BACKGROUND
some plants of middle latitudes require vernalization treatment  to induce their flowering.
this adaptation appeared to protect flower from destruction by cold temperature during winter.
besides being an important trait for adaptation, the requirement of vernalization is also of a great agronomical importance.
in crops such as common wheat , a vernalization requirement distinguishes winter varieties from spring ones.
spring growth habit is a potential advantage in cold climates, with short vegetation season and cold winter, for agriculture which main task is to achieve rich harvest.
in continental middle latitude areas  the field is occupied for two vegetation seasons by winter forms and only for one season - by spring ones.
so spring forms allow obtaining two harvests instead of one of winter growth habit forms, which, moreover, do not overwinter every year.
due to this reason, the processes underlining vernalization and genes responsible for this agronomical feature have been of interest for many years  <cit> .
increasing knowledge about the molecular genetics of growth habit  would contribute to a better understanding of the evaluation of adaptation in crops.
this data would promote wheat breeding for specific environments.
in recent years, researchers made some breakthroughs in understanding of the molecular mechanisms of vernalization in wheat, a plant of prime agronomic importance.
sequences of three vernalization genes vrn <dig>  vrn <dig>  vrn <dig> responsible for spring-winter growth habit have been determine  <cit> .
the predicted model shows that the vrn <dig> gene is dominant for spring growth habit and it is the main initiating factor of the flowering regulatory cascade that is upregulated by vrn <dig>   <cit> .
whereas vrn <dig> gene is dominant for winter growth habit, it has been suggested that mediated protein  <cit> .
in a complementary study, it was shown that nucleotide polymorphisms or insertions-deletions on a and d copies of the wheat ft gene can also explain variations for heading date  <cit> .
investigation of the photoperiod influence on the regulation of vernalization genes showed that down-regulation of the vrn <dig> by switching from ld  to sd  treatment has no effect on the regulation of vrn <dig> until plants are transferred again to ld  <cit> .
these data suggest the existence of one more vrn <dig> repressor.
it has been demonstrated that functional difference between the dominant vrn <dig> and recessive vrn <dig> alleles in cultivated diploid t. monococcum l.  is due to deletions in the promoter region   <cit> .
vrn-a <dig> alleles of the common wheat  and the tetraploid t. turgidum l.  have also nucleotide deletions as well as insertions of mobile elements in vrn <dig> promoter and large deletions inside the first intron  <cit> .
at the same time, the dominant vrn-b <dig> and vrn-d <dig> genes are caused only by large deletions in the first intron  <cit> .
it is more likely that vrn <dig> promoter and the first intron regions contain the regulatory sites for different protein interactions.
carg-boxes are generally recognized by mads-box genes and considered as the critical regulatory site for the response of vrn <dig> to sd.
recently, this hypothesis was refuted  <cit> .
however, damages in either intron or promoter sites are sufficient to accelerate flowering under ld  <cit> .
these data suggest the existence of interactions between these two mechanisms in the regulation of the vrn <dig> gene expression.
most of the wild triticeae dum.
species have a winter growth habit, suggesting that the recessive vrn <dig> allele is the ancestral feature.
in contrast, the majority of cultivated polyploid wheats are spring species  <cit> .
only t. aestivum has the similar number of spring and winter forms http://www.vir.nw.ru and adaptability of landraces differed depending on their growth habit and vrn genotype  <cit> .
their growth habit is due to at least one dominant vrn <dig> allele.
this allele could be either inherited from ancestral diploids or could result from selection of independent mutations that appeared during the adaptation to various growing condition after domestication.
moreover, genetic and molecular experiments show that there are multiple alleles of dominant vrn <dig>  and their manifestation in vernalization response is also different  <cit> .
it means that some mechanisms exist, for which evolutionary background is unknown.
in the present study, molecular variability of wheat vrn <dig> promoter region was analyzed.
in order to get a more detailed insight into complicated molecular basis of spring vs. winter growth habit and associated vernalization genes variability, we studied wild and cultivated wheats together with their close relatives aegilops l. in total,  <dig> accessions of four diploid species  and  <dig> accessions of  <dig> polyploid species belonging to three known sections  were investigated.
seven accessions of goatgrasses belonging to  <dig> species , which participated in the hybridization events in polyploidy wheat evolution, were also studied.
among all  <dig> investigated accessions, eight have winter growth habit, and the remaining - spring growth habit.
the number of genes responsible for spring vs. winter growth habit was estimated in the genetic experiments for ten spring accessions.
RESULTS
genetic analysis of growth habit
for the molecular characterization, it was important to study accessions that possess monogenic control of spring growth habit.
genetic experiments were carried out to demonstrate genetic control of growth habit in some spring wheat and goatgrass species.
the obtained data are given in tables  <dig> and  <dig>
* - values for significance of p =  <dig>  is  <dig>  and p =  <dig>  is  <dig> ; spring accessions are underlined
bwe - cv.
t. dicoccum black winter emmer, i: bs1e - black spring vrn-a1
emmer, i: bs2e - black spring vrn-b <dig> emmer, kristall - introgressive spring line of t. durum winter cv.
kristall with dominant gene vrn-d <dig>
it was established that spring growth habit of most of examined species was controlled by a single dominant gene.
in the table  <dig> the segregation ratio  <dig> to  <dig> is reliable while the value of pierson chi-square test for the segregation ratio  <dig> to  <dig> is higher than the value of significance  <dig> .
three investigated tetraploid species were crossed with near-isogenic test lines, which were characterized with specific dominant vrn-a <dig> , vrn-b <dig>  alleles.
the design of near-isogenic lines is described in methods section.
in crosses with t. dicoccum cv.
black winter emmer , which has recessive vrn-a <dig> and vrn-b <dig> alleles, it was shown that spring growth habit of these species was controlled monogenic, because the segregation ratio  <dig> to  <dig> in f <dig> generation is reliable.
for t. turanicum  monogenic control was shown indirectly from the next two crosses, where the digenic segregation  was obtained for all three species.
it means that dominant allele responsible to spring growth habit is not allelic to vrn-a <dig> and vrn-b <dig> alleles of the tested lines.
in tetraploid wheat species, the dominant gene of the accessions k- <dig> and k- <dig> was temporarily designated as vrn-t and their genotypes were specified as vrn-a1vrn-a <dig> vrn-b1vrn-b <dig> vrntvrnt.
it is an unknown allele, which is nonallelic to vrn-a <dig> and to vrn-b <dig>  the number and allelity of dominant genes vrn <dig> in t. timopheevii  zhuk.
was not determined because all accessions of this species had only spring growth habit  <cit> .
the generalized information concerning genetic control of growth habit is represented in table  <dig>
triticum l.
section urartu dorof.
et a.filat.
auau
section monococcon dum.
abab
section dicoccoides flaksb.
bbauau
vrn-a1u2
vrn-a1u1
vrn-b1a2
section triticum
bbauaudd
vrn-b1
vrn-b1
vrn-a1u2
vrn-b1
vrn-a1a1
vrn-a1u1
vrn-b1
5r
vrn-b1
vrn-a1u3
vrn-b1
section timopheevii a. filat.
