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Article

Combination of Vrn Alleles Assists in Optimising the Vernalization Requirement in Barley (Hordeum vulgare L.)

by
Raushan Yerzhebayeva
1,*,
Tamara Bazylova
1,
Gaziza Zhumaliyeva
1,
Sholpan Bastaubayeva
1,
Askar Baimuratov
1,
Burabai Sariev
1,
Galym Shegebayev
1,
Namuk Ergün
2 and
Yuri Shavrukov
3
1
Kazakh Research Institute of Agriculture and Plant Growing, Almaty District, Almalybak 040909, Kazakhstan
2
Central Research Institute for Field Crops, Ankara 06170, Turkey
3
College of Science and Engineering, Biological Sciences, Flinders University, Adelaide, SA 5042, Australia
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(13), 1389; https://doi.org/10.3390/agriculture15131389 (registering DOI)
Submission received: 16 May 2025 / Revised: 17 June 2025 / Accepted: 23 June 2025 / Published: 28 June 2025
(This article belongs to the Section Crop Genetics, Genomics and Breeding)
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Abstract

Vernalization genes (Vrn) play a key role in plant adaptation to various geographic locations and their allelic diversity can have fundamental importance for breeding programs. In the current study, 340 barley genotypes were studied, including germplasm accessions and advanced breeding lines. For phenotype evaluation in South-Eastern Kazakhstan, the transition of barley plants from vegetative to reproductive stages was estimated in field trials with spring- and winter-sown seeds. For molecular analysis, 10 previously described molecular markers were studied in three barley vernalization loci: Vrn-H1, Vrn-H2 and Vrn-H3. The comparison between molecular results and phenotypes for plant development confirmed 211 spring genotypes, 56 winter and 28 facultative. Vrn-H1 haplotypes 1A and recessive allele vrn-H3 were in the majority. Best spring and winter high-yielding advanced breeding lines were identified. Based on Vrn allele combination, a breeding line 76/13-4 with facultative type development showed superior results in both winter and spring sowings, presenting a new prospective barley cultivar that can be grown equally either in spring or winter sowing conditions. The presented results can be used for barley marker-assisted selection predicting crosses with favourable combinations of Vrn alleles for prospective breeding line development.

1. Introduction

Cultivated barley (Hordeum vulgave L.) is the fourth most important grain crop production-wise after rice wheat and maize. Barley grains are widely used for feeding animals, foods and malt production in the brewing industry [1]. Barley is characterized by high ecological plasticity with early maturity and tolerance to drought and salinity [1,2,3]. It has led to its cultivation throughout diverse climates in mild temperate regions with an altitude of 350–4050 m above sea level [1,2]. Most of the world’s barley is produced in regions where grain crops such as rice and maize cannot grow well [2]. According to FAO, the area under barley cultivation in the world in 2023 was 46.3 M hectares, with production of 145.7 M tons [4].
Rapid growth and high yield of barley cultivars in various environments is strongly determined by optimal allele combinations of key genes, including primarily the loci Vrn (Vernalization) and Ppd (Photoperiod), which control plant response to vernalization and day length. Favourable combinations of Vrn and Ppd alleles significantly and positively affect the rate of plant development, crop structure, frost and winter hardiness, vernalization requirement and early flowering to escape hot temperatures [5,6,7,8].
Three loci are known to determine the requirement for vernalization: Vrn-H1 (=HvVrn1) on chromosome 5H, Vrn-H2 (=HvVrn2) on chromosome 4H and Vrn-H3 (=HvVrn3) on chromosome 1H. Gene Vrn-H1 represents a central regulator of cold temperature vernalization, with a transition to tillering and final induction of flowering [9,10]. This gene encodes the AP1-like MADS-box Transcription Factor (HvBM5A), and it has been shown that Vrn-H1 had much higher expression in spring barley compared to winter type barley at the seedling and tillering stages [11]. Spring and winter types of barley are distinct as different haplotypes of Vrn-H1 based on the presence or absence of a key regulatory element located in the ‘hot-spot’ of the first intron [12,13]. This ‘hot-spot’ is believed by researchers to be involved in cold-activated transcriptional control of Vrn-H1 expression. All winter barley genotypes have ‘initial’ or ‘original’ haplotypes of vrn-H1 without any deletion or rearrangement in this ‘hot-spot’ of the first intron, and they require vernalization for the transition to the reproductive stage. In contrast, the presence of deletions in the first intron of the Vrn-H1 gene resulted in the appearance of several novel and diverse dominant alleles of Vrn-H1 [14,15]. For example, a new allele Vrn-H1-11 was identified during exome sequencing of 403 barley accessions, mostly formally bred cultivars and landraces, with 8283 bp deletion in the first intron, and it was linked to a reduced plant vernalization requirement and spring growing habit [15]. This and other dominant alleles cause the spring type of barley plants, which do not require vernalization. It was confirmed that this spring haplotype of Vrn-H1 with a high expression level was followed by the reduction in the vernalization requirement in barley [12,13,16,17,18,19].
The second gene Vrn-H2, a repressor of flowering, encodes a protein containing zinc finger and ZCCT domains, and it delays the transition from vegetative to reproductive stage in winter-type barley if they have not passed through a vernalization treatment [20]. In winter genotypes, the Vrn-H2 locus was identified as a cluster with three similar ZCCT-H genes (Ha, Hb, and Hc) and considered to be a dominant allele [20]. In contrast, the recessive allele vrn-H2 was associated with accelerated heading and the spring type of barley plants. The vrn-H2 allele had a deletion of all three ZCCT-H genes [12,20].
The third gene Vrn-H3, also known as HvFT1, is located in chromosome 7HS, and it represents an ortholog of the Flowering Locus T gene in Arabidopsis thaliana [21,22]. Vrn-H3 is a major integrator of photoperiod and vernalization signals in the apical meristem, leading to the transition of barley plants to the reproductive state and flowering. The expression of Vrn-H3 can be activated in long day conditions and after completion of a vernalization period in winter-type barley [11,19,21,23]. The spring-type allele, Vrn-H3a, which confers an extremely early flowering habit, is distributed mainly in spring barley cultivars originating from places with high latitudes, where it is likely of some adaptive importance [24].
To combine high productivity and frost tolerance, winter and spring barley are often crossed. The resulting facultative barley genotypes have flexibility for sowing in spring or autumn and increased tolerance to winter conditions. Facultative barley can be very important as an additional crop to grow in unfavourable weather conditions. However, the identification of facultative forms is not so simple and usually based on phenotyping evaluation. Only genetic identification using modern methods of molecular markers can guarantee genotyping and identification of facultative barley.
In this study, known DNA markers are described and used for haplotype identification in three major loci, Vrn-H1, Vrn-H2 and Vrn-H3, in various barley accessions and breeding lines. It was used to prove the central dogma for breeders that the allele combination in all three Vrn genes in barley has to be optimised for accurate prediction of barley plant growth and development. This was successfully realised in six crosses and hybrid populations derived from various barley inbred lines [14]. Therefore, the identification and optimisation of allele combinations in all three vernalization genes is important for better design of new cultivars with specific requirements for vernalization.
The aim of this study is to carry out DNA identification of a barley germplasm collection including cultivars and developed breeding lines for vernalization genes Vrn-H1, Vrn-H2 and Vrn-H3 to accurately determine winter, spring or facultative type of plant development and predict their vernalization requirement.

