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Article

Winter Wheat Vernalization Alleles and Freezing Tolerance at the Seedling and Jointing Stages

by
Fangfang Liu
1,2,
Wenxin Cao
1,2,
Qiqi Zhang
1,2,
Yao Li
1,2,
Heng Zhou
1,2 and
Yingxiu Wan
1,2,*
1
Crop Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230001, China
2
Anhui Key Laboratory of Crop Quality Improvement, Hefei 230031, China
*
Author to whom correspondence should be addressed.
Plants 2025, 14(9), 1350; https://doi.org/10.3390/plants14091350
Submission received: 11 March 2025 / Revised: 24 April 2025 / Accepted: 25 April 2025 / Published: 30 April 2025
(This article belongs to the Special Issue Abiotic Stress of Crops: Molecular Genetics and Genomics—2nd Edition)
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Abstract

:
This study explores the relationship between allelic variation of the vernalization genes (VRN) and the freezing tolerance at the seedling and jointing stages of winter wheat growth. It provides a basis for molecular marker development for freezing tolerance breeding of winter wheat. A total of 435 wheat accessions were used to identify and evaluate the freezing tolerance at the seedling stage using field tests, while 192 wheat accessions were used to evaluate the freezing tolerance at the jointing stage in climate chamber tests. The VRN genes of the wheat accessions were detected using allele-specific markers of the VRN-A1, VRN-B1, VRN-D1 and VRN-B3 loci, and the relationship between VRN genotype and freezing tolerance at the two developmental stages was tested. There were significant differences in freezing tolerance between the wheat accessions. Assessing the freezing tolerance of 52 wheat accessions at both the seedling and jointing stages revealed no significant correlation between tolerance at these two stages. The genotypic analysis found that Vrn-D1 was the most frequent dominant allele in winter wheat, while no accession contained the dominant alleles Vrn-A1 and Vrn-B3. Notably, freezing tolerance showed stage-specific genetic regulation; seedling-stage freezing tolerance strongly correlated with vernalization gene allelic combinations (p < 0.05), whereas jointing-stage freezing tolerance exhibited no such association. The presence of all recessive alleles vrn-A1, vrn-B1, vrn-D1 and vrn-B3 was required for strong seedling-stage freezing tolerance. The VRN-D1 marker was effective for screening freezing tolerance materials under the premise that vrn-A1 and vrn-B1 alleles are recessive at winter wheat seedling stage.

