1. Introduction
The vernalization process is pivotal in wheat, serving as a crucial determinant for distinguishing between winter and spring varieties [
1]. The common methods for winter–spring identification in China primarily involve spring sowing in the field. In the Huanghuai region, the heading rate during the second stage of spring sowing is a fundamental indicator for determining winter and spring wheat varieties [
2,
3]. The determination of wheat’s spring and winter varieties is regulated by the activation and inhibition mechanisms of vernalization genes
Vrn1 and
Vrn2 [
4]. The control of vernalization is regulated by one or several dominant alleles at the
Vrn-1 loci [
3], with homologous genes located on the long arms of chromosomes 5A, 5B, and 5D, respectively [
5,
6]. The photoperiodic genes, namely
Ppd-D1 (
Ppd1),
Ppd-B1 (
Ppd2), and
Ppd-A1 (
Ppd3), are located on chromosomes 2D, 2B, and 2A, respectively [
7]. The presence of both the photoperiodic gene
Ppd-D1a and the vernalization gene
Vrn-D1 alleles in winter wheat, under winter conditions and specific genotypes, substantially improves the consistency of flowering timing [
7].
Varieties containing the
Vrn-B1 gene are typically classified as spring or weak spring varieties, while those with recessive alleles for
Vrn-A1,
Vrn-B1, and
Vrn-D1 are predominantly winter or semi-winter varieties [
8]. The dominant
Vrn-D1 allele is associated with weak winter or weak spring characteristics [
9]. The combination of recessive alleles
Vrn-A1,
Vrn-B1,
Vrn-D1, and
Vrn-B3 results in winter or semi-winter phenotypes [
10]. Combining vernalization gene markers with traditional winter–spring identification methods may enhance the accuracy of variety classification. The photoperiodic recessive gene
Ppd-D1b is typically found only in winter varieties. The photoperiodic gene
Ppd-D1b, associated with the dominant vernalization gene
Vrn-D1, influences varieties to exhibit winter or semi-winter traits, whereas spring varieties typically carry the photoperiod-insensitive alleles [
11]. Cultivars with the
Ppd-D1a allele tend to initiate flowering earlier and can mature under both long- and short-day conditions, whereas most
Ppd-D1b cultivars do not mature under short days [
12]. The influence of these genes on vernalization and photoperiodic traits may not accurately represent the winter–spring and photoperiodic characteristics of all varieties. Breeding selections and identifications for vernalization and photoperiodic traits should primarily rely on phenotypic assessments [
13,
14].
Rye [
15], an annual or biennial herbaceous plant, boasts significant cold tolerance, drought resistance, high forage yield, and rich nutritional content, serving as a vital green fodder and grazing feed for livestock, particularly cattle and sheep, during the winter and spring seasons [
15,
16,
17].
Wintergrazer-70 (
Secale cereale L. ‘
Wintergrazer-70’) was introduced from the United States in 1978 and has since been officially registered as a variety in China. It is primarily cultivated in North China, Northeast China, parts of Northwest China, the Jianghuai River Basin, and the Yungui Plateau, making it an excellent forage crop for winter idle fields in warm temperate regions [
18,
19].
Ganyin No1 (
Secale cereale L. ‘
Ganyin No1’) was a new variety screened from
Wintergrazer-70 by spring planting in high-altitude areas of the Qinghai–Tibet Plateau [
20]. This variety is characterized by high yield, cold resistance, disease tolerance, a short growth period, and the ability to thrive in low fertility. It exhibits broad adaptability and is particularly suited for spring planting in alpine regions ranging from 2000 to 4700 m in altitude [
21,
22,
23,
24].
This study aimed to determine the spring and winter characteristics of Ganyin No1 and establish the optimal sowing time above 3000 m a.s.l. in Tibet. Field planting trials were conducted in Linzhou County, within the Tibet Autonomous Region of China. Subsequently, the spring and winter characteristics of Ganyin No1 were identified in Henan Province. Subsequently, genes associated with vernalization regulation and the photoperiod were screened using RNA sequencing and RT-PCR data. These findings provide a solid foundation for further research into the vernalization process and the elucidation of the molecular mechanisms underlying rye’s seasonal traits.