et dorof.
ggabab
vrn-a1u1
vrn-a1f2
vrn-a1u3
aegilops l.
section sitopsis  zhuk.
ss
section vertebrata zhuk.
emend.
kihara
dd
allelic variability at the vrn <dig> promoter region of diploid species
variability at the primer annealing site
in the present work,  <dig> diploid wheat accessions of four known species  and  <dig> goatgrass accessions of two species  were studied .
initial pcr screening was provided with primer pair ap1_prodel_f <dig>  ap1_prodel_r <dig> .
the pcr product of the expected size of  <dig> bp has been obtained in the majority of the studied wheat accessions and in one goatgrass species, ae.
speltoides .
no pcr products have been found in ae.
squarrosa accessions.
out of  <dig> wheat accessions, ten showed pcr products of the lower size, which can be explained by deletions in the promoter region.
the heterogeneity of diploid genomes and their sequence variability was observed previously  <cit> .
all available vrn <dig> promoter sequences belonging to different wheat genomes  were extracted from genbank and aligned together with primer sequences.
there were a  <dig> bp deletion in d genome near the region complementary to the reverse primer , and a duplicated fragment  near this region in a genome .
therefore, a new primer pair was developed  for amplification of d genome.
to check for possible minor deviations connected with spring growth habit, all fragments obtained in pcr using both primer pairs were directly sequenced.
the resulting sequences were submitted to genbank: gq451714-gq <dig>
the presence of the specific deletions in spring wild and cultivated accessions
among  <dig> investigated spring diploid wheats,  <dig>  had  <dig> bp or  <dig> bp deletions in vrn <dig> promoter region comparing with winter ones .
both deletions covered the specific area of mads-box, and were previously described as associated with spring growth habit in the diploid cultivated wheat t. monococcum  <cit> .
spring t. boeoticum accessions were not screened previously as well as t. urartu.
investigation of the vrn <dig> promoter mutations in both species may provide important information about mechanisms involved in the origin of the spring forms in these wild natural species.
among four spring t. urartu accessions, none had specific deletions within the vrn <dig> promoter region compared to winter forms.
however, one accession was almost identical to the a genome sequences of the polyploid wheats .
this recessive allele was named vrn-a1u and was not found in all analyzed t. boeoticum and t. monococcum sequences .
the closest relatives of triticum l. species are aegilops l. species, which also have spring and winter forms.
in order to address the question whether flowering initialization in spring goatgrasses has common characters with that of diploid wheats, we obtained vrn <dig> promoter regions from six spring and one winter aegilops accessions.
there were some nucleotide substitutions differentiating goatgrasses from wheats .
no significant changes, which could distinguish spring and winter accessions, have been found.
all investigated promoter sequences of the ae.
squarrosa accessions contained the specific d genome  <dig> bp deletion  and some d genome specific nucleotide substitutions.
allelic variation at the vrn <dig> promoter region in polyploid wheats
tetraploid wheat species
vrn <dig> promoter regions from g genomes of spring t. timopheevii  and t. araraticum jakubz.
were amplified using primer specific for b genome.
we analyzed also a tetraploid line determined by karyological analysis , which was found in an accession of t. urartu from lebanon.
sequencing showed a close relatedness of the obtained g genome to the corresponding b genome .
the most interesting was the presence in t. timopheevii g genome and two clones of t. turanicum jakubz.
b genome of several identical substitutions and large insertions in - <dig> position upstream from the start codon.
the insertions were  <dig> bp long in g genome and  <dig> bp in b genome .
we named these vrn <dig> promoter variants vrn-g1a and vrn-b1a.
these insertions were similar to a  <dig> bp foldback element  observed in vrn-a1a allele and named "spring" .
however, the site of insertion was different.
it was not in the place of the carg box  and was flanked by another host direct duplication  .
more detailed analysis of the mobile elements which were found in vrn-a <dig>  vrn-b <dig> and vrn-g <dig> promoter regions of tetra- and hexaploid wheat species showed the presence of the terminal inverted repeats  and predicted open reading frames between both repeats .
the observed transposon element, which is  <dig> bp in length with  <dig> bp tir, could belong to the superfamily tc1/mariner.
the vrn1promoter regions from a genomes of spring tetraploids also contain different promoter damages and share some variability.
in addition, a  <dig> bp deletion was found in the region - <dig> and - <dig> nucleotide from start codon.
this deletion was located in the same region where the foldback element insertion occurred in the g genome .
this vrn <dig> promoter allele was called vrn-a1f.
species of the section dicoccoides, t. turanicum  and t. dicoccum  schuebl.
also had a common  <dig> bp deletion in the region - <dig> - <dig> upstream from the start codon.
this deletion included the repetitive region and was observed previously in vrn-a1b and vrn-a1d alleles of spring tetraploid t. durum desf.
and t. dicoccoides correspondingly  <cit> .
we observed also a  <dig> bp deletion in a genome of t. durum , odessa) that was similar to vrn-a1e alleles of t. dicoccum.
vrn <dig> promoter from a genome of t. dicoccum  displayed identity with vrn-a1a allele observed previously in t. aestivum near-isogenic line triple dirk d  and contained the insertion of a foldback element.
hexaploid wheat species
promoter sequences of vrn-a <dig>  vrn-b <dig> and vrn-d <dig> alleles from two winter cultivars  and a, b and d genome sequences of spring mute mironovskaya yarovaya were determined.
the spring mute was found in cultivar mironovskaya  <dig>  vrn <dig> promoter fragments of winter cultivars and b, d genome of spring mironovskaya yarovaya contained a common for certain genomes variability observed in recessive alleles of winter wheat near-isogenic line triple dirk c   <cit> .
after cloning and sequencing of a genome fragment of spring sample, clones with and without specific deletions were observed.
the observed deletions included  <dig> bp del in - <dig> - <dig> region and  <dig> bp del in - <dig> - <dig> region that were common for the vrn-a1f allele described above in tetraploid species .
three t. aestivum cultivars pyrothrix  <dig>  osijek and jupateko with "weak" vrn <dig> alleles   <cit>  were also included in the research.
no differences from recessive alleles were found in vrn <dig> promoter sequences from a, b and d genomes of spring cvs.
osijek and pyrothrix 28; as well as from b and d genomes of spring cv.
jupateko.
after amplification of the vrn <dig> promoter region from a genome of the cv.
jupateko, two pcr products were visualized on electrophoresis.
sequencing of two larger pcr products revealed the presence of the foldback element insertions that was common for vrna1-a allele  <cit> .
a  <dig> bp foldback element insertion differed moderately from those previously described.
the site of insertion and flanking direct repeats  were similar for both elements.
however, analysis of the foldback element of spring cv.
jupateko indicated an absence of direct repeats inside .
these direct repeats were the sites of deletions in the duplicated promoter sequences of vrn-a1a allele.
therefore, we detected only one variant of the promoter with mobile element insertion in this species.
the smaller pcr products in cv.
jupateko had most common character with vrn-a1f allele observed in k- <dig> t. timopheevii.