2. Materials and Methods

2.1. Plant Material

In this study, 340 barley accessions were used with presumed winter and spring developmental habits, and diverse origins. From them, the following accessions were selected and used in KRIAPG field trail tests: 186 barley germplasm originated from Genebanks, including the local Kazakh Research Institute of Agriculture and Plant Growing (KRIAPG, Almaty region, Kazakhstan), the International Center for Agricultural Research in the Dry Areas (ICARDA, Beirut, Lebanon), and the Barley Breeding Department of Central Research Institute for Field Crops (Ankara, Türkiye). Additionally, 93 developed breeding lines, designated as Group 1 (generations F7–8), and 61 advanced breeding lines from Group 2, (generation F9–10), were selected, providing a total of 154 breeding lines with winter and spring types based on their previous phenotype evaluation. The full list of the studied barley accessions and their origins is presented in Supplementary Table S1.
For molecular analyses, the following barley cultivar varieties were used as positive controls. Winter type cultivars: Aydin, Avci-2002 and Siberia with the genotype vrn-H1/Vrn-H2/vrn-H3; facultative type cultivars: Bulbul-89 and Tarm-92 with the genotype vrn-H1/vrn-H2/vrn-H3 [5], and spring type cv. Arna with the genotype Vrn-H1/vrn-H2/vrn-H3 [25].

2.2. Research Field Experiments and Field Trials

Field experiments with barley cultivars were carried out at KRIAPG in the period 2023–2024. The field site was in the hill zone of South-Eastern Kazakhstan (Almaty region), at an altitude of 740 m above sea level, 43°15′ N and 76°54′ E.
Seeds of all barley accessions were sown in two experiments, in autumn (October 2023) for winter growing, and in spring (April 2024) for summer growing. In Group 1, plants were grown in three-row plots, 1 m × 0.5 m = 0.5 m2, whereas in Group 2, field plots were seven rows, 5 m × 1 m = 5 m2, with similar density of 300–350 plants per m2 in both tests. All these field tests were carried out in triplicate with a completely randomized plot design. The set-up of experiments and all agronomic procedures were carried out at the same time and style as regular field trials.
General plant growing, measuring and recording were made according to the following recommendations [26]. For yield measurement, seeds from each plot were collected at harvesting, threshed and weighed. Seed yield was re-calculated for ‘t/ha’ in each plot. Thousand seed weight (TSW) was measured for 1000 counted seeds and recorded after weighing on scale (RV3102, Ohaus Adventurer, Shanghai, China) with two-decimal accuracy.

2.3. Meteorological Conditions of the Experimental Spot

According to Köppen’s classification [27], the climate of the Almaty region is ‘Dfa’, which can be described as continental, with hot summers. The average annual temperature is 6.5 °C, and the amount of precipitation for the entire season reaches 891 mm [28]. The soils are light chestnut, and the total humus content in the arable layer is low, ranging from 1.6–1.9%. The soil is slightly alkaline with a pH of 7.8, and the content of clay particles reaches 34.9%.
Meteorological conditions during the study period (precipitation and average air temperature) were collected by an automatic weather station, located 800 m from the experimental site. The amount of precipitation for the winter barley growing season, October 2023–June 2024, was 758.0 mm, compared to 615.9 mm in long-term previous observations (1991–2020). In contrast, precipitation for the spring barley growing season, April–July 2024 was 337.4 mm, which was very similar to the 325.8 mm long-term observations.
The average temperature during the autumn–winter growing period was 13.4 °C, 6.8 °C, −1.0 °C, −1.2 °C, and −4.0 °C in each of five months, from October 2023 to February, 2024, respectively. The average temperature during the spring–summer growing period was registered as 5.4 °C, 12.8 °C, 17.6 °C, 24.5 °C, and 25.0 °C in each month, from March to July 2024, respectively.

2.4. Assessment of Developmental Stages in Barley Plants

In this study, the Zadoks decimale system was used in phenotyping evaluation for stages of plant development [29], including major stages: germination (09, leaf just at coleoptile tip); tillering (25, main shoot and 5 tillers); inflorescence emergence (59, emergence of inflorescence completed); anthesis (65, anthesis half-way); and ripening (92, caryopses hard). In barley plots, the transition of plants from vegetative to reproductive stage was estimated as ‘earing’, ‘half-earing’ or ‘no earing’, if all plants, 50% or no plants in the entire plot showed the occurrence of spikes, respectively.

