Comparative assessment of the copy number of satellite repeats in the genome of Triticeae species

Satellite repeats are a significant component of the genome of Triticeae and play a crucial role in the speciation. They are a valuable tool for studying these processes. Pseudoroegneria species play a special role among grasses, as they are considered putative donors of the St-genome in many polyploid species. The aim of this study was to compare the copy number of satellite repeats in the genomes of Triticeae species. Quantitative real-time PCR was applied to determine the copy numbers of 22 newly discovered satellite repeats revealed in the whole-genome sequences of Pseudoroegneria species and one additional repeat previously identified in the genome of Aegilops crassa. The study focused on seven species of Pseudoroegneria, three species of Thinopyrum, Elymus pendulinus, Ae. tauschii, Secale cereale, and Triticum aestivum. Based on the copy number level and coefficients of variation, we identified three groups of repeats: those with low variability between species (medium-copy CL82), those with medium variability (low- and medium-copy CL67, CL3, CL185, CL119, CL192, CL89, CL115, CL95, CL168), and those with high coefficients of variation (CL190, CL184, CL300, CL128, CL207, CL69, CL220, CL101, CL262, CL186, CL134, CL251, CL244). CL69 exhibited a specific high copy number in all Pseudoroegneria species, while CL101 was found in both Pseudoroegneria and Th. junceum, CL244 in Th. bessarabicum, CL184 in P. cognata and S. cereale. CL95, CL128, CL168, CL186, CL207, and CL300 exhibited higher copy numbers in P. cognata compared to other species; CL3, CL95, CL115, CL119, CL190, CL220, CL207, and CL300 in P. kosaninii; CL89 in P. libanotica; CL134 in P. geniculata. Our assessment of the copy number of new satellite repeats in the St-genome and the analysis of their amplification specificity between species can contribute to the molecular-genetic and chromosome markers used for evolutionary, phylogenetic, and population studies of Triticeae species


Introduction
Triticeae is an economically important tribe of the Poaceae family, comprising approximately 500 species of annual and perennial herbaceous plants (NCBI database: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi).Wheat, rye, barley, and fodder grasses are among the species of this tribe that play a significant role in providing food for humanity and have also become an integral part of animal diets (Hod kinson, 2018).
The interest in studying phylogenetic relationships within the Triticeae tribe is largely driven by the potential of wild wheat species to serve as valuable sources of economically important genes for the improvement of cultivated cereals.For example, Thinopyrum and Dasypyrum serve as gene donors for resistance to various diseases (Yang et al., 2005;Luo P.G. et al., 2009;Salina et al., 2015;Wang S. et al., 2019;Li L.F. et al., 2022;Guo et al., 2023).By crossing wheat and Agro pyron, it is possible to increase head productivity (Zhang J. et al., 2016).Representatives of Pseudoroegneria are drought resistant and are used as pasture grasses (Wu et al., 2023b).
The Triticeae tribe consists of approximately 100 annual and 400 perennial species, which carry one (in diploids) or several (in polyploids) of 13 genomes (Wang, Lu, 2014).Re presentatives of Pseudoroegneria carry the Stgenome, and it is believed that this genus was the donor of the Stgenome for Elymus and some Thinopyrum species (Mahelka et al., 2011;Dobryakova, 2017;Linc et al., 2017;Lei et al., 2018;Chen N. et al., 2020;Agafonov et al., 2021).Plants of the genus Agropyron, at all ploidy levels (2n = 2x/4x/6x), are distinguished by the presence of a Pgenome (Zhang Y. et al., 2015).Genome J (=E) is mainly composed of diploids Thi nopyrum bessarabicum (genome J = J b ) and Th.elongatum (genome E = J e ).The Jgenome is evolutionarily close to the Dsubgenome of common wheat, and the most likely donor of the Dsubgenome is Aegilops tauschii (Baker et al., 2020).This may explain why the chromosomes of the Dsubgenome in the introgressive lines of common wheat, developed with the aim of improving it through hybridization with Thinopyrum, show the highest frequency of introgressions from the Jgenome (Chen Q. et al., 2001;Liu Z. et al., 2007;Cui et al., 2018).