thus, two different vrn-a <dig> alleles were detected.
both alleles could be the cause of spring growth habit.
we also found promoter damages in the a genomes of two other investigated intrageneric t. aestivum: substitution line saratovskaya/vietnamskaya 5r and line nulli5b-tetra5d chinese spring.
the investigated promoter regions contained vrn-a1b and vrn-a1f alleles correspondingly .
both alleles could be a reason of spring growth habit and likely inherited from tetraploid species during cultivation and breeding programs.
variability of different clones inside a genome
investigation of a genome sequences revealed at least two different variants of vrn <dig> promoter in some cloned samples according to the presence of indels.
at the same time all cloned b and d genome sequences were identical in the presence or absence of indels.
to exclude any doubt in affiliation with a specific genome, we provided the phylogenetic analysis, which allowed the correlation of each sequence into the genome .
each of three accessions of diccoccoides section  had two different variants of the promoter in a genome.
one of them was intact as in winter vrn-a <dig> allele, and another had a deletion or insertion of the transposon .
vrn <dig> promoters of the remaining three examined species , t. turgidum k-16156) were sequenced directly without cloning and contain only one variant of sequence judging from the absence of superpositions in the chromatograms.
in section timopheevii four clones of t. araraticum  were similar if indels are taken into consideration.
t. timopheevii  had four clones similar to those of t. araraticum  containing vrn-a1f allele and one clone with intact vrn-a <dig> allele.
absence of the previously determined a genome specific  <dig> bp insertion in the region - <dig> - <dig> were observed in some clones of the tetraploid species.
only a genome had variability at this region while b, g and d genomes as well as genomes of the examined goatgrasses were conservative and had deletions in this place.
all obtained vrn-a1f allele had no  <dig> bp insertion.
seven clones of vrn <dig> promoter region from a genome were sequenced in tetraploid line discovered in t. urartu collection under ac.n.pi <dig> and later was determined as tetraploid according to karyological and molecular analyses.
four different promoter variants were found.
at the same time, no positive result was obtained in pcr with b/g genome specific primers for vrn <dig> promoter region and developed previously for acc- <dig> and pgk- <dig> genes  <cit> .
three spring cultivars of hexaploid t. aestivum also contain at least two vrn <dig> promoter variants in a genomes .
vrn <dig> promoter region of a genome of cv.
mironovskaya yarovaya and line nulli5b-tetra5d chinese spring included the recessive vrn-a <dig> and the dominant vrn-a1f alleles.
cultivar jupateko had in addition the third allele differed from others by a transposon insertion.
predicted regulatory elements inside vrn <dig> promoter region
in the present work, we performed the analysis of the cis-regulatory elements  inside the most variable region of the vrn <dig> promoter.
this information helps to investigate the consequences of the damages in the promoter of the main initiation factor of flowering, and possibly is related to the mechanism of growth habit changing.
previously, a specific carg-box common for all mads-box protein family was found in the region where different in size deletions and transposon insertion occurred in spring diploid and polyploid wheat species.
it was suggested that carg-box could be involved in the regulation of vrn <dig> by interacting with other mads-box proteins  <cit> .
our analysis showed that this region also included recognition site for hmg <dig>  a high mobility globular protein, with a high probability .
recently, an increased expression of wheat homolog hmg1b protein after cold exposure of crown tissue was showed  <cit> .
the hmgb <dig> is thought to interact with histones and could participate in modifying chromatin structure.
some other regulatory sites were found in the region of  <dig> bp and  <dig> bp deletions common for vrn-a1b and vrn-a1f alleles, correspondingly .
we also observed the recognition site  for bzip proteins.
the vrn <dig>  protein was shown to upregulate vrn <dig> expression via interaction with tafdl members of bzip transcription factor family  <cit> .
moreover, it appears that tafdl <dig> protein can interact in vitro with acgt elements of the promoter of the meristem identity gene vrn <dig>  including g-box and g/c hybrid of g- and c-boxes .
it is interesting that both transposon insertions observed in b and g genomes contain additional g-box motif  .
near the region of the g/c hybrid box we found the binding motifs for myod-like protein, a member of the bhlh family of transcription factors  .
members of the bhlh family in animals have been shown to regulate the determination and differentiation of a variety of cell types, including skeletal muscle, neurons, and hematopoietic cells.
recent detailed study into the physical interaction among the bhlh proteins of arabidopsis that control stomatal development reflected that a similar mechanism functions in animal muscle and plant stomata  <cit> .
it is known also that markers of myogenic specification such as myod and myogenin gene, which encode transcription factors of the basic helix-loop-helix family, interact with proteins belonging to the mads-box transcription factors  <cit> .
it is likely that examined bhlh proteins  modulate cell cycle regulatory protein activity in a similar fashion in both animal and plant cells.
a binding motif was also found for gata <dig> protein  .
gata transcription factors are a group of dna binding proteins broadly distributed in eukaryotes.
four classes of these proteins are described in plants  <cit> .
some gata transcription factors are downstream effectors of floral homeotic gene action in arabidopsis  <cit> .
it appears that two mads-box gene products, apetala <dig>  and pistillata  can directly interact with two members of gata transcription factor family.
altogether they regulate a variety of light and nutrient responsive genes participating into the transcriptional cascades controlling the specification of floral organ identities.
in particular, apetala <dig> gene  is directly regulated by ap <dig> and pi in arabidopsis  <cit> .
transcription factor binding motifs described here are found in the vrn <dig> promoter region where indels could induce flowering without cold treatment.
this could indicate a possibility that wheat homologs of the hmg <dig>  bzip, bhlh and gata <dig> proteins are involved in the vernalization response.
further experimental approaches are necessary to support this suggestion.
discussion
triticum genetic material
notwithstanding the short evolutionary time since the domestication, some significant morphological changes were accumulated in cultivated plants.
as a result of an intensive cultivation, many wheat varieties with different agronomical important characteristics were selected.
however, in many cases the molecular pathway behind the appearance of a sample with an unusual character in the stable population is still unknown.
winter growth habit is assumed to be a more ancestral character than the spring one.
it is not known whether the ancient farmers found spring forms in nature or in their plots.
however, they started to cultivate spring forms intensively.
among the first wheat species cultivated by people, t. monococcum and t. dicoccum, most have spring forms according to the last investigation of  <dig> and  <dig> accessions correspondingly  <cit> .
the winter forms came back to occupy considerable areas in the middle latitudes at the beginning of the 20th century.
but in the continental middle latitude areas  the field is occupied by winter forms for two vegetation seasons and by spring forms - for one season only.
this is due to the agronomical aspects of spring forms - sowing in spring when winter is too cold.
in these countries it is impossible to use the intermediate forms, which are used in southern europe and mediterranean region, because of climate, when spring crop can not survive in winter.
it appears that, when both forms are used, winter ones are often less economically beneficial.
for this purpose, mutants with spring alleles of vrn <dig> in different genomes  were developed.
multiple alleles have been described within the vrn <dig> locus, based on their different effects on vernalization requirement and flowering time  <cit> .