2.5. Molecular Analyses

Seeds were germinated in Petri dishes with moistened paper towels. After 14 days of germination, the biggest leaf was collected from each of the three typical seedlings and bulked as the representative sample for each studied barley accession. DNA was extracted from fresh representative samples using the method of Dellaporta [30].
For allele identification of three vernalisation genes, Vrn-H1, Vrn-H2 and Vrn-H3, standard PCR was used with a Thermal Mastercycler Pro (Eppendorf, Hamburg, Germany). The sequences of corresponding primers and conditions of PCR amplification are present in Supplementary Table S2. The PCR cocktail mix for amplification in 20 µL reactions consisted of the following components in their final concentrations: 5 ng of template DNA, 1xTaqBuffer with (NH4)2SO4, 0.2 mM of each dNTP, 0.25 mM of each primer, 2.5 mM MgCl2, 2.5% DMSO, 1 U of Taq polymerase E-3050, and total volume adjusted with nuclease-free water (all reagents were purchased from Biosan, Novosibirsk, Russia).
For the identification of allelic variation in the Vrn-H3 gene, a Cleaved amplified polymorphic sequences (CAPS) marker was used. CAPS markers are based on restrictions of PCR products by specific endonucleases, where recognition sites can be changed with SNP [31]. The amplified PCR products of the HvFT1 marker were digested with Ksp22I endonuclease. The restriction was carried out in a 15 μL total volume and included 50% of PCR product, 1×Reaction Buffer, 10 U of Ksp22I restriction enzyme (SibEnzyme, Novosibirsk, Russia), and volume adjusted with purified nuclease-free water. Digests were incubated at 37 °C for 4 h. The amplified PCR products, including those after digestion, were separated in an 8% polyacrylamide gel and visualized and imaged using a UV Transilluminator (Quantum ST-4, Vilber, France). DNA marker Step50 Plus (BiolabMix, Novosibirsk, Russia) was used for molecular weight identification of PCR products.

2.6. Statistical Treatments

Statistical data processing was carried out with R software, version 4.4.1 (Race for Your Life), and the program JASP, version 0.19.3.0 [32]. The chi-square independence criterion was used to determine the correlation between DNA marker results for allelic variation in Vrn loci and the transition of barley genotypes from the vegetative stage to heading and flowering in field trials [33]. Cohen’s kappa (κ) coefficient of agreement was used to assess the level of agreement between molecular results of DNA markers [34]. A one-way analysis of variance (ANOVA) was carried out for multiple comparisons of seed yield in plots and TSW with the post-hoc Tukey test.

3. Results

Allelic variations in vernalization loci Vrn-H1, Vrn-H2 and Vrn-H3 were identified using allele-specific primers in 183 barley from germplasm collections and 157 advanced breeding lines with winter, spring and facultative growth types in experiments carried out at KRIAPG. The transition from the vegetative stage to reproductive with flowering in spring-sown barley accessions was also assessed to determine whether these genotypes required a vernalization treatment.

3.1. Field Trial Tests for Phenotypic Evaluation in Barley Plants

Field experiments were conducted to assess the need for low-temperature exposure to initiate a transition to flowering in barley accessions. In the current study, 340 barley germplasm accessions in total were grown in field conditions with winter and spring sowing. During winter 2023 sowing, in the conditions of South-Eastern Kazakhstan, all barley genotypes successfully grew until spring with 85–95% of winter survival range in 340 accessions, and initiated earing with subsequent flowering.
In contrast, the spring 2024 sowing test of 340 barley accessions showed only 246 of them (74%) transitioning to heading and flowering, including cultivars used as positive controls, Arna, Bulbul-89 and Tarm-92. The remaining 94 samples stayed in the tillering phase until the end of July (harvesting time), including cultivars Aydin and AVCI-2002, used as positive controls for winter barley genotypes.
In the barley germplasm collection, out of 84 genotypes with a presumed winter type of development, 61 accessions did not start the earing and flowering phase, including 33 advanced breeding lines (Figure 1 and Supplementary Table S1). These barley genotypes seem to have a winter growth type and require vernalization. Thus, out of all 340 barley accessions studied during spring 2024 sowing, 94 genotypes including 61 from the germplasm collection, 33 advanced breeding lines and 2 control winter barley cultivars, Aydin and AVCI-2002, remained in tillering without transition to the reproductive stage. This indicates that the identified accessions represent winter barley with a requirement for vernalization.

3.2. Genetic Polymorphism of the Vrn-H1 Gene

To identify the allelic variation of the Vrn-H1 gene, three markers were used, described in Supplementary Table S2 [35,36]. Two cultivars, AVCI-2002 and Siberia served as control genotypes with known alleles of Vrn-H1. According to [35], marker 1 allowed the identification of the winter allele (R) upon amplification of either 830 bp or 344 bp fragments. In barley genotypes with a spring allele (D), such amplification cannot occur due to the presence of deletions in this locus, indicated as a ‘null-allele’.
In the present study, from PCR using marker 1, these fragments were recorded in 126 barley accessions, whereas 830 bp fragments were identified in 115 accessions and 344 bp fragments were found only in 11 genotypes (Supplementary Table S1). The PCR results showing the amplification of fragments associated with winter barley haplotypes of advanced breeding lines are presented in Figure 2.
Marker 2, according to [36], can amplify a fragment for 435 bp in the first intron. The presence of bands with this size correlated with a winter growth habit whereas the absence of this band indicated a spring growing type. Using PCR with primers for marker 2, 164 barley accessions were identified with a 435 bp amplified fragment in the studied genotypes, i.e., the winter allele of Vrn-H1, designated as R (Supplementary Table S1).
The next marker 3 was used to detect the spring type allele associated with fragments with a size of 574 bp or 616 bp [35]. In the current study, 326 accessions were recorded with these two fragments, showing a clear majority (96%) of the allele distribution in the studied accessions.
The applied statistical analysis with the chi-square criterion showed the associations between genotypes with the allelic variation of Vrn-H1 locus and their transition from vegetative to reproductive stage-producing ears and start flowering among spring sowing barley.
The analysis was performed for each DNA marker separately (Table 1). A high correlation was found between the results of the first and second markers for determining of winter or spring allele of the Vrn-H1 gene and heading occurrence during spring sowing. The highest probability of coincidence of the marker results and heading of genotypes was established for marker 1, χ2 =171.5, p < 0.001 (Table 1). However, analysis of the results for all three markers 1–3 showed that there were some inconsistencies in the results for determining of winter or spring-type allele of the Vrn-H1 locus. The markers did not fully confirm each other. Statistical analysis of nominal data was performed to estimate the results of three markers for winter-type allele R and spring-type allele D. The assessment with Cohen’s kappa agreement coefficient showed that the first and second markers had the highest Cohen’s kappa agreement coefficient κ = 0.66 with SE = 0.04, and it was characterized as significant.
In contrast, the agreement results between markers 1 and 3 (κ = 0.07, SE = 0.03) and between markers 2 and 3 (κ = 0.06, SE = 0.02) were not significant. The average Cohen’s kappa agreement coefficient for all three markers was satisfactory, κ = 0.26.
The final determination of the allelic variation (winter or spring-type) of the Vrn-H1 gene in the studied barley accessions for genotype formula was made by comparing and matching the results of all three markers 1–3. In the result, 123 genotypes were identified as carrying the winter recessive allele R whereas 217 other genotypes had the spring dominant allele D of Vrn-H1 locus. Full details of genetic polymorphism for alleles of the Vrn-H1 gene are presented in Supplementary Table S1.