1. Introduction

Wheat is one of the most important food crops, providing approximately 20% of the calories and protein for human nutrition. China is one of the two largest wheat producers and consumers in the world, accounting for 17% of global production and 16% of consumption. A high and stable wheat production is important in ensuring national food security [1]. Wheat is mainly grown in temperate climates. Under the intensification of climatic instability, low-temperature freezing damage occurs frequently, and has become a major meteorological factor that affects wheat’s safe production.
Freezing stress can inhibit various physiological and biochemical activities in wheat, including water use, cell membrane stability, photosynthesis, secondary metabolite synthesis, and plant hormone content [2]. In wheat production, freezing stresses are generally classified into two categories that are distinct in terms of phenology: winter freezing damage, which occurs at the seedling stage and impairs seedling establishment and vegetative growth, and spring freezing damage, which takes place from the jointing stage to the heading stage and disrupts reproductive processes. Both types of freezing damage can lead to reduced yield and lower grain quality [3,4,5]. The jointing stage (the anther connective tissue formation phase, ACFP) is widely recognized as the most suitable period for identifying the spring freezing tolerance of wheat. During this stage, the wheat stems are tender. When exposed to temperatures below 0 °C, it is extremely likely to cause the death of the main stem and large tillers. The Yellow and Huai Valley Wheat Region is the largest wheat-producing area in China. Frequent occurrence of jointing-stage freezing damage in this region causes a substantial reduction in final grain yield by 30–50% in severe cases, affecting nearly 42% of wheat sown areas. Therefore, it is important to study the jointing-stage freezing tolerance.
The freezing tolerance in wheat is a complex quantitative trait, and has been extensively studied. There are two evolutionary adaptive mechanisms: cold acclimation and low temperature vernalization enable wheat to resist cold at the seedling stage. Cold acclimation involves a series of accumulative physiological and biochemical processes that enhance tissue cold tolerance, mediated by various cold-responsive genes including COR (cold-regulated), LTI78 (low-temperature induced 78), and LEA (Late embryogenesis abundant) genes [6]. Low temperature vernalization is the physiological requirement for the transition of winter wheat from vegetative to reproductive growth [7,8], and is also an adaptation for wheat to avoid freezing damage to reproductive organs. Vernalization response is determined by vernalization genes. Allelic variation at four VRN loci (VRN1, VRN2, VRN3, and VRN4) is well characterized. Winter habit is a recessive trait and the dominant Vrn alleles function in circumventing or reducing vernalization requirement. VRN1, which plays the most important role in vernalization, encodes a MADS-box family protein homologous to Arabidopsis APETALA1 that acts as a floral activator and is expressed in leaves and shoot apical meristem [9]. VRN2 inhibits flowering by regulating the expression of VRN1 and VRN3 [10]. VRN3 is an orthologue of Arabidopsis flowering factor FLOWER LOCUS T (FT), and its presence leads to early flowering [11]. VRN4 regulates the vernalization process of wheat by interacting with VRN1 and VRN3 [12]. VRN1 and VRN3 promote flowering and circumvent or reduce the requirement for long-term low temperatures to induce vernalization. There are three VRN1 loci in wheat: VRN-A1, VRN-B1, and VRN-D1 located in chromosome arms 5AL, 5BL, and 5DL, respectively [13]. VRN-A1 is the most sensitive to temperature and has epistatic effects on VRN-B1 and VRN-D1 [14]. The VRN-B3 loci are located on group seven chromosomes [11].
Numerous studies conducted to dissect the genetic architecture of freezing tolerance have highlighted the importance of VRN loci in freezing tolerance. The presence of a dominant Vrn1 allele significantly reduces freezing tolerance. Accessions with dominant alleles at two or three VRN1 loci generally have weak freezing tolerance, whereas genotypes with recessive alleles at all three loci have strong freezing tolerance [15,16,17]. The VRN1 gene may enhance the freezing tolerance of wheat by interacting with cold-regulated genes [18]. Genes Fr-A1, Fr-B1 and Fr-D1 for frost tolerance are located at or near the VRN1 loci in homeologous group five chromosomes [19,20]. FR1 and VRN1 synergistically regulate the expression of Cor regulated by CBF transcription factors that can enhance the freezing tolerance of wheat [21]. Transcript analysis showed that VRN1 alleles directly regulate CBF genes and repress their expression, thereby reducing freezing tolerance during reproductive growth [22,23,24]. The recessive allele vrn-A1 increases freezing tolerance 2.1-and 2.4-fold in both winter and spring wheat compared to the dominant allele Vrn-A1 [25]. Futhermore, the copy number of vrn-A1 also influences the freezing tolerance of hexaploid wheat [26]. Spring freezing tolerance also shows complex associations with VRN1, where Vrn-A1 and Vrn-D1 increase spring freezing susceptibility, while Vrn-B1 enhances freezing tolerance [27]. Although primarily involved in flowering regulation, Vrn-B3 also participates in freezing tolerance-related signaling pathways. These studies have revealed complex relationships between VRN loci and freezing tolerance in wheat. However, there is still a knowledge gap in understanding the relationship between the combination of vernalization gene alleles rather than single loci and the multi-stage freezing tolerance of wheat.
VRN1 and VRN-B3 genes have been cloned, and molecular markers developed from them [28,29] are widely used in breeding [30,31,32]. In this study, we used 575 wheat accessions to identify the allelic status of genes Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3, investigate the freezing tolerance at the seedling stage using field tests, and at the jointing stage in climate chamber tests, and analyze the effects of various alleles on freezing tolerance across different growth stages. The results were expected to identify freezing-tolerant germplasm and molecular markers for breeding.