however, characterization of these alleles at the molecular level is necessary.
the number of dominant vrn genes in common wheat is important since it modifies spring growth habit, and allows for the development of commercial cultivars with different vegetative periods.
studying the unique collection of spring wild and domesticated diploid and polyploid wheats not examined before allowed us to observe a greater variability, especially in tetraploid and hexaploid species.
we described three additional vrn <dig> promoter variants vrn-a1f, vrn-a1g and vrn-a1h.
moreover, we showed that vrn <dig> promoter region of b and g genomes can also contain damages such as the insertion of a transposable element.
the duplication of the vrn <dig> promoter together with the first exon was also demonstrated.
inheritance of vrn <dig> gene from spring wild diploids to cultivated diploids
it is known that wild diploid wheats were the donors of a genome in polyploid wheat species.
diploid species are the most ancient in the triticum genus so their genome  has the longest history in the genome composition of the modern wheat species.
the majority of the wild wheats have a winter growth habit, suggesting that the recessive vrn-a <dig> allele with an intact vrn <dig> promoter is the ancestral character.
we studied the number of wild and cultivated diploid species, and established that vrn <dig> promoter deletions that resulted in the elimination of repressor influence in cultivated species t. monococcum are also common for wild species t. boeoticum.
therefore, it is possible to conclude that spring forms originated in wild populations and later were selected by man for culture.
new alleles may result from selection of independent mutations that appeared during the domestication.
it is likely that a shorter  <dig> bp deletion appeared first and then increased to  <dig> bp deletion.
both variants were inherited by cultivated diploid forms and could be inherited also by polyploids.
species of the genus aegilops are closely related to wheats; they have common geographic range, and about  <dig>  million years ago a first polyploid wheat appeared as a result of hybridization of wheats with goatgrasses  <cit> .
similar to wild diploid spring triticum species, there are early-flowering forms among aegilops species, which evolved in two intraspecific lineages, middle asian and transcaucasian  <cit> .
the existence of longitudinal and latitudinal clines in the onset of flowering at the species level was observed; however, mechanism controlling switch from vegetative to generative growth habit is unknown.
to answer the question whether these mechanisms have common characters with that of diploid wheats we analyzed vrn <dig> promoter regions of several aegilops accessions.
spring ae.
squarrosa accessions originated from the northern part of afghanistan and iran while in other regions only winter forms were usually found  <cit> .
however, no indel events were found inside the investigated region.
therefore, taking into account the genealogical and ecological framework complexity of flowering time diversification in the investigated aegilops accessions we expect the existence of another regulatory mechanism controlling flowering time in goatgrass species.
increasing genetic variability in spring tetraploids within a, b, and g genomes
in contrast with wild triticum species, the majority of cultivated polyploid wheat species have spring growth habit  <cit> .
spring forms are due to at least one dominant mutation in vrn <dig> gene that could be inherited or appeared independently during domestication or historical selection process.
four different variants of vrn <dig> promoter in a genomes of diploid species were observed .
the main difference between sequences is the presence or absence of a specific  <dig> bp deletion that was not observed only in one spring accession of t. urartu and inherited by a genome of many polyploids.
in comparison with diploid species three other new vrn <dig> promoter variants due to deletions appeared in tetraploids.
one of them represents vrn-a1e allele and contains  <dig> bp deletion in place where specific  <dig> and  <dig> bp deletions occurred in diploids.
possibly there was a deletion expansion in tetraploids but independent appearance is also likely due to the existence of flanking direct repeats, which may be the reason of nonhomologous recombination .
there are many different repeat regions inside vrn <dig> promoter and most of the deletions flanked by short direct repeats .
it was shown that unequal recombination by such repeats can play the main role in the origin of novel alleles in vrn <dig> gene  <cit> .
the vrn-a1e allele could have originated from the allele containing  <dig> bp insertion and observed only in one wild diploid species t. urartu.
another vrn-a1b allele found in tetraploids contains a  <dig> bp deletion .
its origin is also related to t. urartu vrn <dig> promoter containing  <dig> bp insertion .
the described deletion most likely happened in species of dicoccoides section.
this allele is not found in wheat species from monococcon dum.
and timopheevii sections.
at the same time, vrn-a1f allele likely occurred in timopheevii section within species with g genomes .
moreover, its origin is related to diploid vrn <dig> promoter containing  <dig> bp deletion.
we found this allele also in t. turanicum ; this, however, is likely a case of hybridization event that may occur between tetraploid species from different wheat sections.
the vrn <dig> promoter variant found in t. dicoccum accession  had all common characters of vrn-a1a allele as well as the transposon insertion .
taking into consideration the origin of bs1e line, as the near-isogenic line of cv.
black winter emmer whose vrn-a <dig> allele was replaced with vrn-a1a of hexaploid wheat triple dirk  by backcrossing, this proves its derivation from t. aestivum near-isogenic line tdd.
we found and described also two different alleles in b and g genomes of tetraploid species that contain a transposon insertion .
the indel events in these genomes were observed for the first time, and both species contained also vrn <dig> promoter damages in a genomes.
investigated hexaploid accessions of section triticum inherited all alleles from tetraploid species except one with transposon insertion that most likely happened at this ploidy level .
evidence of the vrn <dig> promoter duplication
in the present work, two and more vrn <dig> promoter copies were detected and distinguished by the presence of indel events within a genome of several spring polyploid species.
there are three possible explanations of these results:  nonspecific duplication of different genomes,  heterozygous plant material with two different chromatids in one genome,  duplication of the investigated region.
the first possibility was tested by the presence of the nucleotide substitutions and indel events specific to different genomes.
for more reliable results the phylogenetic tree was constructed that demonstrated relatedness of each clone to certain genomes.
results showed an absence of nonspecificity and supported attributes of each clone to genome which amplification product was cloned.
the genetic experiment and clearance of each examined lines exclude the second possible case.
all accessions with two and more vrn <dig> promoter variants contained one intact promoter that was observed in winter plants.
however no splitting was detected in five generations of each line.
the most likely explanation is the duplication of the investigated region that occurred in polyploid species.
previously it was supposed the duplication of the promoter region with the first exon in vrn-a1a allele  <cit> .
in most cases we found two copies of the promoter that differed by the presence of deletions.
exceptions were accessions of t. dicoccoides  and cv.
jupateko of common wheat .
in most polyploid vrn <dig> promoter variants, an ancestral form is an intact winter allele found only in t. urartu accession.
the second vrn <dig> promoter sequences that was found in diploids and contained  <dig> bp del was inherited by some tetraploids of section timopheevii.
in the genome of ancestor species of this group additional  <dig> bp deletion occurred.
it is most likely that duplication of the promoter region occurred before deletion formation and transposon insertion.
moreover, deletions occurred in polyploid species independently from diploid species.
therefore, spring forms, which resulted from indels, were not inherited from spring diploid species.
the obtained data demonstrated that quite complicated molecular alterations occurred in the studied region of spring polyploid species.
two main underlying causes of such dynamic evolutionary changes seem to be polyploidy and intensive breeding of agronomic characters.