At present, the origin, relationships, and proximity of ge nomes within the Triticeae tribe remain controversial and un certain.The challenges associated with studying Triticeae genomes are primarily due to the significant differences be tween the polyploid subgenome and the ancestral genome of the di ploid parent organism.These differences arise from the mo di fications that occur during evolution.Additionally, the diploid ancestor of the donor organism may have become extinct or has not yet been found (Jakob, Blattner, 2010;Liu Q.L. et al., 2020;Sha et al., 2022).Perennial polyploid species, for example, Th. intermedium and Th.ponticum, may have an unbalanced genome or chromosomal translocations (Kruppa, MolnarLang, 2016;Liu Y. et al., 2023).This could be associated with the transition to vegetative reproduction, which does not involve sexual processes for seed formation and therefore does not require stable meiosis (Comai et al., 2005;Husband et al., 2013).The same species are characte rized by recombinant subgenomes, the origin of which is still unclear (Wang R.R.C. et al., 2015;Liu Y. et al., 2023).Discus sions continue regarding potential donors of the Y sub genomes in Elymus and Roegneria (Yan et al., 2011;Liu Q.L. et al., 2020;Wu et al., 2021), as well as the maternal form in the occurrence of Thinopyrum, Roegneria, Elymus, Kengyilia, and other polyploids (Mahelka et al., 2011;Luo X. et al., 2012;Zeng et al., 2012;Lei et al., 2018;Chen N. et al., 2020).
In addition, there is a problem of correlation between the species identification of a particular specimen based on botani cal traits (often influenced by the environment) and that based on molecular genetic and cytogenetic characteristics (Wang, Lu, 2014;AlSaghir, 2016;Rodionov, 2022).For example, this issue arises in Elymus, as demonstrated by studies con ducted by A.V. Rodionov et al. (2019), V. Lucia et al. (2019), and L. Tan et al. (2021).Another example is the relationship between Th. elongatum and Th.bessarabicum that bear fairly similar genomes, but differ in botanical characteristics (Gre wal et al., 2018;Dai et al., 2021;Chen C. et al., 2023).The problem is compounded by the fact that natural spontaneous hybrids are often found (Chen C. et al., 2022;Luo Y.C. et al., 2022;Wu et al., 2023a).The study of phylogenetic relation ships deepens our understanding of evolutionary processes in plants and speciation, and helps to improve biosystematics.The acquired knowledge will enhance the efficiency of uti lizing genetic resources from wild species by improving the understanding of their proximity to the genomes of cultivated crops and the potential for obtaining valuable introgressions.
The genome of Triticeae grasses is characterized by a large size, which complicates wholegenome deep sequencing and makes assembly difficult (Rabanus-Wallace, Stein, 2019).A significant portion of the Triticeae genome is composed of repetitive DNA, known as repeats.The repeatome include mobile elements, gene clusters (specifically the 5S and 45S rRNA genes), and satellite repeats (Dvořák, 2009;Shcherban et al., 2015;Gao et al., 2023;Vershinin et al., 2023).
Tangible progress has been made in the study of Triticeae genomes, owing to the invention of wholegenome sequencing methods and bioinformatic algorithms for analyzing the data obtained (RabanusWallace, Stein, 2019;Gao et al., 2023).The rapid growth in the volume of information on genome wide sequences of Triticeae has significantly accelerated and simplified the search for repetitive DNA that can be used as chromosomal markers (Du et al., 2017;Said et al., 2018;Tang et al., 2018;Chen J. et al., 2019;Kroupin et al., 2019aKroupin et al., , 2022;;Lang et al., 2019a;Liu Q.L. et al., 2020;Wu et al., 2021Wu et al., , 2022)).Due to the significant presence of repetitive DNA in the Triticeae genomes, information about satellite repeats can be obtained even through sequencing with low coverage.This greatly simplifies the process of searching for repeated sequences (NavajasPerez, Paterson, 2009;Kroupin et al., 2019b;ŠatovicVukšic, Plohl, 2023).
A wellestablished method for quantifying the number of copies of repetitive DNA, including satellite repeats, is quantitative realtime PCR (qPCR) (Harpke, Peterson, 2007;NavajasPérez et al., 2009;Baruch, Kashkush, 2012;Feliciello et al., 2015;Divashuk et al., 2016Divashuk et al., , 2019Divashuk et al., , 2022;;Pereiera et al., 2018;Shams, Raskina, 2018).Compared to Southern blot or dot-blot hybridization on a membrane, or fluorescent in situ hybridization on chromosomes, qPCR has proven to be an easier-to-use, accurate, and effective method for assessing the copy number of the target sequence.This method allows researchers to identify the number of copies of satellite repeats in the genome and the variability between genomes (Kalendar et al., 2020;Pös et al., 2021).