2. Results

2.1. Analysis of Freezing Tolerance Traits at the Seedling Stage and at the Jointing Stage

The accession panel (Table S1) showed considerable variation in freezing tolerance across environments (Table 1). The freezing tolerance grades ranged from one to five, with standard deviations between 0.89 and 0.99, and coefficients of variation (CV) ranging from 34.78% to 46.04% (Table 1). These findings indicated that the three experimental environments effectively differentiated freezing tolerance among genotypes. Histograms showed that the phenotypic distributions for the three environments were normally distributed (Figure 1). The majority of accessions exhibited freezing tolerance grades of one to three across all three environments, indicating a higher proportion of accessions with strong to moderate freezing tolerance. Highly significant correlations were observed among freezing tolerance phenotypes across three environments, with correlation coefficients ranging from 0.699 to 0.773 (Table 2), which demonstrated that the phenotypes were stable across environments.
A total of 435 wheat accessions were collected from four ecological wheat production zones in China: the Northern Winter Wheat Region (NWWR), the Yellow and Huai Valley Winter Wheat Region (YHVWWR), the Middle and Lower Reaches of the Yangtze River Winter Wheat Region (MLRYRWWR), and the Southwest Winter Wheat Region (SWWR). Significant regional variations in freezing tolerance were observed among these accessions. The NWWR accessions (n = 13) exhibited the highest freezing tolerance with an average damage level of 1.7, followed by YHVWWR accessions (n = 268) at 2.0. In contrast, MLRYRWWR (n = 128) and SWWR (n = 26) accessions showed comparatively lower freezing tolerance, with average damage levels of 3.0 and 3.3, respectively. Statistical analysis revealed that the freezing damage grades of NWWR and YHVWWR accessions were significantly lower than those of MLRYRWWR and SWWR accessions (p < 0.05; Table S2), clearly demonstrating superior freezing tolerance in wheat varieties from the northern regions (NWWR and YHVWWR) compared to their southern counterparts (MLRYRWWR and SWWR).
The dead stem rates of 192 accessions showed significant variation, ranging from 0.01 to 1.00 (Table S3) with a mean of 0.16 and a coefficient of variation of 52.24%. The −6 °C/6 h treatment at the jointing stage effectively distinguished differences in freezing tolerance among accessions at this growth stage. Following the dead stem grading standards from Liu et al. [33], freezing tolerance was divided into five tolerance levels (Figure 2A) with the majority in level five (Figure 2B). There were 17 accessions with grade one, and the average dead stem rate was 0.08. There were 26 accessions with grade two, and the average dead stem rate was 0.22. There were 23 accessions with grade three, and the average dead stem rate was 0.37. There were 35 accessions with grade four, and the average dead stem rate was 0.54. There were 91 accessions with grade five, and the average dead stem rate was 0.90 (Table S3). In summary, the freezing tolerance of 192 wheat accessions is obviously different, and the proportion of extremely weak accessions is the largest.
The relationship between seedling-stage and jointing-stage freezing tolerance of 52 accessions grown in the Yellow and Huai Valley Wheat Region is shown in Table 3. Some accessions such as Handan 6172, Huaimai 22, Jimai 22, Yannong 21 and Huaimai 29 had strong freezing tolerance at both growth stages. Accessions such as Annong 1124, Chuanmai 42 and Nemai 8 had strong freezing tolerance at jointing, but weak seedling freezing tolerance in winter; accessions Liangxing 99, Jinan 17 and Guomai 8 had strong seedling freezing tolerance, but weak tolerance in spring. Correlation analysis indicated that freezing tolerance at the two growth stages was not significant.

2.2. Association of Seedling Freezing and Vernalization Genotype

At VRN-A1 locus, screening of the genotyping of the panel with PCR primer set Vrn1-AF/Int1R indicated that all 435 accessions had the 734-bp fragment for the vrn-A1 or Vrn-A1c alleles. Amplification with primer set Intr1-AF2/AR3 produced no PCR product whereas primer Intr1-CF/ABR produced a fragment of 1068-bp (Figure 3A). Results from the three independent PCRs indicated that all 435 wheat accessions carried the recessive vrn-A1 allele (Table S1).
Genotyping of the VRN-B1 locus by PCR primer sets Intr-BF/BR3 and Intr-BF/BR4 (Figure 3B) indicated that 18 accessions had a 709-bp fragment, indicative of the Vrn-B1 allele, While vrn-B1 was detected in all other accessions (Table S1).
Genotyping of the VRN-D1 locus with PCR primer sets VRN4-B-INS-F/R and VRN4-B-NOINS-F/R (Figure 3C) indicated that 178 accessions had the 1671-bp fragment, indicative of Vrn-D1, while vrn-D1 was detected in all other accessions (Table S1).
At VRN-B3 locus, amplification with primer set VRN4-B-INS-F/R detected no PCR product that identifies Vrn-B3; however, all accessions produced a 1140-bp fragment when amplified with primer set VRN4-B-NOINS-F/R (Figure 3D), indicative of vrn-B3.
Molecular marker detection results indicated that the highest frequency is the dominant Vrn-D1 allele, accounting for 40.92% of the tested accessions, and a higher frequency is the dominant Vrn-B1 allele, accounting for 4.14% of the tested accessions among 435 wheat accessions. We did not find the dominant Vrn-A1 allele and dominant Vrn-B3 allele (Figure 4). Characterization of the allelic combination of vernalization genes at Vrn-A1, Vrn-B1, Vrn-D1 and Vrn-B3 loci revealed that there was a total of four types of allelic variation compositions. Among them, there were 242 accessions that possessed the recessive vrn-A1/vrn-B1/vrn-D1/vrn-B3 allelic variant combinations (accounting for 55.63%). There were 190 accessions carrying one dominant allelic variation; 175 out of 190 accessions had vrn-A1/vrn-B1/Vrn-D1/vrn-B3 allelic variant combinations (accounting for 40.23%), while 15 accessions possessed vrn-A1/Vrn-B1/vrn-D1/vrn-B3 allelic variant combinations (accounting for 3.45%; Table 4). Only 3 accessions possessed two dominant allelic variations, which were vrn-A1/Vrn-B1/Vrn-D1/vrn-B3 allelic variant combinations (accounting for 0.69%). These suggested that the recessive allelic combination of vrn-A1/vrn-B1/vrn-D1/vrn-B3 was predominant, but the combination of vrn-A1/vrn-B1/Vrn-D1/vrn-B3 was prevalent in winter wheat.
A significant association was observed between the Vrn-D1 allele and reduced freezing tolerance in three environments. The Vrn-D1 allele was positively correlated with freezing tolerance grade with correlation coefficients of 0.288, 0.280, and 0.503, respectively (p < 0.01; Table 3). A Mann–Whitney U test also showed that the average freezing tolerance grade of Vrn-D1 and vrn-D1 genotypes was significantly different (p < 0.01; Figure 5). The Vrn-B1 allele had no significant correlation with the freezing tolerance grade surveyed in 2017 and 2018, but showed a significant positive correlation with freezing tolerance grade in 2021 although the 0.141 correlation coefficient was very low (Table 3).
The average freezing tolerance level of lines carrying alleles Vrn-B1 and Vrn-D1 was 3.00, which was higher than accessions carrying only Vrn-B1 (2.87) or Vrn-D1 (2.78). The average freezing damage grade of lines with all four recessive alleles was 2.05 and significantly different from lines with one or two dominant alleles (p < 0.05; Table 4). Co-presence of recessive genes at all four loci was prerequisite for strong freezing tolerance in seedling.