CONCLUSIONS
in the present study we investigated variability in the promoter region of vernalization gene vrn <dig> in spring wheat accessions.
a greater variability in the vrn <dig> promoter region of polyploid species in comparison with diploid ones was observed.
in diploid species, damages of vrn <dig> promoter leading to spring growth habit were inherited by cultivated forms from the wild.
a complicated evolutionary process including duplication of vrn <dig> promoter was found in a genome of polyploids.
different indel events likely occurred in spring polyploid species independently from spring diploid species.
specific transposon insertions found in a genome as well as in b and g genomes of spring tetraploid species pointed to active transposition in polyploid genome.
some transcription element binding sites were observed in silico in the regions of indel events.

 some plants of middle latitudes require vernalization treatment to induce their flowering. this adaptation appeared to protect flower from destruction by cold temperature during winter. besides being an important trait for adaptation, the requirement of vernalization is also of a great agronomical importance. in crops such as common wheat, a vernalization requirement distinguishes winter varieties from spring ones. spring growth habit is a potential advantage in cold climates, with short vegetation season and cold winter, for agriculture which main task is to achieve rich harvest. in continental middle latitude areas the field is occupied for two vegetation seasons by winter forms and only for one season - by spring ones. so spring forms allow obtaining two harvests instead of one of winter growth habit forms, which, moreover, do not overwinter every year. due to this reason, the processes underlining vernalization and genes responsible for this agronomical feature have been of interest for many years. increasing knowledge about the molecular genetics of growth habit would contribute to a better understanding of the evaluation of adaptation in crops. this data would promote wheat breeding for specific environments. in recent years, researchers made some breakthroughs in understanding of the molecular mechanisms of vernalization in wheat, a plant of prime agronomic importance. sequences of three vernalization genes vrn vrn vrn responsible for spring-winter growth habit have been determine. the predicted model shows that the vrn gene is dominant for spring growth habit and it is the main initiating factor of the flowering regulatory cascade that is upregulated by vrn. whereas vrn gene is dominant for winter growth habit, it has been suggested that mediated protein. in a complementary study, it was shown that nucleotide polymorphisms or insertions-deletions on a and d copies of the wheat ft gene can also explain variations for heading date. investigation of the photoperiod influence on the regulation of vernalization genes showed that down-regulation of the vrn by switching from ld to sd treatment has no effect on the regulation of vrn until plants are transferred again to ld. these data suggest the existence of one more vrn repressor. it has been demonstrated that functional difference between the dominant vrn and recessive vrn alleles in cultivated diploid t. monococcum l. is due to deletions in the promoter region. vrn-a alleles of the common wheat and the tetraploid t. turgidum l. have also nucleotide deletions as well as insertions of mobile elements in vrn promoter and large deletions inside the first intron. at the same time, the dominant vrn-b and vrn-d genes are caused only by large deletions in the first intron. it is more likely that vrn promoter and the first intron regions contain the regulatory sites for different protein interactions. carg-boxes are generally recognized by mads-box genes and considered as the critical regulatory site for the response of vrn to sd. recently, this hypothesis was refuted. however, damages in either intron or promoter sites are sufficient to accelerate flowering under ld. these data suggest the existence of interactions between these two mechanisms in the regulation of the vrn gene expression. most of the wild triticeae dum. species have a winter growth habit, suggesting that the recessive vrn allele is the ancestral feature. in contrast, the majority of cultivated polyploid wheats are spring species. only t. aestivum has the similar number of spring and winter forms http://www.vir.nw.ru and adaptability of landraces differed depending on their growth habit and vrn genotype. their growth habit is due to at least one dominant vrn allele. this allele could be either inherited from ancestral diploids or could result from selection of independent mutations that appeared during the adaptation to various growing condition after domestication. moreover, genetic and molecular experiments show that there are multiple alleles of dominant vrn and their manifestation in vernalization response is also different. it means that some mechanisms exist, for which evolutionary background is unknown. in the present study, molecular variability of wheat vrn promoter region was analyzed. in order to get a more detailed insight into complicated molecular basis of spring vs. winter growth habit and associated vernalization genes variability, we studied wild and cultivated wheats together with their close relatives aegilops l. in total, accessions of four diploid species and accessions of polyploid species belonging to three known sections were investigated. seven accessions of goatgrasses belonging to species, which participated in the hybridization events in polyploidy wheat evolution, were also studied. among all investigated accessions, eight have winter growth habit, and the remaining - spring growth habit. the number of genes responsible for spring vs. winter growth habit was estimated in the genetic experiments for ten spring accessions.  genetic analysis of growth habit for the molecular characterization, it was important to study accessions that possess monogenic control of spring growth habit. genetic experiments were carried out to demonstrate genetic control of growth habit in some spring wheat and goatgrass species. the obtained data are given in tables and * - values for significance of p = is and p = is; spring accessions are underlined bwe - cv. t. dicoccum black winter emmer, i: bs1e - black spring vrn-a1 emmer, i: bs2e - black spring vrn-b emmer, kristall - introgressive spring line of t. durum winter cv. kristall with dominant gene vrn-d it was established that spring growth habit of most of examined species was controlled by a single dominant gene. in the table the segregation ratio to is reliable while the value of pierson chi-square test for the segregation ratio to is higher than the value of significance. three investigated tetraploid species were crossed with near-isogenic test lines, which were characterized with specific dominant vrn-a, vrn-b alleles. the design of near-isogenic lines is described in methods section. in crosses with t. dicoccum cv. black winter emmer, which has recessive vrn-a and vrn-b alleles, it was shown that spring growth habit of these species was controlled monogenic, because the segregation ratio to in f generation is reliable. for t. turanicum monogenic control was shown indirectly from the next two crosses, where the digenic segregation was obtained for all three species. it means that dominant allele responsible to spring growth habit is not allelic to vrn-a and vrn-b alleles of the tested lines. in tetraploid wheat species, the dominant gene of the accessions k- and k- was temporarily designated as vrn-t and their genotypes were specified as vrn-a1vrn-a vrn-b1vrn-b vrntvrnt. it is an unknown allele, which is nonallelic to vrn-a and to vrn-b the number and allelity of dominant genes vrn in t. timopheevii zhuk. was not determined because all accessions of this species had only spring growth habit. the generalized information concerning genetic control of growth habit is represented in table triticum l. section urartu dorof. et a.filat. auau section monococcon dum. abab section dicoccoides flaksb. bbauau vrn-a1u2 vrn-a1u1 vrn-b1a2 section triticum bbauaudd vrn-b1 vrn-b1 vrn-a1u2 vrn-b1 vrn-a1a1 vrn-a1u1 vrn-b1 5r vrn-b1 vrn-a1u3 vrn-b1 section timopheevii a. filat. et dorof. ggabab vrn-a1u1 vrn-a1f2 vrn-a1u3 aegilops l. section sitopsis zhuk. ss section vertebrata zhuk. emend. kihara dd allelic variability at the vrn promoter region of diploid species variability at the primer annealing site in the present work, diploid wheat accessions of four known species and goatgrass accessions of two species were studied. initial pcr screening was provided with primer pair ap1_prodel_f ap1_prodel_r. the pcr product of the expected size of bp has been obtained in the majority of the studied wheat accessions and in one goatgrass species, ae. speltoides. no pcr products have been found in ae. squarrosa accessions. out of wheat accessions, ten showed pcr products of the