In order to comprehend the potential of using them as tools for evolutionary and phylogenetic studies of wild representatives of the Triticeae tribe, as well as for studying wide hybrids using molecular biology and cytogenetics methods, it is crucial to first determine the copy number of satellite repeats in the genomes of Stgenome carriers.Therefore, we have chosen Pseudoroegneria species with different ploidy levels as our research subject.
To assess the specificity of satellite repeats for the St-ge nome, we included Thinopyrum species in the experiment, which contain the Jgenome that is common among Triticeae.We also selected Th. intermedium with the J r J vs Stgenomic formula, serving as a carrier of the Stsubgenome and St specific repeats in the recombinant J vs genome.To explore the potential of utilizing the identified satellite repeats for the characterization of distant wheat and rye hybrids, Triticum aestivum and Secale cereale accessions were included in the study.In addition, due to the evolutionary proximity of the J and Dgenomes, we included the Ae.tauschii accession.
E. pendulinus was also used, carrying both the Stsubgenome targeted by our work and the Ysubgenome of unknown origin, which is common among Elymus sensu lato species.The ex periment also included a satellite repeat of CL244, which we obtained as a result of analyzing the wholegenome nucleotide sequen ce of Ae. crassa (D1 X cr ), a carrier of the Dgenome variant (Kroupin et al., 2022).Despite this, CL244 was not found in Ae. tauschii, showed a small number of hybridiza tion sites on the chromosomes of T. aestivum and Ae.crassa, while on the chromosomes of the J b genome in Th. bessara bicum, bright signals were observed indicating a high level of its abundance.

Materials and methods
The Triticeae species with various genomic compositions, as listed in the Table, served as the material for our study.
The young leaves of the plants were frozen in liquid nitro gen.Genomic DNA was then isolated using the CTAB pro tocol (Rogers, Bendich, 1985).This DNA was used for sub sequent sequencing and quantitative PCR (qPCR).The con centration and purity of the isolated DNA were tested using Qubit 4 (Thermo Fisher Scientific, USA) and electrophoresis in a 0.8 % agarose gel.
Shotgun sequencing libraries were synthesized using the Swift 2S Turbo DNA Library Kit (Swift Bioscience, USA) in accordance with the manufacturer's protocol.To assess the quality of the libraries, a test run was conducted on the MiSeq device (Illumina, Inc., USA).The libraries were then converted and sequenced using DNBSEQG400 on one track.The initial amount of DNA was 25 ng.The fragments were approximately 350 bp long and were indexed at both ends using the Swift 2S Turbo Unique Dual Indexing Kit (Swift Bioscience).Sequencing was performed on Illumina Next Seq (Illumina, Inc.), using the NextSeq 500/550 Mid Output Kit v.2.5 (Illumina, Inc.).
Bioinformatic analysis was conducted on the processing and assembly of the reads of nucleotide sequences that involved a sequence of satellite tandem repeats.The uniqueness of these sequences was evaluated in comparison to previously published methods described in P.Y.Kroupin et al. (2022).The primer sequences for the identified monomers of satellite repeats are provided in Supplementary Material 1 1 .
Quantitative realtime PCR was conducted using DNA from the accessions listed in the Table as a template, with three replicates.The amplification was carried out using a CFX realtime amplifier system (Bio-Rad Laboratories, Inc., USA) and a reaction mixture of RealTime PCR Mix containing the Eva Green fluorophore (Synthol Ltd., Russia) in accordance with the manufacturer's protocol.A singlecopy gene, VRN1, was used as the reference gene.Primer concentration in mixtures consisted of 10 ng/μl, while DNA concentration was 0.4 ng/ μl.Amplification was performed according to the following pro gram: preincubation -10 min at 95 °С; followed by 40 cycles: denaturation -10 s at 95 °С; primer annealing -30 s at 60 °С.
