop a spring growth habit.The nucleotide sequences of both deletions in the first intron of the Vrn-D1 gene identified in spring accessions K-992 and KU-2009 as well as the sequences of the deletions in the first intron of the VRN-1 genes found in the spring wheat and barley plants contain the RIP-3 region [11,68,69].It was previously shown that nucleotide substitutions in this region can cause a weaker binding of the protein TaGRP2 to the nucleotide sequence [13], which results in a weaker plant response to vernalization [70].The Vrn-D4 gene located in the D genome of common wheat is a copy of the Vrn-A1 gene [12].It has an allelic variant with three nucleotide substitutions in the RIP-3 region, which forms a very weak bond with TaGRP2 and determines the expression of this gene even though there are no deletions in the first intron [13].Based on the available data, it can be assumed that the RIP-3 region is a key sequence required for binding the GRP2 family repressor protein to the pre-mRNA genes of the VRN-1 locus in cereal plants.Our data indirectly agree with the finding, since the sequence of the 3273 bp deletion found in the first intron of the Vrn-D1 gene in spring accession K-992 includes the RIP-3 region but does not completely cover the previously identified deletions conferring a spring growth habit on cereals [11,68,69].
When grown in field conditions, K-992 plants formed inflorescences earlier than KU-2009 plants, while the expression level of the Vrn-D1 gene in K-992 became significantly different from that of the KU-2009 allele as early as starting from week 3 of development.Such a relationship between the time of onset of VRN-1 expression and the heading time has been shown previously [20] and now was additionally demonstrated in our study.The difference in the time of heading and the time of appearance of the first transcripts of the Vrn-D1 gene between K-992 and KU-2009 is unquestionable; however, this is most likely not due to the difference in the allelic variants of the Vrn-D1 gene.
It may be noted that deletions in the first intron of the Vrn-D1 gene in all spring accessions analyzed in this study were detected, but there was not a single spring accession containing other known alleles of the Vrn and Ppd-D1 genes that differed from the wild types (Table 2).At the same time, several spring accessions with an allelic variant of the Vrn-D1 gene identical to that in KU-20-6 were significantly superior to the spring control in vegetative period duration (Table 1).The difference in vegetative period duration observed among the spring accessions of Ae. tauschii with the same 5437 bp deletion in the first intron of the Vrn-D1 gene has been described previously [62] and was confirmed in our study.Consequently, the presence of the same allelic variant of the Vrn-D1 gene in different spring accessions of Ae. tauschii did not form the same length of the growing season.
Spring accessions K-992 and KU-20-6 have Vrn-D1 alleles but do not differ significantly from each other in the duration of the vegetative period (Tables 1 and 2).Monogenic control of the growth habit in K-992 has been shown previously [71], which completely excludes the presence of other Vrn alleles in it, except for the allele of the Vrn-D1 gene that we discovered.Thus, taking into account all the above evidence, it can be assumed that the plants of the accessions we studied have different undescribed allelic variants of the genes that have an impact on vegetative period duration rather than growth habit.However, as entries in Table 1 confirm, the 3273 bp deletion in the first intron of Vrn-D1 accounts for the development of a spring growth habit by the Ae.tauschii accession.The effect of this deletion on the expression time of the Vrn-D1 gene and the earliness of plants can only be speculated and still remains to be confirmed.

Plant Material, Growth Conditions, Assessment of Vernalization Requirements and Determination of Heading Time
A total of 15 accessions of Ae. tauschii from various regions were taken as the material for the study.The seed progeny was obtained from each accession to assess the growth habit and heading time.A total of 10 seeds were sown per accession at the hydroponic greenhouse of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences without preliminary vernalization.Plants were grown at a temperature of 23-25 • C and a long day (16 h).KU-20-6 with the dominant allele of the Vrn-D1 gene was used as the spring control (plants that head without vernalization) [62].K-1740 with the intact allele of the Vrn-D1 gene was used as the winter control (plants that do not head without vernalization).Plants that did not ear without vernalization were transferred to a cold chamber and grown at 3-4 • C and 16 h photoperiod for 50 days, after which the growing conditions were set to standard (23-25 • C and 16 h photoperiod) and the plants were observed to be heading (such plants were referred to as winter plants).
The number of days from planting to heading was recorded for each plant individually.Based on the data obtained, the average value for each accession was calculated.The significance of the differences in vegetative period duration was confirmed by the difference in the sample means between the accessions and controls for each group (spring type/winter type) compared to the smallest significant difference (LSD 0.05 ) calculated by one-way analysis of variance.Accession voucher, place of collection, growth habit and heading time for each accession are presented in Table 1  Isolation of total DNA was carried out using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol.For extraction of DNA, 50-100 mg of leaves taken from plants of each accession were used.The quantity and quality of isolated DNA were determined using the NanoDrop2000 (Thermo Scientific, Waltham, MA, USA) spectrophotometer and electrophoretic separation in 1% agarose gel containing ethidium bromide (0.5 mg/mL) in 1 × TAE.The primer pairs used for PCR amplification and annealing temperatures are provided in Supplementary Materials Table S1.