2.3. Association of Jointing Freezing and Vernalization Genotype

PCR screening of the 192 wheat accessions from the Yellow and Huai Valley Wheat Region using primer set Vrn1-AF/Int1R indicated that all produced the 734-bp fragment, whereas there was no PCR product with primer set Intr1-AF2/AR3and 1068-bp fragment with primer set Intr1-CF/ABR. The combined PCR results indicated that all these accessions had vrn-A1. Screening with PCR primer sets Intr-BF/BR3 and Intr-BF/BR4 indicated that three wheat accessions (Bainong 3217, Huaimai 30, and Shan7859) had Vrn-B1 allele characterized by a 709-bp fragment with primer Intr-BF/BR3; the remaining 189 accessions had vrn-B1. Seventy one accessions (59%) harbored Vrn-D1 allele, and 121 carried vrn-D1. PCR results showed that all 192 accessions carried vrn-B3. The average dead-stem score of accessions carrying Vrn-D1 was 0.62; the average score for vrn-D1 accessions was 0.61, indicating that the VRN-D1 locus had no obvious relationship with freezing tolerance at the jointing stage.

3. Discussion

Freezing stress on seedlings at the beginning of winter and at jointing in spring can injure wheat plants and negatively impact growth, development, and yield. Although the occurrence of frost damage is influenced by various factors, genetic variation among accessions plays a crucial role. In this study, we systematically evaluated the freezing tolerance of wheat at the seedling stage and the jointing stage. Currently, the evaluation of seeding-freezing tolerance of wheat in China follows the industry standard of the People’s Republic (NY/T 1301–2007) [34] which divides freezing symptoms into five grades. Using this assessment method, we phenotyped 435 wheat accessions in three different environments. The highly significant correlations (ranging from 0.699 to 0.773) among the freezing tolerance phenotypes across these environments (Table 2) indicate that the genetic basis for seedling-freezing tolerance is stable and heritable. This stability allows for reliable selection of freezing-tolerant germplasm during breeding.
Yellow and Huai Valley Wheat Region is in the transitional zone between north and south where frequent non-anticipated temperature fluctuations in spring can affect the wheat crop. Hence accessions with strong freezing tolerance especially at the jointing stage would be beneficial to production. The jointing-stage freezing tolerance of wheat is influenced by multiple factors, such as the occurrence-period, intensity and duration of low temperature. Because the occurrence period and intensity of low temperature in field are not consistent among years, it is difficult to get reliable and repeatable results. Artificial simulation identification is characterized by a remarkably short cycle and high repeatability, which enables researchers to efficiently obtain consistent and reliable results [35]. In this study, we applied this pot-planting and artificial simulation approach to evaluate the jointing-freezing tolerance of 192 wheat accessions from the region. Using the dead stem rate as an evaluation index, we found significant differences among different wheat accessions. The −6 °C/6 h treatment at the jointing stage effectively distinguished the genetic differences in freezing tolerance, which is consistent with previous studies [33]. However, compared with the seedling stage, a higher proportion (65.63%) of accessions showed weak freezing tolerance at the jointing stage, indicating that breeding for jointing-stage freezing tolerance is more challenging.
Assessing the freezing tolerance of 52 wheat accessions at both the seeding and jointing stages revealed no significant correlation between tolerance at these two stages, a finding consistent with previous reports by Zhong et al. [36]. This suggests that there may be different genetic mechanisms for the regulation of freezing tolerance at these two stages in wheat. In this study, only 12.19% of accessions showed a lack of seedling freezing tolerance, whereas 65.63% lacked tolerance at jointing. These findings demonstrate that freezing tolerance at the seedling stage is more amenable to selection during breeding compared to that at the jointing stage. Breeding for jointing-freezing tolerance is more challenging and future breeding programs should prioritize enhancing spring freezing tolerance, particularly during critical developmental phases such as the jointing stage.