Statistical analysis, including the calculation of the ave rage values of Cq, standard deviation, and the corresponding number of copies relative to the reference gene VRN1, was per formed using BioRad CFX and Manager 3.1 software.To assess the similarity of copy numbers among repeats, we have introduced the concept of "repeat copy number pattern", a set of copy number values for a specific repeat within the set of species being studied.To assess the similarity of copy number among the species under investigation, we have introduced the concept of "species copy number pattern", a set of values of the copy number of the satellite repeats being studied for a particular species.
Pearson's correlation coefficients (r) were calculated using Statistica 12 software (StatSoft, USA) to determine the relationship between repeat copy number patterns and spe cies copy number patterns.The significance level was set at p < 0.05.Diagrams were constructed using the principal component analysis method for satellite repeats and the studied species.The diagrams were based on the data obtained from Statistica 12 software, which included information on the copy number of satellite repeats.The coefficient of variation of the satellite repeatability values between species was calculated using Microsoft Excel (USA).
The characteristics of the identified satellite including its length and the most similar sequences in the NCBI database are presented in Supplementary Material 1.After comparing the nucleotide sequences of the 22 repeats with those previ ously published in the NCBI, we did not find any matches for nine of them (CL69, CL89, CL95, CL168, CL185, CL207, CL251, CL262, CL300).For the remaining 13, the level of identity among similar published sequences ranged from 70 to 98 %.This indicates that they are different from previously published sequences (see Supplementary Material 1).
Two satellite repeats showed similarities to repeats found in common wheat: CL119 was similar to the pTa465 clone (77 % identity), and CL101 was similar to the Spelt1like subtelomeric repeat (80 % identity).Three repeats showed similarities to the following known satellites: CL220 to CL219, which was detected in the Ae.crassa genome (82 %), CL134 to CL97 from the Th.bessarabicum genome (71 %)

ГЕНЕТИКА РАСТЕНИЙ / PLANT GENETICS
Relative copy number of the satellite repeats in the studied species of the Triticeae tribe expressed as a decimal logarithm.and CL186 to ACRI_TR_CL80 from the A. cristatum nome (70 %).The three repeats show similarities to microsatellites: CL128 has similaritiy to L15 identified in the P. stipifolia genome (84 %), CL190 shows similarity to P523 from the genome of Ae. tauschii (81 %), and CL82, to pTa451 from common wheat (88 %).Four repeats we found showed similarity to the following mobile elements: CL184, which has a 98 % similarity to the Cassandra retrotransposon from the rye genome, CL67 and CL115, which have a 91 and 78 % similarity to retrotransposons from the barley genome Cereba and Sandra5, respectively, and CL192, which has a similarity to transposon XJ from the Ae.tauschii genome (70 %).The CL3 repeat was most similar to the E-gene-specific marker Th. elongatum 516 (79 %).

Assessment of the copy number of satellite repeats using qPCR
The data obtained on the relative copy numbers of 23 satellite repeats in 14 species, calculated in relation to the reference singlecopy VRN1 gene, are shown in Supplementary Mate rial 2.1.All of the repeats we studied differed in terms of copy numbers and the coefficient of variation between the species.Since the order of the obtained copy numbers varied signifi cantly, the results were presented in the form of a decimal logarithm for the convenience of comparing their abundance (see the Figure and Supplementary Material 2.2).Hereafter, we will simply refer to the decimal logarithm of relative copy number as "copy number".Since the copy number rate varied from 0 to 5, the repetitions were classified into the following groups based on their copy number: low (≤ 2), medium (> 2, < 4), and high (≥ 4).Since the coefficient of variation ranged from 0 to 0.6, we assumed that the variability was low when its value was less than 0.1, medium when it fell between 0.1 and 0.25, and high when it exceeded 0.25.
CL82 turned out to be the least variable repeat: its abun dance was mediumcloser to high and ranged from 2.9 to 3.8.
The medium coefficient of variation for the copy number of satellite repeats in all studied accessions (from 0.16 to 0.25) was observed in nine specific repeats, namely CL67, CL3, CL185, CL119, CL192, CL89, CL115, CL95, and CL168 (listed in ascending order based on their coefficient of varia tion).The average copy number values in Pseudoroegneria species were 2-11 % higher compared to the entire studied collection.However, CL67 had the highest copy number in the rye genome (3.2).CL89 showed the highest value in P. libanotica (2.8), CL3, CL119, and CL115 had the highest values in P. kosaninii (2.7, 3.1, and 2.7, respectively), CL95 had the highest value in both P. cognata and P. kosaninii (2.1), and CL168 had the highest value in P. cognata (2.9).The re maining repeats of this group were generally characterized by a lowtomedium level of copy number across all accessions.The minimum copy number was observed in CL192, ranging from 1.2 to 2.5, while the maximum copy number was found in CL185, ranging from 1.8 to 3.2.