Analysis of the Expression Level of the Vrn-D1 Gene, Isolation of Total RNA, cDNA Synthesis, Real-Time PCR Amplification and Evaluation of the Expression Level
To analyze the expression level at the experimental field of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, plants of three accessions were grown: spring accession K-992, spring accession KU-2009 and winter accession K-1740.Ten seeds of each accession were sown and not exposed to low positive temperatures (they were not vernalized).The number of days from sowing to heading in the spring plants was recorded for each plant individually, after which the mean sample value was calculated for each spring accession and the significance of differences was assessed using the Student's t-test.Winter plants were used as the control with a low basal value of the Vrn-D1 expression.Each week of plant growth from planting to heading, a leaf of the main shoot was picked.Since several leaves were formed on the shoots of Ae. tauschii, it was possible to obtain fresh material for RNA isolation throughout the entire vegetative period.The collection was made in the middle of the day to avoid the influence of circadian rhythms.Leaves of all accessions were frozen in liquid nitrogen and stored at −80 • C until use.
Total RNA was isolated from leaves using the LRU-100-50 kit (Biolabmix, Russia) according to the manufacturer's instructions.Leaves of three plants taken from each accession (three biological replicates) once per week were taken for 12 weeks.The concentration of isolated RNA was measured using the NanoDrop2000 (Thermo Scientific, USA) spectrophotometer and the quality of RNA was checked by electrophoretic separation in 1% agarose gel.cDNA was synthesized using the RNAscribe RT kit (Biolabmix, Russia), according to the manufacturer's instructions, after which it was diluted 5 times and used as a template for real-time PCR.
Real-time PCR was performed in the CFX96 Real-Time Touch amplifier (Bio-Rad, Hercules, CA, USA) with SYBR Green (Biolabmix, Russia) in three technical replicates.To study the relative expression level of the Vrn-D1 gene, we used three endogenous control genes: Actin (ACTIN), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Sadenosylmethioninedecarboxylaseproenzyme (SAMD).The primers for the ACTIN and SAMD genes were as in previous studies [25,72].For amplification of the GAPDH gene region, we developed original primers.For amplification of the Vrn-D1 gene, we also designed new primers that amplified the region of the seventh and eighth exons of the gene, similar to the pair of primers designed to study the expression of the Vrn-D1 gene in common wheat [73].The sequences of the primer pairs used for real-time PCR are presented in additional file 1: Table S2.The annealing temperature was the same for all primer pairs.Primer efficiency was tested by a series of dilutions of the cDNA template.A melt-curve protocol was performed for each primer pair to detect specific products.The expression level was calculated relative to the geometric mean of the expression levels of three endogenous control genes (ACTIN, GAPDH, SAMD) [74].The values obtained were used to plot the change in the relative expression level of the Vrn-D1 gene in three accessions.The significance of the differences in expression level was confirmed using the Student's t-test.

Conclusions
In this work, we explored the allelic composition of the Vrn and Ppd genes in 15 accessions of Ae. tauschii.Mutant alleles were found only for the Vrn-D1 gene, including a previously undescribed allelic variant with a 3273 bp deletion in the first intron.We found that the expression level of the new allele was significantly higher than that of the wild-type gene when the plants were grown without vernalization.Furthermore, the location of the detected deletion narrows the size of the DNA sequence in the first intron, in which the deletions lead to a spring growth habit in Ae. tauschii.The new allele can be introgressed into common wheat to enhance the biodiversity of a spring growth habit and vegetative period duration of wheat cultivars.

Figure 1 .
Figure 1.Diagram of interaction between VRN loci in leaves.The VRN-1 and VRN-D4 loci suppress the VRN-2 locus, which leads to the appearance of florigen (VRN-3 locus).The VRN-3 genes' protein is then transported through the phloem to the shoot apical meristem where it induces the meristemidentity genes and the initiation of the reproductive stage.

Figure 1 .
Figure 1.Diagram of interaction between VRN loci in leaves.The VRN-1 and VRN-D4 loci suppress the VRN-2 locus, which leads to the appearance of florigen (VRN-3 locus).The VRN-3 genes' protein is then transported through the phloem to the shoot apical meristem where it induces the meristemidentity genes and the initiation of the reproductive stage.