2. Materials and Methods
2.1. Test Site Profile
The field experiment was conducted at the Linzhou Grassland Ecosystem Observation and Research Station of the Tibet Autonomous Region (91°11′ E, 29°54′ N), situated in Lhasa City. The station is located in the Lhasa River Valley’s agricultural region, central Tibet, at an elevation of 3759 m above sea level. The region has a plateau temperate monsoon semi-arid climate, with an average annual temperature of 5.8 °C and annual rainfall ranging from 300 to 510 mm. Precipitation predominantly occurs between June and September, and the frost-free period is approximately 100–120 days.
The springiness and winterness identification site was located in the Cognition Park of Longzihu Campus, Henan Agricultural University, Zhengzhou City, Henan Province (113°82′ E, 34°79′ N). Located at an elevation of 84 m above sea level, this site experiences a continental monsoon climate typical of the northern temperate zone, characterized by frequent shifts between cold and warm air masses. The region experiences distinct seasons—spring, summer, autumn, and winter—with an average annual temperature of 15.6 °C. Precipitation averages approximately 1100 mm annually, predominantly in the summer season.
2.2. Experimental Materials
The study utilized Wintergrazer-70 and Ganyin No1 rye as experimental materials, sourced from the Lhasa Agro-Ecological Experimental Station, Chinese Academy of Sciences. The test fertilizer was a spring barley or winter wheat-specific compound fertilizer, with a total nutrient content of at least 45% (N-P2O5-K2O = 22-13-10), produced by China Qinghai Province Golmud city Golmud Shengnong Compound Fertilizer Co., Ltd.
2.3. Field Conditions
The 2021 sowing trial employed a randomized block design, following the principles of rigorous experimental design and statistical analysis. In 2021, five sowing treatments were established on different dates: 26 June (Group A), 6 July (Group B), 16 July (Group C), 26 July (Group D), and 6 August (Group E). Each treatment was replicated three times, with experimental plots measuring 16 m2 (4 m × 4 m). The experiment utilized line seeding with row spacings ranging from 23 to 25 cm. A basal fertilizer containing 300 kg/hm2 of compound fertilizer was applied. Standard irrigation and weeding practices were employed.
The springiness and winterness identification tests were conducted across eight dates in 2023: 18 February, 25 February, 4 March, 11 March, 15 March, 19 March, 23 March, and 27 March. Each treatment comprised three replicates. On 27 April, five leaf samples from each treatment group were randomly collected and sent to BGI for RNA sequencing. The experiment utilized line seeding with a row spacing of 25 cm. Diammonium phosphate was applied at a rate of 300 kg/hm2 as the base fertilizer. Standard irrigation and weeding practices were employed.
2.4. Content and Method of Determination
Forage yield (fresh): The fresh weight yield per 1 m2 of grass was determined by randomly sampling each plot. Samples were collected at a height of 2 cm to 3 cm above the ground during mowing.
Fresh/dry ratio: Thirty plants from each plot were randomly sampled, cut at a height of 2 cm to 3 cm above the ground, weighed for fresh weight, and then baked in a 65 °C oven for 48 h until a constant weight was achieved. The dry weight was then measured to calculate the fresh/dry ratio.
Hay yield: The water content of the forage was determined using the fresh/dry ratio, and the hay yield was calculated based on its moisture content.
Spring sowing heading rate: A primary indicator for assessing the winter–spring characteristics of wheat varieties.
RNA-seq was employed to sequence the cDNA library of rye using the BDA DNBSEQ platform. High-quality reads were obtained using the filtering software SOAPnuke v1.4.0, which removed reads with low quality, contaminated joints, and a high proportion of unknown bases (Ns). The clean reads were assembled de novo using Trinity v2.0.6, and the transcripts were clustered using TGICL to eliminate redundancy, resulting in the rye Unigene library. The quality of the assembled transcripts was evaluated using the single-copy orthologous gene database BUSCO. Candidate coding regions within the Unigene library were identified using TransDecoder v3.0.1 software, and homologous sequences from the Pfam protein database and HMMs from the Hmmscan were aligned using BLAST to predict the coding regions.
The qRT-PCR analysis employed actin as the internal reference gene. The gene-specific primers are detailed in
Supplementary Table S4. The Roche 480 system (Roche, Switzerland) was employed for this study. The qRT-PCR assay was performed using the PerfectStart
TM M Green qPCR SuperMix kit, sourced from All-Gold Biotechnology Co., Ltd. (Bei**g, China). The PCR program was as follows: initial denaturation at 94 °C for 30 s; denaturation at 94 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 15 s, for 45 cycles; and final extension at 50 °C for 10 s, followed by a temperature ramp from 60 °C to 95 °C to plot a melting curve. Relative gene expression was quantified using the 2
−ΔΔCt method following four replicates.