3.3. Genetic Diversity of the Vrn-H2 Gene

The vrn-H2 locus of winter genotypes consists of a complex cluster including three similar ZCCT-H genes (Ha, Hb and Hc). Therefore, six diagnostic markers were selected and tested that determine the allelic variation of the Vrn-H2 locus [12,20,21,37,38]. From six selected markers, five were aimed to identify the winter type of the locus, a cluster consisting of three ZCCT-H genes (Ha, Hb and Hc).
Molecular marker 4 can amplify a 307 bp fragment of the ZCCT-Ha gene and a 273 bp fragment of the ZCCT-Hb gene [20], and this is a dominant marker. Amplification of the indicated fragments should occur in genotypes with winter-type alleles, but it is absent in spring-type barley genotypes, as they have a deletion of the ZCCT-H gene family. In the current study, simultaneous amplification of two fragments 273 bp + 307 bp was recorded in 81 genotypes out of the 340 studied. They were identified as barley genotypes with the dominant winter-type allele D [20]. The absence of PCR products was recorded as the recessive R spring-type allele vrn-H2 (Figure 3 and Supplementary Table S1).
The assessment of the presence of specific alleles of the cluster with three ZCCT genes (Ha, Hb and Hc) was continued using the next three molecular markers 5–7 (Table 1) [21,37]. Based on the results of molecular markers 5–7, 35 genotypes were identified with amplified PCR products in the coding regions of ZCCT-Ha (600 bp), ZCCT-Hb (600 bp) and ZCCT-Hc (200 bp) and, therefore, containing all three ZCCT genes. These barley genotypes were characterized as a dominant winter-type allele of Vrn-H2. The other 29 barley accessions contained only two ZCCT genes.
The molecular marker 8 was also used to identify winter-type alleles of Vrn-H2, group ZCCT-Ha and -Hb [12]. When marker 8 was used, the amplification of a 1500 bp fragment confirmed the presence of a winter-type allele of the ZCCT-H group in 52 barley genotypes. The absence of PCR products was recorded as recessive R spring-type allele vrn-H2.
The last molecular marker 9, is closely associated with the Vrn-H2 locus and proximal gene HvSnf2 [38], and it was aimed at detecting the spring-type allele of the Vrn-H2 gene. PCR with marker 9 amplified a 375 bp fragment in 302 barley accessions, 88.8% of the total number of studied genotypes.
The received results for all six molecular markers to detect winter and spring-type alleles of the Vrn-H2 locus also recorded inconsistencies. Statistical analysis of the nominal data of these six molecular markers using Cohen’s kappa coefficient of agreement showed significant agreement at the level of κ = 0.62, SE = 0.05 between markers 5 and 8, and moderate agreement between markers 6 and 7, 6–8 and 5–6 (κ = 0.43–59, SE = 0.04–0.05). The average Cohen’s kappa coefficient agreement for five markers aimed to detect winter-type allele was satisfactory, κ = 0.34, but was reduced for all six used markers, κ = 0.22.
The chi-square statistical analysis of DNA markers showed the highest probability of coincidence of marker results and earing of plants during spring sowing, established for molecular marker 4, χ2 = 66.3, p < 0.001 and for marker 7, χ2 = 39.3, p < 0.001 (Table 1).
The final determination of the allelic diversity (winter or spring-type) of the Vrn-H2 gene in studied barley accessions for the genotype formula was made by comparing and matching the results of two and a larger number of results for all six molecular markers with corresponding primers. In the result, 234 genotypes were identified as carrying recessive spring-type allele vrn-H2 and the remaining 106 other barley accessions had dominant winter-type allele Vrn-H2. More details about genetic polymorphism for alleles of the Vrn-H2 gene are presented in Supplementary Table S1.

3.4. Genetic Variability of the Vrn-H3 Gene

For the analysis of genetic polymorphism in the vernalization gene Vrn-H3, a CAPS marker was used, designated as marker 10 in the current study (Table 1) [22]. The amplified PCR products using molecular marker 10 showed fragments with identical lengths of 350 bp. However, a different pattern of PCR products was found after the treatment with endonuclease Ksp22I. The dominant spring-type D allele can be identified with three fragments of digested PCR products of 138, 142 and 69 bp. In contrast, the recessive winter-type R allele showed only two digested PCR fragments of 138 and 211 bp in length. In the current study, among the 340 barley germplasm studied, only eight genotypes were identified with the dominant D allele of Vrn-H3, and the rest of the 332 accessions showed the recessive R allele (Figure 4). The relatively rare eight genotypes with the dominant allele of Vrn-H3 originated from ICARDA (7 accessions) and one barley advanced breeding line from KRIAPG.

3.5. Barley Germplasm Classification for Vernalization: Comparison of Genotyping and Phenotyping Results

In the current study, based on the results of DNA markers for identification of allelic diversity in Vrn-H1, Vrn-H2 and Vrn-H3 loci, the formulas of all studied 340 barley germplasm accessions were determined. The following genotypes were identified as spring-type: 173 germplasm with formula DRR; 43 accessions with DDR formula; 1 genotype with formula DRD; and 1 genotype with formula DDD.
The second group of genotypes was identified as winter-type: 59 accessions with formula RDR; and 4 genotypes with RDD formula. The last group of genotypes was identified as facultative: 58 accessions with the formula RRR and 2 genotypes with the RRD formula (Supplementary Table S1).
The non-parametric chi-square test estimated the correlation between the genotypic analysis of Vrn-H genes in studied barley accessions and the actual phenotyping results for heading during spring sowing (Table 2). The significance level of the chi-square coefficient was consistently high χ2 = 197.6, df = 2, p = < 0.001. Most of the results for genetic analysis corresponded to its phenotypic evaluation.
At the same time, the comparison of phenotypic results with genotype analysis revealed a number of discrepancies. This included seven genotypes (2.1%), defined as winter genotypes that eared during spring sowing. Additionally, 31 barley accessions (9.2%) were identified as facultative and another 7 genotypes (2.0%) were determined as spring, but these barley plants did not transfer to earing and flowering stages in spring sowing.