The distribution frequencies of vernalization gene dominant alleles vary among different regions. In our study, among 435 winter wheat samples, the dominant allele frequencies were Vrn-D1 (40.92%) > Vrn-B1 (4.14%), with no detection of Vrn-A1 or Vrn-B3. In the 192 accessions from the Yellow and Huai Valley Wheat Region, Vrn-D1 was even more predominant at 58.68%, followed by Vrn-B1 at 1.56%. Comparative analysis with previous studies showed notable differences: Zhang et al. [37] reported higher detection rates for Vrn-B1 (18.2%) and Vrn-A1 (13.8%) in broader geographical samples, though Vrn-D1 (45.3%) remained predominant. Jiang et al. [38] documented a Vrn-D1 frequency (56.12%) closely aligned with our Yellow and Huai Valley data. These discrepancies primarily stem from sample heterogeneity—prior studies included accessions from northern spring wheat regions, whereas our investigation strictly focused on winter wheat germplasm. By integrating geographical distribution patterns [39], we discerned that the high-frequency occurrence of Vrn-A1/B1 in spring wheat regions directly explains their absence in our winter wheat samples. This robustly confirms that winter wheat accessions are predominantly characterized by the Vrn-D1 dominant allele, with Vrn-B1 as a secondary component-a distribution pattern consistent with China’s wheat ecoregionalization mechanisms.
We also analyzed the relationship between vernalization gene alleles and freezing tolerance. At the seedling stage, we found that the dominant gene Vrn-D1 was positively correlated with the freezing damage grade (Table 3), negatively regulating the freezing tolerance of wheat. The result is strongly associated with Zhang et al. [40], who found the recessive vrn-D1 allele was more effective than dominant Vrn-D1 allele in improving winter tolerance of wheat. The average freezing tolerance level of accessions carrying Vrn-B1 and Vrn-D1 was higher than that of accessions carrying only one of these dominant alleles. Moreover, the average freezing damage grade of accessions with all four recessive alleles (vrn-A1/vrn-B1/vrn-D1/vrn-B3) was significantly lower (2.05) compared to accessions with one or two dominant alleles (Table 4). This indicates that the recessive VRN1 allelic combination is closely related to strong seedling-freezing tolerance, which is correlated with You et al. [41]. Therefore, under the premise that VRN-A1 site is recessive, VRN-D1 gene markers can be effectively used for screening strong seedling-freezing tolerance wheat materials.
However, at the jointing stage, there was no obvious relationship between the vernalization gene and spring freezing tolerance. Although previous studies have shown that some VRN1 alleles can affect spring freezing tolerance [27], in our study, due to the limited sample size and type, no dominant alleles Vrn-A1 and Vrn-B3 were detected in 192 accessions from the Yellow and Huai Valley Wheat Region, and only two samples contained the dominant Vrn-B1 gene. Despite the high proportion (58.68%) of dominant Vrn-D1, there was no difference in the dead stem rate between the recessive vrn-D1 and dominant Vrn-D1 accessions. This suggests that other genetic or environmental factors may play more important roles in regulating jointing-freezing tolerance.
In this study, freezing tolerance of 435 wheat germplasm at the seedling stage and 192 wheat germplasm at the jointing stage were systematically evaluated, and allelic variation analysis of VRN gene was combined to screen out germplasm resources with significant frost resistance potential. Among the 435 accessions evaluated at the seedling stage, 47 exhibited consistently high freezing tolerance (grade one) in at least two independent environments. Notably, seven accessions—Handan 6172, ENESCO, Shijiazhuang 8, Shijiazhuang 15, Tai 10604, Niavt14, and Gushenmai 9—maintained grade one tolerance across all three testing environments. These accessions carried the recessive allelic combination of vrn-A1/vrn-B1/vrn-D1/vrn-B3. Such accessions can serve as valuable genetic resources for breeding wheat varieties with enhanced seedling freezing tolerance. For the 192 wheat accessions from the Yellow and Huai Valley Wheat Region evaluated at the jointing stage, although most of the accessions (65.63%) showed very weak tolerance (Figure 2B), some accessions such as Anke 20, Fengde Cunmai 5, Gu Shen 6, Huacheng 2019, Anke 237, Fengde Cunmai 1, An 1302, Anke 238, Henong 825, Hengjinmai 8, Anke 2101, Bifeng 1, Handan 6172, Huacheng 3366, Anke 239, Huaimai 22, and Xiaoyan 6 showed relatively strong freezing tolerances at the jointing stage. Although the relationship between vernalization genes and jointing-freezing tolerance was not clear in this study, these accessions can still be considered as potential germplasm for improving spring freezing tolerance in wheat breeding programs. Further research on their genetic mechanisms of freezing tolerance may provide new insights into enhancing wheat’s resistance to spring cold stress.