lower size, which can be explained by deletions in the promoter region. the heterogeneity of diploid genomes and their sequence variability was observed previously. all available vrn promoter sequences belonging to different wheat genomes were extracted from genbank and aligned together with primer sequences. there were a bp deletion in d genome near the region complementary to the reverse primer, and a duplicated fragment near this region in a genome. therefore, a new primer pair was developed for amplification of d genome. to check for possible minor deviations connected with spring growth habit, all fragments obtained in pcr using both primer pairs were directly sequenced. the resulting sequences were submitted to genbank: gq451714-gq the presence of the specific deletions in spring wild and cultivated accessions among investigated spring diploid wheats, had bp or bp deletions in vrn promoter region comparing with winter ones. both deletions covered the specific area of mads-box, and were previously described as associated with spring growth habit in the diploid cultivated wheat t. monococcum. spring t. boeoticum accessions were not screened previously as well as t. urartu. investigation of the vrn promoter mutations in both species may provide important information about mechanisms involved in the origin of the spring forms in these wild natural species. among four spring t. urartu accessions, none had specific deletions within the vrn promoter region compared to winter forms. however, one accession was almost identical to the a genome sequences of the polyploid wheats. this recessive allele was named vrn-a1u and was not found in all analyzed t. boeoticum and t. monococcum sequences. the closest relatives of triticum l. species are aegilops l. species, which also have spring and winter forms. in order to address the question whether flowering initialization in spring goatgrasses has common characters with that of diploid wheats, we obtained vrn promoter regions from six spring and one winter aegilops accessions. there were some nucleotide substitutions differentiating goatgrasses from wheats. no significant changes, which could distinguish spring and winter accessions, have been found. all investigated promoter sequences of the ae. squarrosa accessions contained the specific d genome bp deletion and some d genome specific nucleotide substitutions. allelic variation at the vrn promoter region in polyploid wheats tetraploid wheat species vrn promoter regions from g genomes of spring t. timopheevii and t. araraticum jakubz. were amplified using primer specific for b genome. we analyzed also a tetraploid line determined by karyological analysis, which was found in an accession of t. urartu from lebanon. sequencing showed a close relatedness of the obtained g genome to the corresponding b genome. the most interesting was the presence in t. timopheevii g genome and two clones of t. turanicum jakubz. b genome of several identical substitutions and large insertions in - position upstream from the start codon. the insertions were bp long in g genome and bp in b genome. we named these vrn promoter variants vrn-g1a and vrn-b1a. these insertions were similar to a bp foldback element observed in vrn-a1a allele and named "spring". however, the site of insertion was different. it was not in the place of the carg box and was flanked by another host direct duplication. more detailed analysis of the mobile elements which were found in vrn-a vrn-b and vrn-g promoter regions of tetra- and hexaploid wheat species showed the presence of the terminal inverted repeats and predicted open reading frames between both repeats. the observed transposon element, which is bp in length with bp tir, could belong to the superfamily tc1/mariner. the vrn1promoter regions from a genomes of spring tetraploids also contain different promoter damages and share some variability. in addition, a bp deletion was found in the region - and - nucleotide from start codon. this deletion was located in the same region where the foldback element insertion occurred in the g genome. this vrn promoter allele was called vrn-a1f. species of the section dicoccoides, t. turanicum and t. dicoccum schuebl. also had a common bp deletion in the region - - upstream from the start codon. this deletion included the repetitive region and was observed previously in vrn-a1b and vrn-a1d alleles of spring tetraploid t. durum desf. and t. dicoccoides correspondingly. we observed also a bp deletion in a genome of t. durum, odessa) that was similar to vrn-a1e alleles of t. dicoccum. vrn promoter from a genome of t. dicoccum displayed identity with vrn-a1a allele observed previously in t. aestivum near-isogenic line triple dirk d and contained the insertion of a foldback element. hexaploid wheat species promoter sequences of vrn-a vrn-b and vrn-d alleles from two winter cultivars and a, b and d genome sequences of spring mute mironovskaya yarovaya were determined. the spring mute was found in cultivar mironovskaya vrn promoter fragments of winter cultivars and b, d genome of spring mironovskaya yarovaya contained a common for certain genomes variability observed in recessive alleles of winter wheat near-isogenic line triple dirk c. after cloning and sequencing of a genome fragment of spring sample, clones with and without specific deletions were observed. the observed deletions included bp del in - - region and bp del in - - region that were common for the vrn-a1f allele described above in tetraploid species. three t. aestivum cultivars pyrothrix osijek and jupateko with "weak" vrn alleles were also included in the research. no differences from recessive alleles were found in vrn promoter sequences from a, b and d genomes of spring cvs. osijek and pyrothrix 28; as well as from b and d genomes of spring cv. jupateko. after amplification of the vrn promoter region from a genome of the cv. jupateko, two pcr products were visualized on electrophoresis. sequencing of two larger pcr products revealed the presence of the foldback element insertions that was common for vrna1-a allele. a bp foldback element insertion differed moderately from those previously described. the site of insertion and flanking direct repeats were similar for both elements. however, analysis of the foldback element of spring cv. jupateko indicated an absence of direct repeats inside. these direct repeats were the sites of deletions in the duplicated promoter sequences of vrn-a1a allele. therefore, we detected only one variant of the promoter with mobile element insertion in this species. the smaller pcr products in cv. jupateko had most common character with vrn-a1f allele observed in k- t. timopheevii. thus, two different vrn-a alleles were detected. both alleles could be the cause of spring growth habit. we also found promoter damages in the a genomes of two other investigated intrageneric t. aestivum: substitution line saratovskaya/vietnamskaya 5r and line nulli5b-tetra5d chinese spring. the investigated promoter regions contained vrn-a1b and vrn-a1f alleles correspondingly. both alleles could be a reason of spring growth habit and likely inherited from tetraploid species during cultivation and breeding programs. variability of different clones inside a genome investigation of a genome sequences revealed at least two different variants of vrn promoter in some cloned samples according to the presence of indels. at the same time all cloned b and d genome sequences were identical in the presence or absence of indels. to exclude any doubt in affiliation with a specific genome, we provided the phylogenetic analysis, which allowed the correlation of each sequence into the genome. each of three accessions of diccoccoides section had two different variants of the promoter in a genome. one of them was intact as in winter vrn-a allele, and another had a deletion or insertion of the transposon. vrn promoters of the remaining three examined species, t. turgidum k-16156) were sequenced directly without cloning and contain only one variant of sequence judging from the absence of superpositions in the chromatograms. in section timopheevii four clones of t. araraticum were similar if indels are taken into consideration. t. timopheevii had four clones similar to those of t. araraticum containing vrn-a1f allele and one clone with intact vrn-a allele. absence of the previously determined a genome specific bp insertion in the region - - were observed in some clones of the tetraploid species. only a genome had variability at this region while b, g and d genomes as well as genomes of the examined goatgrasses were conservative and had deletions in this place. all obtained vrn-a1f allele had no bp insertion. seven clones of vrn promoter region from a genome were sequenced in tetraploid line discovered in t. urartu collection under ac.n.pi and later was determined as tetraploid according to karyological and molecular analyses. four different promoter variants were found. at the same time, no positive result was obtained