3.6. Examples of Successful Application of Genotyping for Vrn Genes in Barley Breeding

Evaluation of barley advanced breeding lines from Group 2 using molecular markers enabled the determination of their allele composition in all three Vrn genes and growth type. Out of 17 studied breeding lines, 2 genotypes were identified as spring-type with Vrn genotype formula DDR and DRR; 11 genotypes were winter-type, RDR formula, and 4 genotypes were facultative with RRR formula of Vrn (Supplementary Table S3). Phenological observations of the growth and development of plants from the breeding lines during both winter and spring sowing confirmed the genotyping results. In spring sowing, barley genotypes with spring and facultative type of development initiated ears and flowering without a delay and matured perfectly within 75–78 days of germination, whereas winter genotypes failed to transit to flowering in the same conditions. In winter sowing, all studied breeding lines completed their growth life cycles and produced matured seeds, but they varied in their survival rates for winter hardiness. The single exception was found for winter genotype 60/15-4 with the identified formula RDR confirming their winter-type but plants of this barley breeding line eared during spring sowing. The reason for this mismatch between genotyping and phenotyping remains unclear and needs more attention in future studies (Supplementary Table S3).
Yield and HSW were evaluated in all 17 barley breeding lines both in winter and spring sowing in 5 m2 plots as described in Section 2.2 above. The best spring breeding line 70/08-3 showed higher grain yield (24–17%) and TSW (11–12%) compared to the local standard (check) cv. Bereke-54 in both winter and spring sowing, respectively (Table 3). In a similar way, the best winter breeding line 67/08-6 exceeded the local standard cv. Aydin in grain yield for 40% and TSW for 33% in winter sowing only due to no plants producing ears in spring sowing.
The best barley facultative breeding line 76/13-4 also had very similar results for grain yield and TSW compared to the best spring and winter breeding lines and significantly higher than those of the respective standards in both winter and spring sowing. This dual benefit was accompanied by a very high and improved winter hardiness survival rate (93–95%), and a non-delayed transition to flowering in spring sowing (Table 3 and Table S3).