4. Materials and Methods

4.1. Plant Materials

A total of 435 wheat accessions from different ecological wheat production zones were used for the evaluation of freezing tolerance in seedlings (Table S1). Among them, 268 accessions are from the Yellow and Huai Valley Winter Wheat Region (YHVWWR), 128 are from the Middle and Lower Reaches of the Yangtze River Winter Wheat Region (MLRYRWWR), 26 are from the Southwestern Winter Wheat Region (SWWWR), 13 are from the Northern Winter Wheat Region (NWWR). Since wheat in the Yellow and Huai Valley Wheat Region is frequently affected by freezing injury in spring, we also assessed 192 accessions (52 from the above accessions) from the Yellow and Huai Valley Wheat Region for freezing tolerance at jointing (Table S3).

4.2. Assessing of Freezing Tolerance Traits in Seedlings

A total of 435 wheat accessions were grown in field trial at the Huaibei Experiment Station (116°45′ N, 33°54′ E) of Anhui Academy of Agricultural Sciences (Hefei in Anhui province). They were planted in the conventional autumn season on 26 October 2016, 2017 and 2020, which is during the recommended seeding period for winter wheat in this region. The experimental design was a randomized complete block with two replicates. Plot size was 2-m-long rows with row spacing of 25 cm. All trials were seeded by manual dibbling at a seedling rate of 80 seeds row−1. The total amount of urea applied during the whole growth period of wheat is 390 kg/hm. The application ratio of the base fertilizer to the top dressing is 7:3. Nitrogen fertilizer should be top-dressed during the jointing stage of wheat, and this should be carried out in combination with irrigation.
Meteorological information was provided by the Weather Bureau of Suixi County, Huaibei City. Frost damaging events occurred during 22~25 January 2017 (environment E1), 19~21 January 2018 (environment E2), and 6~9 January 2021 (environment E3). The lowest temperatures were −8 °C, −12 °C, and −12 °C, respectively. Before freezing stress, the diurnal temperature variation was relatively stable, which is generally suitable for the initial growth and development of wheat. Adequate sunlight ensured normal photosynthesis and promoted the growth of seedlings. The soil moisture content was maintained at a relatively optimal level, which provided sufficient water for the plants. Freezing tolerance phenotypes were recorded two weeks after freezing. According to the agricultural industry standard of China (NY/T1301–2007) [31], freezing tolerance was recorded on a five-grade, as follows:
Grade 1 (G1): no freezing damage;
Grade 2 (G2): leaf yellowing;
Grade 3 (G3): 50% leaf death;
Grade 4 (G4): All leaves dead or withered;
Grade 5 (G5): entire plants or most tillers dead.

4.3. Assessing Freezing Tolerance Traits in Spring

This experiment was conducted in 2020–2021 at the experimental station of Anhui Academy of Agricultural Sciences (31.83° N, 117.24° E), Hefei, China. The 192 wheat accessions were used in the experimentation. Wheat accessions were sown in pots of 28 cm diameter × 35 cm height, on 4 November 2021. There were 13 pots for each accession. Each pot was filled with 8 kg soil and 5.00 g compound fertilizer (N:P:K = 15:15:15) incorporated in it. The soil was taken from the field of 0∼20 cm upper plowing layer. Twenty seeds were planted in each pot and seedlings were thinned to ten at the three-leaf stage. All the pots were placed in the field conditions. The field environment for wheat growth is suitable, with stable day-night temperatures, ample sunlight, and appropriate soil moisture, creating favorable conditions for the growth of wheat.
When the plants reached the jointing stage (ACFP), eight pots of uniformly grown wheat plants from each accession were moved to a climate chamber for exposure to −6 °C for 6 h (humidity: 60%; light intensity: 0 µmol m−2 s−1). The pots were moved back to the field after the treatment. The numbers of dead main stem and first and second tillers were assessed after 10 days of low temperature treatment. The dead-stem rate per pot was calculated as: Dead-stem rate = number of dead stems/total number of stems. The mean dead-stem rate was calculated for each accession using 8 replicated pots, and the tolerance grade of wheat accessions were classified based on these mean values. According to the criteria of jointing-freezing tolerance [30], the jointing-freezing tolerance of wheat was divided into 5 grades (Table 5).

4.4. Molecular Marker Detection

Genomic DNA (gDNA) was extracted from young leaves of ten-day-old seedlings using the phenol chloroform method [42]. DNA concentration and quality were checked with NanoDrop2000 (Thermo Scientific, Waltham, MA, USA). DNA samples with a 260 nm/280 nm ratio equal to or higher than 1.8 were considered suitable for further PCR analysis. Nine functional markers (Table S4) specific for Vrn-A1, Vrn-B1, Vrn-D1 and Vrn-B3 alleles [10,26,27] were used to genotype all accessions. Primers were synthesized by Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, China). DNA amplification was carried out in 20-µL reaction volumes, each consisting of 1 µL of 50–100 ng/µL DNA, 1 µL of 10 µmol/L of each primer, 10 µL of 2 × Taq PCR Master Mix (Tsingke Biotechnology Co., Ltd., Beijing, China), and 7 µL of sterilized ddH2O. The annealing temperature and extension time used for the PCR are provided in Supplementary Materials Table S4. PCR products were separated in 1–3% agarose gels depending on the PCR product size (Table S4) and visualized under UV light after staining with ethidium bromide.