Figure 2 .
Figure 2. (A): Electrophoresis of the PCR products of the first intron of the Vrn-D1 gene with the primers AetVrn-D1_Intr1F2 and AetVrn-D1_Intr1R2.The 998 bp long region indicates the presence of an intact intron.(B): Electrophoresis of the PCR products of the first intron of the Vrn-D1 gene with the primers AetVrn-D1_Intr1F1 and AetVrn-D1_Intr1R1.The 747 bp long region indicates the presence of a 5437 bp deletion in the first intron.(C): The scheme of the Vrn-D1 gene and the position of the primers used.Gray rectangles, exons; black rectangles, introns; the 5437 bp deletion is in red.

Figure 2 .
Figure 2. (A): Electrophoresis of the PCR products of the first intron of the Vrn-D1 gene with the primers AetVrn-D1_Intr1F2 and AetVrn-D1_Intr1R2.The 998 bp long region indicates the presence of an intact intron.(B): Electrophoresis of the PCR products of the first intron of the Vrn-D1 gene with the primers AetVrn-D1_Intr1F1 and AetVrn-D1_Intr1R1.The 747 bp long region indicates the presence of a 5437 bp deletion in the first intron.(C): The scheme of the Vrn-D1 gene and the position of the primers used.Gray rectangles, exons; black rectangles, introns; the 5437 bp deletion is in red.

Figure 3 .
Figure 3. (A): Electrophoresis of PCR products of the promoter region of the Ppd-D1 gene with the primers Ppd-D1_F and Ppd-D1_R1.The presence of a 454 bp fragment indicates the absence of deletions upstream of the Ppd-D1 gene promoter.(B): Scheme of the Ppd-D1 gene and the position of the primers used.Gray rectangles, exons; black rectangles, introns; deletion 2089 bp in the promoter region is highlighted in red.

Figure 3 .
Figure 3. (A): Electrophoresis of PCR products of the promoter region of the Ppd-D1 gene with the primers Ppd-D1_F and Ppd-D1_R1.The presence of a 454 bp fragment indicates the absence of deletions upstream of the Ppd-D1 gene promoter.(B): Scheme of the Ppd-D1 gene and the position of the primers used.Gray rectangles, exons; black rectangles, introns; deletion 2089 bp in the promoter region is highlighted in red.

Figure 4 .
Figure 4. Position of deletions of the first intron of two allelic variants of the Vrn-D1 gene in Ae. tauschii.Gray rectangles, exons; black bars, introns; red bars, deletions.Yellow dots indicate the sequence of the RIP-3 region in the first intron.The designated link "Takumi S. et al. 2011" is located in the "References" section under number [62].The critical region is the region of the first intron, where deletions lead to the development of the spring growth habit in wheat and barley [11,68,69].

Figure 4 .
Figure 4. Position of deletions of the first intron of two allelic variants of the Vrn-D1 gene in Ae. tauschii.Gray rectangles, exons; black bars, introns; red bars, deletions.Yellow dots indicate the sequence of the RIP-3 region in the first intron.The designated link "Takumi S. et al. 2011" is located in the "References" section under number [62].The critical region is the region of the first intron, where deletions lead to the development of the spring growth habit in wheat and barley [11,68,69].

Figure 5 .
Figure 5. Change in the relative expression level of accessions with different alleles.Violet color indicates accession K−1740, blue-KU−2009, orange-K−992.Data for all accessions are expressed as mean value ± standard deviation (n = 3).Student's t−test was used for testing of statistical significance (* p < 0.05, ** p < 0.01).(A): comparison of K−1740, KU−2009 and K−992.(B): comparison of K−992 and KU−2009 five weeks before heading.The expression profile is shown from the second week of development for K−992 and from the seventh week for KU−2009.

Figure 5 .
Figure 5. Change in the relative expression level of accessions with different alleles.Violet color indicates accession K−1740, blue-KU−2009, orange-K−992.Data for all accessions are expressed as mean value ± standard deviation (n = 3).Student's t−test was used for testing of statistical significance (* p < 0.05, ** p < 0.01).(A): comparison of K−1740, KU−2009 and K−992.(B): comparison of K−992 and KU−2009 five weeks before heading.The expression profile is shown from the second week of development for K−992 and from the seventh week for KU−2009. .

Table 1 .
Ae. tauschii accessions used in the study, their growth habit (S, spring; W, winter), vegetative period and the significance of differences.
* Significant differences between accessions and controls are indicated.

Table 2 .
Structural changes of the genes that control the duration of the vegetative period of the Ae.tauschii accessions studied.