Discussion
Satellite repeats constitute a significant portion of the Triticeae genome and play a crucial role in the formation and evolu tion of new species.As a consequence, they serve as valuable tools for analyzing these processes (Shcherban, 2015;Salina, Adonina, 2019;Vershinin et al., 2023).The search for new satellite repeats is necessary to understand the phylogenetic relationships and evolution of the Triticeae tribe, which is of significant importance to humans.One of the initial steps in determining the suitability of the identified satellite repeats as tools for such studies is to conduct a comparative assessment of their copy number in related species.
Some of the satellite repeats we found in the Stgenome showed a similar copy number among the studied species.Homologs have been found in the genomes of wheat and barley, suggesting their common origin.CL82 and CL119 showed similarity to pTa451 and pTa465, respectively, which were identified in T. aestivum (Komura et al., 2013).CL67 is similar to the centromeric retrotransposon Cereba (Hudako va et al., 2001) and is conserved in Triticeae (Dvořák, 2009).Although CL3 is 79 % identical to the E-specific repeat 51-6, it did not show any specificity for Thinopyrum species in our study.This suggests that we have discovered an older and less genome-specific variant.
CL69 was distinguished by a high copy number in Pseu doroegneria accessions and a medium copy number in Thi nopyrum and E. pendulinus species.This may indicate its occurrence before the divergence of the St and Jgenomes.CL101 has a medium copy number in Pseudoroegneria and Th.junceum species and could also occur in a common ances tor of the St and Jgenomes.Since CL101 is 80 % identical to the subtelomeric Spelt1like repeat, it is likely that it may have a common origin with Spelt1, which is common in Triti cum and Aegilops.This repeat is characterized by significant variation in copy number between species (Pestsova et al., 1998;Ruban, Badaeva, 2018).The copy number of CL69 and CL101 in individual accessions is relatively high, reaching values of up to 3.9 and 5.3, respectively.This makes them suitable candidates for use as chromosomal markers in the FISH procedure.Further experiments using the FISH method will show whether these repeats can serve as chromosomal markers for identifying the Stsubgenome in polyploid species, such as E. pendulinus, studying recombinant J vs genomes in intermediate wheatgrass and investigating chromosomal rear rangements in wide wheat hybrids.
The highest copy number value in P. cognata and S. cereale was demonstrated by CL184, which shows similarity to the Cassandra retrotransposon found in the rye genome.Cas sandra is found in the genomes of many plant species and is characterized by significant differences in copy number between them (Kalendar et al., 2020).Since one of the mecha nisms by which satellite repeats are propagated throughout the genome is through the movement of retroelements (Gar rido Ramos, 2021;Šatović-Vukšić, Plohl, 2023), it is possible that we have identified a repeat that has been retained as a consequence of Cassandra spreading along the ancestral lineage of the St and Rgenomes.
CL244, previously identified by us in the Ae.crassa ge nome, was characterized by a higher copy number in Th. bes sarabicum than in common wheat, Ae. crassa and Ae.taus chii (Kroupin et al., 2022).In this study, the results were confirmed.However, at the same time, there was a significant variation in copy number between Pseudoreogneria species, which could be attributed to the elimination or accumulation of CL244 during speciation and subsequent evolution.CL244 has terminal localization on chromosome Th.bessarabicum (Kroupin et al., 2022), and can presumably accumulate or be eliminated in various species, similar to the terminal repeats of Spelt1 and Spelt52 in Aegilops and Triticum (Raskina et al., 2011;Ruban, Badaeva, 2018) or pSc200 and pSc250 in rye (Evtushenko et al., 2016).CL220, which is specific to P. kosaninii, exhibited similari ties with CL219, which we had previously identified in the Ae.crassa genome (Kroupin et al., 2022).CL186, specific to P. cognata, showed similarity to ACRI_CL80, which was identified in A. cristatum (Pgenome) (Said et al., 2018).Both repeats probably arose before the divergence of Triticeae ge nomes from a common ancestor and accumulated in separate species at certain periods.Since CL219 and ACRI_CL80 were localized on separate chromosomes, it can be inferred that CL220 and CL186 will also exhibit chromosome-specific localization on the chromosomes of P. kosaninii and P. cog nata, respectively.