4. Discussion

The vernalization genes Vrn-H are very important for barley plant development and the combination of all three known loci, Vrn-H1, Vrn-H2 and Vrn-H3, can have a strong impact on the breeding of this crop. Allelic diversity of these genes can form the basis of broader adaptation of barley plants. By managing favourable allele combinations in these three vernalization loci, breeders can precisely predict flowering time and produce new barley cultivars better adapted to different conditions; for example, spring type for northern regions, winter genotypes for southern areas, and facultative barley plants for locations with intermediate climate conditions [8].
Additionally, the importance of vernalization genes was demonstrated using the example of barley plants with different spring-type alleles of Vrn-H1. Heading started earlier in plants grown under drought compared to well-watered conditions and this significantly affected grain yield in barley with various vernalization genotypes [19]. In another study, frost tolerance was not changed during the introgression of diverse alleles of Vrn-H1 into Spanish barley [18], and in the study of a wide diversity of barley accessions including landraces and wild barley [15] with reduced vernalization requirement.
For Vrn-H1, in the current study, two molecular markers, 1 and 2, were used for targeting the ‘critical region’ of Intron 1, which determines winter-type alleles of the Vrn-H1 gene. The results of both markers were in consensus, confirming each other in 83.3% of studied barley accessions. The Cohen’s kappa coefficient of agreement was significant (κ = 0.66). Previously, in the identification of 52 barley accessions including local cultivars, breeding lines and landraces, the correlation of plant genotyping for the Vrn-H1 gene using two molecular markers, 1 and 2, with spring and winter-type of plant development was 100% [39]. In the current study, 340 barley accessions were studied from both local and international germplasm collections and the application of marker 1 in the studied barley accessions was successful in the identification of two winter barley haplotypes 5C (344 bp) and 1A (830 bp). Barley accessions with Vrn-H1 winter haplotype 5C included six advanced breeding lines (66/08-11, 8/16-11, 1-8, 11/16-2, 22/18-5 and 11-16-5), three winter genotypes from Germplasm barley collections (IBYT-W-3, IBYT-W-321 and IBYT-W-382), and two cultivars from Türkiye (Anka-06 and Bozlak). The majority (91.1%) of the studied barley accessions with winter-type allele Vrn-H1 was represented by haplotype 1A. This finding is similar to previously published reports that winter haplotype 1A was also identified as predominant among 400 barley cultivars distributed over most of Western Europe [40] and in 100 British barley cultivars [35].
According to Cockram et al. [35], the other seven Vrn-H1 haplotypes (1B, 2, 3, 4A, 4B, 5A and 5B) are known as spring barley haplotypes. In this regard, molecular marker 3 was used in the current study to identify spring-type alleles. In our results, two PCR fragments, 616 bp and 574 bp, were found in the present study in almost all tested barley genotypes (95.9%). Similar results were reported earlier with the same specified molecular markers, where the first PCR fragment, 616 bp, was amplified not only in spring haplotypes 1B and 2, but also in winter haplotype 1A [35]. The second PCR product, 574 bp, also was amplified both in spring haplotypes 3, 4A, 4B, 5A and 5B, together with winter haplotype 5C [35].
In the present study, the amplification of fragments of either 574 bp or 616 bp using molecular marker 3 was recorded in all barley advanced breeding lines. These breeding lines easily eared during spring sowing and, therefore, this finding in the current study confirmed the presence of spring-type alleles Vrn-H1. Moreover, in all barley accessions identified as spring genotypes in the current study, the amplification of PCR fragments failed using molecular marker 3.
Overall, molecular markers 1 and 3 confirmed their effectiveness for Vrn-H1 allele genotyping, and it shows a strong consensus with previously published data [35]. The high efficiency of molecular markers 1 and 3 in the identification of spring-type alleles was also demonstrated in the study of 90 cultivars of spring barley of Russian and Belarusian selection, where spring haplotypes with 574 bp or 616 bp fragments were recorded [25].
For Vrn-H2, six diagnostic markers (4–9) were tested to identify the allelic variation of the gene. From that, five molecular markers (4–8) aimed to identify the winter-type alleles of Vrn-H2, with a cluster of three genes, ZCCT-Ha, Hb and Hc. Results were positive for all five markers, indicating the presence of the cluster in 10 genotypes. In the remaining barley accessions, the level of agreement varied from 32 to 53%. The agreement coefficient between markers, calculated using Cohen’s Kappa, was κ = 0.34, indicating a satisfactory level of agreement. However, the efficiency of these markers for practical selection was insufficient. This made it difficult to accurately identify the winter-type allele of the Vrn-H2 gene in our experiments, compared to those published earlier [12,20].
The molecular marker 9, associated with the HvSnf2 gene, proximal to Vrn-H2 [38], aimed to identify the spring alleles of Vrn-H2. The results obtained are consistent with previously published data [12,41]. The amplification of a 1500 bp fragment using molecular marker 8 was observed only in winter genotypes, while a 375 bp fragment amplified with marker 9 was detected in both winter and spring barley genotypes. The presence of the spring-type allele was recorded only in barley accessions with PCR amplification of the 375 bp fragment, but not the 1500 bp fragment.
According to [42], from the three genes of the ZCCT-H cluster in barley, Ha and Hb play a key role in the formation of plants with a winter type of habit. Deletion or mutations in any of them can reduce the requirement of plants for vernalization. At the same time, according to [43], ZCCT-Ha and -Hc genes are the most significant for controlling vernalization requirements in barley plants. In addition to molecular marker 4 in our present study, marker 7 was applied to identify the presence of ZCCT-Hc alleles. The amplification of PCR fragments from the Hc gene showed a significant association with phenotypic evaluation for vernalization and heading barley plants. This finding confirms the significant contribution of the ZCCT-Hc gene in the regulation of plant transition from the vegetative to the reproductive stage and, accordingly, its necessity for vernalization.
Spring-type alleles of the third vernalization gene, Vrn-H3, cause an extremely early start to flowering, and they were found predominantly in exotic barley accessions [14]. A CAPS marker was used to identify the allelic variation in this gene. CAPS markers are widely used for molecular characterization of barley and other crops [44,45], because these markers are codominant and very effective in detecting genetic polymorphism with simple interpretation [46,47]. In our current study, 97.6% of the analysed accessions had the recessive allele vrn-H3, and only eight genotypes had the dominant allele. The obtained data correspond to the results of genotype identification in germplasm collections reported by other researchers: the recessive allele vrn-H3 was found in 75% of 90 barley cultivars used in Russia and Belarus [25], as well as in 100% (94 accessions) of a winter barley germplasm collection [41]. The identified genetic resources of dominant alleles are recommended to be included in crosses to expand genetic diversity and accelerate flowering and maturity time in barley plants.
Genetic analysis in the current study found 59 barley accessions with a facultative growth (RRR genotype). The facultative growth pattern is determined by a deletion in the Vrn-H2 locus and the co-presence of winter-type allele vrn-H1. Such genotypes can have considerable value and importance, as they combine winter hardiness with no mandatory need for vernalization [12,20,43]. In the current study, a delay in earing time by 5–10 days was observed in some facultative forms. Partial earing was observed in nine facultative forms (30–40%).
However, in our studies, 31 of the 59 genotypes with a facultative type of plant development failed to enter the reproductive phase during spring sowing. This may indicate potential inaccuracy in allele identification using DNA markers and, presumably, such ‘conflicting’ genotypes may belong to winter-type barley plants. Additionally, identification errors may occur due to partial deletions of ZCCT-H genes, recombinations or heterogeneity of studied barley germplasm accessions. Sometimes, the use of dominant markers may lead to false negative results, which also can affect the accuracy of data interpretation.
According to some published reports, spring sowing facultative forms demonstrated significant delays in earing time, often had shifted or irregular development, and delayed ear emergence [39,48]. The facultative type of development has an intermediate form between the winter and spring types and, therefore, they still require a certain cold temperature and photoperiod to undergo flowering induction. In spring sowing, especially in short-day zones, this signal may be insufficient, resulting in delayed or even complete absence of heading [48]. Finally, plant response to cold temperature and transition to plant reproduction could result from a more complicated molecular–genetic mechanism, where additional (Ppd genes) and unknown factors can play a significant role in vernalization requirements in different barley genotypes [49,50].
Nevertheless, the successful story of one facultative advanced breeding line, 76/13-4, presents a very important demonstration of modern and relatively simple molecular technology, where marker-assisted selection has shown a very positive contribution to barley breeding. The 76/13-4 breeding line was identified with a combination of alleles in three Vrn genes and it showed high yield and thousand seed weight in both winter and spring sowings. Therefore, it can provide additional benefit for barley farmers, when they decide to sow seeds of facultative breeding line 76/13-4 in winter, spring or in both seasons with significantly higher outcomes for grain yield with larger-sized seeds. It is also significant that this breeding line 76/13-4 has been submitted as a new barley facultative cultivar for agricultural production in Kazakhstan.
In the current study, through genetic and phenotypic analysis, allelic variation of 295 genotypes was established and 56 winter genotypes, 211 spring genotypes and 28 facultative barley genotypes were identified. In those cases where mismatches occurred between genotype and phenotype, further and more careful experiments are required.

5. Conclusions

Based on the presented results, allelic variation was analysed for three vernalization loci in 295 barley accessions. As a result of genotyping, 56 true winter barley, 211 springs, and 28 facultative accessions were identified. Two effective markers, 1 and 3, were identified for the determination of allelic variation in the Vrn-H1 gene. These markers demonstrated a high degree of association with earing phenotype during spring sowing (χ2 = 171.5–102.1, p < 0.001), as well as a significant Cohen’s kappa κ = 0.66, SE = 0.04, and this confirms their practical application in marker-assisted selection. From six molecular markers tested in the analysis of the Vrn-H2 locus, two identified markers, 4 and 7, demonstrated a significant association with the transition of plants to the reproductive stage during spring sowing (χ2 = 66.3–39.3, p < 0.001).
The genotype analysis provided knowledge and scientific information for breeders about genetic polymorphism and allelic diversity in vernalization loci in barley germplasm collections and advanced breeding lines. Barley genotypes with identified rare alleles Vrn-H1 and Vrn-H3 are recommended to breeders for crossing to enrich the genetic diversity of barley breeding material.
In the current study, novel facultative barley breeding lines were identified with high flexibility to grow in different sowing periods. The advanced breeding line, 76/13-4, with a facultative type of development was identified in Group 2 based on the combination of alleles in three Vrn genes. This breeding line showed high yield and TSW in both winter and spring sowings, and is now recommended as a new barley facultative cultivar for agricultural production in Kazakhstan.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15131389/s1. Table S1: The full list of the studied barley accessions, their genotyping with 10 molecular markers and phenotyping evaluation. Table S2: The used molecular markers, sequences of corresponding primers and conditions of PCR amplification. Table S3: Evaluation of 17 barley advanced breeding lines with different growth habits in comparison to standard cultivars (checks) for yield and TSW in winter- and spring-sowing field trials.