in pcr with b/g genome specific primers for vrn promoter region and developed previously for acc- and pgk- genes. three spring cultivars of hexaploid t. aestivum also contain at least two vrn promoter variants in a genomes. vrn promoter region of a genome of cv. mironovskaya yarovaya and line nulli5b-tetra5d chinese spring included the recessive vrn-a and the dominant vrn-a1f alleles. cultivar jupateko had in addition the third allele differed from others by a transposon insertion. predicted regulatory elements inside vrn promoter region in the present work, we performed the analysis of the cis-regulatory elements inside the most variable region of the vrn promoter. this information helps to investigate the consequences of the damages in the promoter of the main initiation factor of flowering, and possibly is related to the mechanism of growth habit changing. previously, a specific carg-box common for all mads-box protein family was found in the region where different in size deletions and transposon insertion occurred in spring diploid and polyploid wheat species. it was suggested that carg-box could be involved in the regulation of vrn by interacting with other mads-box proteins. our analysis showed that this region also included recognition site for hmg a high mobility globular protein, with a high probability. recently, an increased expression of wheat homolog hmg1b protein after cold exposure of crown tissue was showed. the hmgb is thought to interact with histones and could participate in modifying chromatin structure. some other regulatory sites were found in the region of bp and bp deletions common for vrn-a1b and vrn-a1f alleles, correspondingly. we also observed the recognition site for bzip proteins. the vrn protein was shown to upregulate vrn expression via interaction with tafdl members of bzip transcription factor family. moreover, it appears that tafdl protein can interact in vitro with acgt elements of the promoter of the meristem identity gene vrn including g-box and g/c hybrid of g- and c-boxes. it is interesting that both transposon insertions observed in b and g genomes contain additional g-box motif. near the region of the g/c hybrid box we found the binding motifs for myod-like protein, a member of the bhlh family of transcription factors. members of the bhlh family in animals have been shown to regulate the determination and differentiation of a variety of cell types, including skeletal muscle, neurons, and hematopoietic cells. recent detailed study into the physical interaction among the bhlh proteins of arabidopsis that control stomatal development reflected that a similar mechanism functions in animal muscle and plant stomata. it is known also that markers of myogenic specification such as myod and myogenin gene, which encode transcription factors of the basic helix-loop-helix family, interact with proteins belonging to the mads-box transcription factors. it is likely that examined bhlh proteins modulate cell cycle regulatory protein activity in a similar fashion in both animal and plant cells. a binding motif was also found for gata protein. gata transcription factors are a group of dna binding proteins broadly distributed in eukaryotes. four classes of these proteins are described in plants. some gata transcription factors are downstream effectors of floral homeotic gene action in arabidopsis. it appears that two mads-box gene products, apetala and pistillata can directly interact with two members of gata transcription factor family. altogether they regulate a variety of light and nutrient responsive genes participating into the transcriptional cascades controlling the specification of floral organ identities. in particular, apetala gene is directly regulated by ap and pi in arabidopsis. transcription factor binding motifs described here are found in the vrn promoter region where indels could induce flowering without cold treatment. this could indicate a possibility that wheat homologs of the hmg bzip, bhlh and gata proteins are involved in the vernalization response. further experimental approaches are necessary to support this suggestion. discussion triticum genetic material notwithstanding the short evolutionary time since the domestication, some significant morphological changes were accumulated in cultivated plants. as a result of an intensive cultivation, many wheat varieties with different agronomical important characteristics were selected. however, in many cases the molecular pathway behind the appearance of a sample with an unusual character in the stable population is still unknown. winter growth habit is assumed to be a more ancestral character than the spring one. it is not known whether the ancient farmers found spring forms in nature or in their plots. however, they started to cultivate spring forms intensively. among the first wheat species cultivated by people, t. monococcum and t. dicoccum, most have spring forms according to the last investigation of and accessions correspondingly. the winter forms came back to occupy considerable areas in the middle latitudes at the beginning of the 20th century. but in the continental middle latitude areas the field is occupied by winter forms for two vegetation seasons and by spring forms - for one season only. this is due to the agronomical aspects of spring forms - sowing in spring when winter is too cold. in these countries it is impossible to use the intermediate forms, which are used in southern europe and mediterranean region, because of climate, when spring crop can not survive in winter. it appears that, when both forms are used, winter ones are often less economically beneficial. for this purpose, mutants with spring alleles of vrn in different genomes were developed. multiple alleles have been described within the vrn locus, based on their different effects on vernalization requirement and flowering time. however, characterization of these alleles at the molecular level is necessary. the number of dominant vrn genes in common wheat is important since it modifies spring growth habit, and allows for the development of commercial cultivars with different vegetative periods. studying the unique collection of spring wild and domesticated diploid and polyploid wheats not examined before allowed us to observe a greater variability, especially in tetraploid and hexaploid species. we described three additional vrn promoter variants vrn-a1f, vrn-a1g and vrn-a1h. moreover, we showed that vrn promoter region of b and g genomes can also contain damages such as the insertion of a transposable element. the duplication of the vrn promoter together with the first exon was also demonstrated. inheritance of vrn gene from spring wild diploids to cultivated diploids it is known that wild diploid wheats were the donors of a genome in polyploid wheat species. diploid species are the most ancient in the triticum genus so their genome has the longest history in the genome composition of the modern wheat species. the majority of the wild wheats have a winter growth habit, suggesting that the recessive vrn-a allele with an intact vrn promoter is the ancestral character. we studied the number of wild and cultivated diploid species, and established that vrn promoter deletions that resulted in the elimination of repressor influence in cultivated species t. monococcum are also common for wild species t. boeoticum. therefore, it is possible to conclude that spring forms originated in wild populations and later were selected by man for culture. new alleles may result from selection of independent mutations that appeared during the domestication. it is likely that a shorter bp deletion appeared first and then increased to bp deletion. both variants were inherited by cultivated diploid forms and could be inherited also by polyploids. species of the genus aegilops are closely related to wheats; they have common geographic range, and about million years ago a first polyploid wheat appeared as a result of hybridization of wheats with goatgrasses. similar to wild diploid spring triticum species, there are early-flowering forms among aegilops species, which evolved in two intraspecific lineages, middle asian and transcaucasian. the existence of longitudinal and latitudinal clines in the onset of flowering at the species level was observed; however, mechanism controlling switch from vegetative to generative growth habit is unknown. to answer the question whether these mechanisms have common characters with that of diploid wheats we analyzed vrn promoter regions of several aegilops accessions. spring ae. squarrosa accessions originated from the northern part of afghanistan and iran while in other regions only winter forms were usually found. however, no indel events were found inside the investigated region. therefore, taking into account the genealogical and ecological framework complexity of flowering time diversification in the investigated aegilops accessions we expect the existence of another regulatory mechanism controlling flowering