4.5. Statistical Analysis

Phenotypic differences in freezing tolerance among accessions were tested using analysis of variance (ANOVA) in the SPSS software 20.0, and multiple comparisons were made using the least significant difference (LSD) test at p < 0.05.

5. Conclusions

Vernalization genes play an important role in seedling-stage freezing tolerance of wheat. The VRN-D1 molecular marker can be used as an effective tool for screening freezing-tolerant accessions at the seedling stage. However, jointing-stage freezing tolerance did not show a significant association with VRN genotypes, which may involve other low-temperature responsive genes or interactions with environmental factors. Han 6172, Huai Mai 29, and other germplasms with strong freezing tolerance at both the seedling and jointing stages were selected, which provided the core parents for multi-stage resistance breeding. Future studies should further analyze the molecular basis of freezing tolerance at the jointing stage, develop efficient molecular markers, and integrate phenomics with gene-editing technologies to accelerate the cultivation of new wheat varieties exhibiting broad adaptability to climate change.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14091350/s1. Table S1. The Seedling-freezing grade and vernalizing genotypes of 435 wheat accessions. Table S2. The differences in freezing tolerance across different ecological regions. Table S3. The jointing-freezing tolerance and vernalizing genotypes of 192 wheat varieties. Table S4. Primer sequences used to identify VRN-1 and VRN-B3 alleles.