The RNA extraction and cDNA synthesis were performed using the Eastep® Super total RNA kit and GoScriptTM Reverse Transcription kit, both provided by Promeg Bioproducts Co., Ltd. (Shanghai, China). Data analysis was conducted using Excel and R, with GraphPad Prism 8, RStudio 4.3.2, and other software employed for visualization.
4. Discussion
The Henan wheat district test findings suggest that the spring sowing method, which uses the heading rate of stage 2 as a fundamental indicator, is reliable for assessing wheat’s winter and spring characteristics [
30,
31,
32]. A comprehensive sequence classification of wheat spring sowing enabled us to identify the winter–spring traits in both
Wintergrazer-70 and
Ganyin No1 rye varieties. The effect of mRNA on leaf transcriptomics in
Wintergrazer-70 and
Ganyin No1 rye was examined using full-length transcriptomics over multiple planting periods. This analysis uncovered altered expression profiles for the two rye cultivars under various planting conditions. The findings indicated a higher number of differentially expressed genes in Ganyin No1 during the jointing stage and in
Wintergrazer-70 during the tillering stage. For both varieties, the number of differentially expressed genes was relatively low during both the jointing and tillering stages. The observed differences primarily stem from significant variations in physiological and biochemical processes during the same growth period, aligning with results from other studies.
The Gene Ontology (GO) annotation and enrichment analysis of differentially expressed genes revealed significant enrichments in various biological processes, including cellular and metabolic processes. A notable disparity in the growth period was observed between the identical species planted simultaneously and the mutant varieties. Mutation sites in Ganyin No1 rye were not limited solely to the vernalization gene. The differential gene expression profiles, obtained from the GO analysis, identified genes involved in vernalization and photoperiodic regulation. This study encountered limitations, as a subset of differentially expressed genes associated with vernalization and photoperiod was not identified. Further studies using alternative databases may be warranted for screening.
Six major pathways have been identified for regulating flowering time in plants, comprising three external pathways (photoperiodic regulation, vernalization, and ambient temperature) and three endogenous pathways (autonomous, age, and gibberellin pathways) [
33,
34,
35,
36]. This study primarily examines the regulatory pathways of photoperiod and vernalization within the external signaling cascades. The vernalization treatment identified four differentially expressed genes, whereas the photoperiod treatment revealed twenty-eight. Notably, the 15 March treatment group showed a majority of these differentially expressed genes, marking it as a distinct group. Among these, genes associated with vernalization were found to promote rye flowering, and it was found that most photoperiod-related genes also contribute to flowering. However, the Sc7296g5_i1G3 gene seems to inhibit flowering. Variations in these genes are hypothesized to explain the differences in vernalization between the two varieties. The functions of these genes can be further validated, and identifying the differential sites may help address the variation between the two varieties.
The KEGG annotation and enrichment analysis identified plant–pathogen interaction as the predominantly enriched pathway among the differentially expressed genes. Plant resistance against biotrophic pathogens is primarily mediated by salicylic acid, whereas necrotrophic pathogen infections are mainly regulated by jasmonic acid and ethylene, which often exhibit antagonistic effects during plant–pathogen interactions. Nonetheless, notable differences in carbon metabolism were detected between the two rye varieties during the tillering and jointing stages, possibly attributable to variations in nutrient conversion mechanisms influencing biomass production. The growth of plants is regulated by many PGPRs through the production of auxins, gibberellins, and cytokinins. Furthermore, the observed differences in rye at the tillering or jointing stage may stem from an increased demand for auxins and other hormones.
5. Conclusions
In conclusion, our study on the introduction of Wintergrazer-70 and Ganyin No1 rye in Tibet revealed significant differences between the two varieties in terms of summer sowing. Subsequently, identification tests confirmed that Ganyin No1 is a spring variety. RNA-seq identified 26 genes associated with the vernalization process, of which 4 were differentially expressed, all implicated in promoting vernalization. RNA-seq also identified 144 genes associated with the photoperiod, with 28 differentially expressed genes, predominantly involved in promoting flowering. However, the Sc7296g5_i1G3 (Gene8) gene was found to be associated with inhibiting flowering. Its remarkable adaptability to environmental conditions, along with beneficial traits such as resistance to barrenness, lodging, and rapid growth, make Ganyin No1 rye highly suitable for cultivation in the challenging Qinghai–Tibet Plateau region.