time in goatgrass species. increasing genetic variability in spring tetraploids within a, b, and g genomes in contrast with wild triticum species, the majority of cultivated polyploid wheat species have spring growth habit. spring forms are due to at least one dominant mutation in vrn gene that could be inherited or appeared independently during domestication or historical selection process. four different variants of vrn promoter in a genomes of diploid species were observed. the main difference between sequences is the presence or absence of a specific bp deletion that was not observed only in one spring accession of t. urartu and inherited by a genome of many polyploids. in comparison with diploid species three other new vrn promoter variants due to deletions appeared in tetraploids. one of them represents vrn-a1e allele and contains bp deletion in place where specific and bp deletions occurred in diploids. possibly there was a deletion expansion in tetraploids but independent appearance is also likely due to the existence of flanking direct repeats, which may be the reason of nonhomologous recombination. there are many different repeat regions inside vrn promoter and most of the deletions flanked by short direct repeats. it was shown that unequal recombination by such repeats can play the main role in the origin of novel alleles in vrn gene. the vrn-a1e allele could have originated from the allele containing bp insertion and observed only in one wild diploid species t. urartu. another vrn-a1b allele found in tetraploids contains a bp deletion. its origin is also related to t. urartu vrn promoter containing bp insertion. the described deletion most likely happened in species of dicoccoides section. this allele is not found in wheat species from monococcon dum. and timopheevii sections. at the same time, vrn-a1f allele likely occurred in timopheevii section within species with g genomes. moreover, its origin is related to diploid vrn promoter containing bp deletion. we found this allele also in t. turanicum; this, however, is likely a case of hybridization event that may occur between tetraploid species from different wheat sections. the vrn promoter variant found in t. dicoccum accession had all common characters of vrn-a1a allele as well as the transposon insertion. taking into consideration the origin of bs1e line, as the near-isogenic line of cv. black winter emmer whose vrn-a allele was replaced with vrn-a1a of hexaploid wheat triple dirk by backcrossing, this proves its derivation from t. aestivum near-isogenic line tdd. we found and described also two different alleles in b and g genomes of tetraploid species that contain a transposon insertion. the indel events in these genomes were observed for the first time, and both species contained also vrn promoter damages in a genomes. investigated hexaploid accessions of section triticum inherited all alleles from tetraploid species except one with transposon insertion that most likely happened at this ploidy level. evidence of the vrn promoter duplication in the present work, two and more vrn promoter copies were detected and distinguished by the presence of indel events within a genome of several spring polyploid species. there are three possible explanations of these results: nonspecific duplication of different genomes, heterozygous plant material with two different chromatids in one genome, duplication of the investigated region. the first possibility was tested by the presence of the nucleotide substitutions and indel events specific to different genomes. for more reliable results the phylogenetic tree was constructed that demonstrated relatedness of each clone to certain genomes. results showed an absence of nonspecificity and supported attributes of each clone to genome which amplification product was cloned. the genetic experiment and clearance of each examined lines exclude the second possible case. all accessions with two and more vrn promoter variants contained one intact promoter that was observed in winter plants. however no splitting was detected in five generations of each line. the most likely explanation is the duplication of the investigated region that occurred in polyploid species. previously it was supposed the duplication of the promoter region with the first exon in vrn-a1a allele. in most cases we found two copies of the promoter that differed by the presence of deletions. exceptions were accessions of t. dicoccoides and cv. jupateko of common wheat. in most polyploid vrn promoter variants, an ancestral form is an intact winter allele found only in t. urartu accession. the second vrn promoter sequences that was found in diploids and contained bp del was inherited by some tetraploids of section timopheevii. in the genome of ancestor species of this group additional bp deletion occurred. it is most likely that duplication of the promoter region occurred before deletion formation and transposon insertion. moreover, deletions occurred in polyploid species independently from diploid species. therefore, spring forms, which resulted from indels, were not inherited from spring diploid species. the obtained data demonstrated that quite complicated molecular alterations occurred in the studied region of spring polyploid species. two main underlying causes of such dynamic evolutionary changes seem to be polyploidy and intensive breeding of agronomic characters.  in the present study we investigated variability in the promoter region of vernalization gene vrn in spring wheat accessions. a greater variability in the vrn promoter region of polyploid species in comparison with diploid ones was observed. in diploid species, damages of vrn promoter leading to spring growth habit were inherited by cultivated forms from the wild. a complicated evolutionary process including duplication of vrn promoter was found in a genome of polyploids. different indel events likely occurred in spring polyploid species independently from spring diploid species. specific transposon insertions found in a genome as well as in b and g genomes of spring tetraploid species pointed to active transposition in polyploid genome. some transcription element binding sites were observed in silico in the regions of indel events. 
some transcription factor recognition sites including hybrid c/g-box for tafdl protein known as the vrn gene upregulator were predicted inside the variable region. variability of the vrn promoter region of the unique collection of spring polyploid and wild diploid wheat species together with diploid goatgrasses were investigated. sequence analysis indicated great variability in the region from - to - nucleotide positions of the vrn promoter region. it was shown that vrn promoter region of b and g genome can also contain damages such as the insertion of the transposable element. at the same time, the duplication of the promoter region was observed in a genomes of polyploid species. the spring t. urartu and goatgrass accessions have another mechanism of flowering activation that is not connected with indels in vrn promoter region. we can conclude that supposed molecular mechanism of the vrn gene activating in cultivated diploid wheat species t. monococcum is common also for wild t. boeoticum and was inherited by t. monococcum. it was shown that deletions leading to promoter damage occurred in diploid and polyploid species independently. dna transposon insertions first occurred in polyploid species.
 variability of the vrn promoter region of the unique collection of spring polyploid and wild diploid wheat species together with diploid goatgrasses were investigated. accessions of wild diploid and tetraploid species were studied for the first time.  sequence analysis indicated great variability in the region from - to - nucleotide positions of the vrn promoter region. different indels were found within this region in spring wheats. it was shown that vrn promoter region of b and g genome can also contain damages such as the insertion of the transposable element. some transcription factor recognition sites including hybrid c/g-box for tafdl protein known as the vrn gene upregulator were predicted inside the variable region. it was shown that deletions leading to promoter damage occurred in diploid and polyploid species independently. dna transposon insertions first occurred in polyploid species. at the same time, the duplication of the promoter region was observed in a genomes of polyploid species.  we can conclude that supposed molecular mechanism of the vrn gene activating in cultivated diploid wheat species t. monococcum is common also for wild t. boeoticum and was inherited by t. monococcum. the spring polyploids are not related in their origin to spring diploids. the spring t. urartu and goatgrass accessions have another mechanism of flowering activation that is not connected with indels in vrn promoter region. all obtained data may be useful for detailed insight into origin of spring wheat forms in evolution and domestication process. 