We have identified repeats that vary in copy number between Pseudoroegneria species with varying levels of ploidy.For example, the octaploid P. kosaninii is characterized by a high copy number of CL115, CL190, and CL220, while the tetraploid P. geniculata has a high copy number of CL134.The observed differences in the abundance of the repeats may be attributed to polyploidization processes.This is because tandem repeats in the centromeric and terminal regions have a significant impact on chromosome recognition and divergence
Comparison of the repeat copy number patterns helped determine which of them have similar copy numbers among the studied accessions (see Supplementary Materials 2.3 and 2.4).CL3, CL115, CL119, CL190, and CL220 were grouped together because they exhibited the highest levels of copy number in P. kosaninii.CL95, CL207, and CL300 are more specific to P. cognata and P. kosaninii.In CL128, CL168, and CL186, the maximum copy number was observed in P. cognata.A comparison of the species copy number patterns allowed for a general understanding of which accessions are characterized by similar repeat copy numbers.
The overall clustering of Pseudoroegneria species (see Supplementary Materials 2.5 and 2.6) indicates that, in ge neral, they exhibit similar copy numbers of repeats that are different from those of other species.The copy number pat tern of E. pendulinus (2n = 28, StStYY) differed from that of Pseudoroegneria, suggesting that the St-specific repeats we discovered could be valuable for analyzing the Stsubgenome of E. pendulinus and other Elymus sensu lato accessions.Thi nopyrum and common wheat exhibited different copy number patterns.CL244 and CL69 can be utilized to identify wheat wheatgrass hybrids and detect introgressions from all three studied wheatgrass species into the wheat genome.Similar ly, CL134 and CL251 can be used for this purpose in Th. jun ceum.
L. Wang et al. (2017) found a repeat of St 2 80 in the genome of P. libanotica, hybridizing along the entire length of the St (sub)genome chromosomes and only in the terminal regions of the E, H, P, and Y(sub)genomes.Q.L. Liu et al. (2020) identified mobile elements in the genome of P. stipifolia, in cluding dispersed repeats S13, S158, and S21, which showed varying intensity between the chromosomes of the St and Y-subgenomes.They also found S5, which had a specific point localization and differed between the chromosomes of the St and Ysubgenomes.D. Wu et al. (2022) created chro mosomal markers STlib_96, STlib_98, and STlib_117 based on satellite repeats of P. libanotica.However, the possibility of using these markers for the analysis of allopolyploids with the Stgenome is not reported.
Our assessment of the copy number of satellite repeats found in the St-genome and the determination of their amplification specificity between species can enhance the range of molecu lar genetic and cytogenetic markers utilized in studying the Triticeae tribe.The copy number of satellite repeats can vary significantly between species, populations, and even within them (Wang Q. et al., 2010;Belyaev, Raskina, 2013;Pollak et al., 2018;Tao et al., 2021).The satellite repeats identified in the present study can be useful, among other purposes, for population studies of Pseudoroegneria and Triticeae species.

Conclusion
Based on the data from wholegenome sequencing of Pseu doroegneria accessions, we identified 22 satellite repeats.In the genomes of 14 representatives of the Triticeae tribe, we determined their copy number, including CL244, which was previously discovered in Ae. crassa.As a result of the evalua tion, the studied repeats were classified according to the level of abundance and variability between species.The satellite repeats identified in the present study can be used as molecular genetic markers for evolutionary, phylogenetic, and population studies of Triticeae.They also have the potential to serve as cytogenetic markers for in situ hybridization.

949 ГЕНЕТИКА РАСТЕНИЙ / PLANT GENETICS
Comparative assessment of the copy number of satellite repeats in the genome of Triticeae species 1 GRIN -germplasm resources information network of the Agricultural Research Service of the US Department of Agriculture (USDA-ARS Germplasm Resources Information Network). 2VIR -N.I.Vavilov All-Russian Institute of Plant Genetic Resources. 3NGC -P.P. Lukyanenko National Grain Center.