Author Contributions

Supervision and writing—original draft preparation, R.Y.; methodology and data curation, T.B.; investigation and visualization, G.Z.; investigation and software, G.S.; investigation, A.B. and B.S.; project administration, S.B.; resources, N.E.; writing—review and editing, Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan, Grant No. АР19678544 “Production of facultative forms of barley based on methods of breeding and biotechnology for growing plants on rainfed and non-irrigated fields of Kazakhstan”, and Program No. BR-22885305 “Breeding and genetic technology for the development of long-term storage systems, restoration, monitoring and rational use of agrobiodiversity, as a basic basis for improving selection programs in the Republic of Kazakhstan”.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in the manuscript are available in the Supplementary Materials.

Acknowledgments

We want to thank Carly Schramm for critical comments during the editing of the manuscript. Additionally, special thanks to the staff and students of our Research Institute and University for their support in this research and help with the manuscript preparation.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Agriculture 15 01389 g001
Figure 1. Evaluation of barley plants for the transition from tillering to heading stage in a spring sown field in 0.5 m2 three-row plots in the Almaty region, South-Eastern Kazakhstan (A) Advanced breeding line 40/16-10 remained in the tillering stage; (B) Germplasm accession ICARDA-738 showing the development to the earing stage. The images were taken 40 days after seed sowing.
Figure 1. Evaluation of barley plants for the transition from tillering to heading stage in a spring sown field in 0.5 m2 three-row plots in the Almaty region, South-Eastern Kazakhstan (A) Advanced breeding line 40/16-10 remained in the tillering stage; (B) Germplasm accession ICARDA-738 showing the development to the earing stage. The images were taken 40 days after seed sowing.
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Figure 2. Identification of allelic variation of Vrn-H1 gene in barley advanced breeding lines using molecular marker 1: R1, winter allele with 344 bp band; R2, winter allele with 830 bp bands; D, spring allele with no amplified bands. M, Molecular weight marker (Step50 Plus); C1, barley cv. Siberia, haplotype 5C; C2, winter barley cv. AVCI-2002, haplotype 1A; and following breeding lines: 1, 15/18-4; 2, 87/13-2; 3, 66/15-2; 4, 2-42-20; 5, 14/09-4; 6, 22/18-5; 7, 77/12-5; 8, 1-4; 9, 10/16-1; 10, 20/18-3; 11, 14/09-2; 12, Aidyn, local standard (check); 13, 40/15-1; 14, 15/18-2; 15, 14/18-5; 16, 11/16-5.
Figure 2. Identification of allelic variation of Vrn-H1 gene in barley advanced breeding lines using molecular marker 1: R1, winter allele with 344 bp band; R2, winter allele with 830 bp bands; D, spring allele with no amplified bands. M, Molecular weight marker (Step50 Plus); C1, barley cv. Siberia, haplotype 5C; C2, winter barley cv. AVCI-2002, haplotype 1A; and following breeding lines: 1, 15/18-4; 2, 87/13-2; 3, 66/15-2; 4, 2-42-20; 5, 14/09-4; 6, 22/18-5; 7, 77/12-5; 8, 1-4; 9, 10/16-1; 10, 20/18-3; 11, 14/09-2; 12, Aidyn, local standard (check); 13, 40/15-1; 14, 15/18-2; 15, 14/18-5; 16, 11/16-5.
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Figure 3. Identification of barley breeding lines for allelic diversity in Vrn-H2 gene using molecular marker 4: R winter allele, no amplification of any bands; D, spring allele, two bands 273 and 307 bp. M, Molecular weight marker (Step50 Plus); C1, negative control spring barley cv. Arna; C2, positive control winter barley cv. AVCI-2002; and the following breeding lines: 1, 2805; 2, 40/16-10; 3, 67/08-13; 4, 71/12-8; 5, D1-8; 6, 70/08-4; 7, 70/08-3; 8, 8/16-11; 9, 66/08-11; 10, 10/16-2; 11, 76/13-1; 12, 76/13-4; 13, 60/15-4; 14, 89/08-3; 15, 71/13-13; 16, 14/16-2; 17, 67/08-6.
Figure 3. Identification of barley breeding lines for allelic diversity in Vrn-H2 gene using molecular marker 4: R winter allele, no amplification of any bands; D, spring allele, two bands 273 and 307 bp. M, Molecular weight marker (Step50 Plus); C1, negative control spring barley cv. Arna; C2, positive control winter barley cv. AVCI-2002; and the following breeding lines: 1, 2805; 2, 40/16-10; 3, 67/08-13; 4, 71/12-8; 5, D1-8; 6, 70/08-4; 7, 70/08-3; 8, 8/16-11; 9, 66/08-11; 10, 10/16-2; 11, 76/13-1; 12, 76/13-4; 13, 60/15-4; 14, 89/08-3; 15, 71/13-13; 16, 14/16-2; 17, 67/08-6.
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Figure 4. Identification of barley germplasm collection for allelic variability in Vrn-H3 gene using molecular marker 10. PCR products after restriction with Ksp22I: D, spring allele with three bands, 138, 142 and 69 bp; R, winter alleles with two bands, 211 and 138 bp. M, Molecular weight marker (Step50 Plus); C1, positive control cv. Aidyn; C2, positive control cv. Astana-2000, and the following barley accessions: 1, Saida; 2, P-24; 3, Armelle; 4, MacKey-8; 5, MacKey-2; 6, Manchurian (CI-739); 7, MacKey-14; 8, CI-9214; 9, P-ll; 10, Canadian Lake Shore (K-25282); 11, MacKey-4; 12, Robust; 13, Pirkka; 14, P-01; 15, GRU-22639; 16, Р-21; 17, CI-9214 (Platz0); 18, CI-9825; 19, P-07. Genotype No. 10 seems to be a heterozygote of the Vrn-H3 gene or an admixture of two genotypes.
Figure 4. Identification of barley germplasm collection for allelic variability in Vrn-H3 gene using molecular marker 10. PCR products after restriction with Ksp22I: D, spring allele with three bands, 138, 142 and 69 bp; R, winter alleles with two bands, 211 and 138 bp. M, Molecular weight marker (Step50 Plus); C1, positive control cv. Aidyn; C2, positive control cv. Astana-2000, and the following barley accessions: 1, Saida; 2, P-24; 3, Armelle; 4, MacKey-8; 5, MacKey-2; 6, Manchurian (CI-739); 7, MacKey-14; 8, CI-9214; 9, P-ll; 10, Canadian Lake Shore (K-25282); 11, MacKey-4; 12, Robust; 13, Pirkka; 14, P-01; 15, GRU-22639; 16, Р-21; 17, CI-9214 (Platz0); 18, CI-9825; 19, P-07. Genotype No. 10 seems to be a heterozygote of the Vrn-H3 gene or an admixture of two genotypes.
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Table 1. Results of statistical treatment with chi-square (χ2) assessing the associations between the results of 10 DNA marker analyses for allelic diversity in Vrn loci and the transition to heading and flowering of barley accessions in spring-2024 sown field trials. D, dominant allele; R, recessive allele; df, degrees of freedom; p probability level.
Table 1. Results of statistical treatment with chi-square (χ2) assessing the associations between the results of 10 DNA marker analyses for allelic diversity in Vrn loci and the transition to heading and flowering of barley accessions in spring-2024 sown field trials. D, dominant allele; R, recessive allele; df, degrees of freedom; p probability level.
GeneMolecular Markers
and Primers		AlleleEaring
Genotypes		No Earing GenotypesTotal
Genotypes		χ2dfp
Vrn-H1Marker 1
(HvBM5A intron1 F3b/R3b)		D2077214171.51<0.001
R3987126
Marker 2
(HvBM5.84/85 F/R)		D1697176102.11<0.001
R7787164
Marker 3
(HvBM5A exon2F1/R1)		D2438332618.91<0.001
R31114
Vrn-H2Marker 4
(HvZCCT.06F/07R)		D30518166.31<0.001
R21643259
Marker 5
(ZCCTH.14F/19R)		D46348011.51<0.004
R20060260
Marker 6
(ZCCTb.8F/11R)		D4328716.21<0.01
R20366269
Marker 7
(ZCCT.HcF/HcR)		D716213339.31<0.001
R17532207
Marker 8
(HvZCCT.001/002)		D3022526.51<0.01
R21572287
Marker 9
(HvSnf2.01F/2.03R)		D2117386.21<0.01
R22577302
Vrn-H3Marker 10
(HvFT1-F/R)		D2689.22<0.002
R24488332
Table 2. Classification and chi-square (χ2) test result of barley germplasm accessions by genotyping for plant development and phenotyping for transfer to the reproductive stage during spring sowing in the conditions of South-Eastern Kazakhstan. df, degrees of freedom; p probability level.
Table 2. Classification and chi-square (χ2) test result of barley germplasm accessions by genotyping for plant development and phenotyping for transfer to the reproductive stage during spring sowing in the conditions of South-Eastern Kazakhstan. df, degrees of freedom; p probability level.
Genotyping
Classification		Phenotyping Evaluation, Number of Accessions
EaringNon-EaringTotal
Spring2117218
Winter75663
Facultative283159
Total24694340
Chi-square resultχ2 = 197.6; df = 2; p =< 0.001
Table 3. Comparison of best barley selected breeding lines with different growth habits and standard cultivars (checks) for yield and TSW in winter- and spring-sown field trials. Values followed by the same letters are not significantly different at probability p < 0.05.
Table 3. Comparison of best barley selected breeding lines with different growth habits and standard cultivars (checks) for yield and TSW in winter- and spring-sown field trials. Values followed by the same letters are not significantly different at probability p < 0.05.
Growth
Habit		Accession NamePedigreeFormula Vrn-HWinter SowingSpring Sowing
Yield
t/ha		TSW, gYield
t/ha		TSW, g
Spring[Standard] Bereke-54Krasnovodopad. Breeding StationDDR4.1 a42.6 a2.3 a39.6 a
70/08-3Bulbul × Yuzhno-
Kazakhstansky-43		DRR5.1 b47.2 ab2.7 ab44.4 b
Winter[Standard] AydinVavilon//Zazjan
/80-5151		RDR4.0 a38.0 a--
67/08-6АI-19 × Yuzhno-
Kazakhstansky-43		RDR5.6 b50.4 b--
Facultative76/13-4946 × 579 NBat-2RRR5.3 b49.4 b3.4 b44.6 b
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Yerzhebayeva, R.; Bazylova, T.; Zhumaliyeva, G.; Bastaubayeva, S.; Baimuratov, A.; Sariev, B.; Shegebayev, G.; Ergün, N.; Shavrukov, Y. Combination of Vrn Alleles Assists in Optimising the Vernalization Requirement in Barley (Hordeum vulgare L.). Agriculture 2025, 15, 1389. https://doi.org/10.3390/agriculture15131389