Author Contributions

Conceptualization, Y.W.; methodology, F.L. and W.C.; formal analysis, F.L. and Q.Z.; resources, H.Z. and Y.L.; data curation, F.L. and Y.L.; writing—original draft preparation, F.L.; writing—review and editing, F.L. and W.C.; supervision, W.C. and Q.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by National Key R and D Program of China (2024YFD1201100), the First Level Youth Talent Program of Anhui Academy of Agricultural Sciences (2022), the joint research of improved wheat variety of Anhui (2021-), the China Agriculture Research System of MOF and MARA (CARS-03-76).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We are grateful to Zhaoshi Xu and Maoyun She for their excellent technical assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Distributions of freezing tolerance of 435 wheat accessions in three environments.
Figure 1. Distributions of freezing tolerance of 435 wheat accessions in three environments.
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Figure 2. The freezing tolerance of wheat accessions at the jointing stage. (A) Growth performance of different tolerance grades of wheat accessions. (B) Frequency of freezing tolerance of 192 wheat accessions. Freezing tolerance was rated on a one to five grade: G1 = the strongest tolerance, G5 = the weakest tolerance.
Figure 2. The freezing tolerance of wheat accessions at the jointing stage. (A) Growth performance of different tolerance grades of wheat accessions. (B) Frequency of freezing tolerance of 192 wheat accessions. Freezing tolerance was rated on a one to five grade: G1 = the strongest tolerance, G5 = the weakest tolerance.
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Figure 3. Allelic variation detected in the VRN-A1, VRN-B1, VRN-D1 and VRN-B3 loci among nine wheat accessions. Amplification with (A): primers Vrn1-AF/Int1R (uppermost), Intr1-AF2/AR3 (middle) and Intr1-CF/ABR (lowermost); (B): primers of Intr-BF/BR3 and Intr-BF/BR4; (C): primers Intr1-DF/DR3 and Intr1-DF/DR4; (D): primers of VRN4-B-INS-F/R and VRN4-B-NOINS-F/R. M, DL2000; 1, Yannong 19; 2, Yangmai 158; 3, Huaimai 20; 4, Su 553; 5, Huaimai30; 6, Bainong207; 7, Annong 1124; 8, Luo 1106; 9, Qian 110209.
Figure 3. Allelic variation detected in the VRN-A1, VRN-B1, VRN-D1 and VRN-B3 loci among nine wheat accessions. Amplification with (A): primers Vrn1-AF/Int1R (uppermost), Intr1-AF2/AR3 (middle) and Intr1-CF/ABR (lowermost); (B): primers of Intr-BF/BR3 and Intr-BF/BR4; (C): primers Intr1-DF/DR3 and Intr1-DF/DR4; (D): primers of VRN4-B-INS-F/R and VRN4-B-NOINS-F/R. M, DL2000; 1, Yannong 19; 2, Yangmai 158; 3, Huaimai 20; 4, Su 553; 5, Huaimai30; 6, Bainong207; 7, Annong 1124; 8, Luo 1106; 9, Qian 110209.
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Figure 4. Frequency of alleles of vernalization genes in 435 wheat accessions.
Figure 4. Frequency of alleles of vernalization genes in 435 wheat accessions.
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Figure 5. Comparison of freezing tolerance between the Vrn-D1 and vrn-D1 genotypes. ** p < 0.01.
Figure 5. Comparison of freezing tolerance between the Vrn-D1 and vrn-D1 genotypes. ** p < 0.01.
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Table 1. Phenotypic variation of freezing tolerance of 435 wheat accessions.
Table 1. Phenotypic variation of freezing tolerance of 435 wheat accessions.
YearsMinimum
Temperature (°C)		Grade RangeMeanSDCV (%)
2017−81–52.150.9946.04
2018−121–52.410.9840.76
2021−121–512.560.8934.78
SD: Standard deviation, CV: Coefficient of variation, Freezing tolerance was rated on a one to five scale: one = the strongest tolerance, five = the weakest tolerance.
Table 2. Correlations of phenotypic data and genotypes of 435 wheat accessions in different environments.
Table 2. Correlations of phenotypic data and genotypes of 435 wheat accessions in different environments.
Freezing ToleranceGene Type
201720182021Vrn-D1Vrn-B1
20171
20180.773 **1
20210.699 **0.702 **1
Vrn-D10.288 **0.280 **0.503 **1—
Vrn-B10.0500.0780.141 **—1
** p < 0.01; — Not determined.
Table 3. The freezing grade of 52 winter wheat accessions.
Table 3. The freezing grade of 52 winter wheat accessions.
AccessionSeedling-Freezing Grade
(In the Field)		Jointing-Freezing Grade
(In the Climate Chamber)
201720182021
Fengdecunmai 53221
Henong 8252221
Handan 61721111
Huaimai 221121
Xiaoyan 62121
Annong11243342
Bainon g32172222
Chuanmai 423442
Jimai 221122
Huaimai 302332
Huaimai 291122
Huiyan 221232
Neimai 85442
Yannong 211122
An12431222
Bainong 2073322
Huaimai 181123
Huaimai 201123
Luyuan 5022333
Yannong 191123
Huaimai 251133
Shijiazhuang 81113
Yangnuomai 15553
Zhengmai 3662223
Su 5531224
Aikang 581224
Fanmai 52234
Yanzhan 41102234
Jimai 731124
Zhengmai 90231224
Wanmai 381124
Zhoumai 182324
Yannong 9992224
Jinan 171124
Zhongmai 8953224
Luanxuan 9883225
Xinong 8892225
Qianmai 183325
Guomai 81125
Huiyan 771235
Xinmai 182325
Jimai 202225
Kaimai 183325
Yangmai 203435
Liangxing 991125
Mianmai 393235
Guoshengmai 13445
Yangmai 1584345
Wanmai 521225
Ligao 61225
Guinong 7752215
Neimai 8365445
Table 4. Effects of vernalization genotype on freezing grade.
Table 4. Effects of vernalization genotype on freezing grade.
Gene TypeFreezing Grade
(Mean ± SD)		Number of AccessionsFrequency (%)
vrn-A1 + vrn-B1 + vrn-D1 + vrn-B32.05 ± 0.69 a 124255.63
vrn-A1 + vrn-B1 + Vrn-D1 + vrn-B32.78 ± 0.90 b17540.23
vrn-A1 + Vrn-B1 + vrn-D1 + vrn-B32.87 ± 0.77 b153.45
vrn-A1 + Vrn-B1 + Vrn-D1 + vrn-B33.00 ± 0.33 b30.69
1 Different lowercase letters indicate significant differences at p < 0.05.
Table 5. Evaluation criteria of jointing-freezing tolerance.
Table 5. Evaluation criteria of jointing-freezing tolerance.
Tolerance GradeDead Stem RateTolerance Type
10.00~0.13Extremely strong
20.14~0.28Strong
30.29~0.42Moderate
40.43~0.65Weak
50.66~1.00Extremely weak
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Liu, F.; Cao, W.; Zhang, Q.; Li, Y.; Zhou, H.; Wan, Y. Winter Wheat Vernalization Alleles and Freezing Tolerance at the Seedling and Jointing Stages. Plants 2025, 14, 1350. https://doi.org/10.3390/plants14091350

AMA Style

Liu F, Cao W, Zhang Q, Li Y, Zhou H, Wan Y. Winter Wheat Vernalization Alleles and Freezing Tolerance at the Seedling and Jointing Stages. Plants. 2025; 14(9):1350. https://doi.org/10.3390/plants14091350

Chicago/Turabian Style

Liu, Fangfang, Wenxin Cao, Qiqi Zhang, Yao Li, Heng Zhou, and Yingxiu Wan. 2025. "Winter Wheat Vernalization Alleles and Freezing Tolerance at the Seedling and Jointing Stages" Plants 14, no. 9: 1350. https://doi.org/10.3390/plants14091350

APA Style

Liu, F., Cao, W., Zhang, Q., Li, Y., Zhou, H., & Wan, Y. (2025). Winter Wheat Vernalization Alleles and Freezing Tolerance at the Seedling and Jointing Stages. Plants, 14(9), 1350. https://doi.org/10.3390/plants14091350

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