AMA Style

Yerzhebayeva R, Bazylova T, Zhumaliyeva G, Bastaubayeva S, Baimuratov A, Sariev B, Shegebayev G, Ergün N, Shavrukov Y. Combination of Vrn Alleles Assists in Optimising the Vernalization Requirement in Barley (Hordeum vulgare L.). Agriculture. 2025; 15(13):1389. https://doi.org/10.3390/agriculture15131389

Chicago/Turabian Style

Yerzhebayeva, Raushan, Tamara Bazylova, Gaziza Zhumaliyeva, Sholpan Bastaubayeva, Askar Baimuratov, Burabai Sariev, Galym Shegebayev, Namuk Ergün, and Yuri Shavrukov. 2025. "Combination of Vrn Alleles Assists in Optimising the Vernalization Requirement in Barley (Hordeum vulgare L.)" Agriculture 15, no. 13: 1389. https://doi.org/10.3390/agriculture15131389

APA Style

Yerzhebayeva, R., Bazylova, T., Zhumaliyeva, G., Bastaubayeva, S., Baimuratov, A., Sariev, B., Shegebayev, G., Ergün, N., & Shavrukov, Y. (2025). Combination of Vrn Alleles Assists in Optimising the Vernalization Requirement in Barley (Hordeum vulgare L.). Agriculture, 15(13), 1389. https://doi.org/10.3390/agriculture15131389

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