Functional Analysis of the “Green Revolution” Gene Photoperiod-1 and Its Selection Trends During Bread Wheat Breeding

Flowering is central to the transformation of plants from vegetative growth to reproductive growth. The circadian clock system enables plants to sense the changes in the external environment and to modify the growth and development process at an appropriate time. Photoperiod-1 (Ppd-1), which is controlled by the output signal of the circadian clock, has played an important role in the wheat “Green Revolution.” In the current study, we systematically studied the relationship between Ppd-1 haplotypes and both wheat yield- and quality-related traits, using genome-wide association analysis and transgenic strategies, and found that highly appropriate haplotypes had been selected in the wheat breeding programs. Genome-wide association analysis showed that Ppd-1 is associated with significant differences in yield-related traits in wheat, including spike length (SL), heading date (HD), plant height (PH), and thousand-grain weight (TGW). Ppd-1-Hapl-A1 showed increased SL by 4.72–5.93%, whereas Ppd-1-Hapl-B1 and Ppd-1-Hapl-D1 displayed earlier HD by 0.58–0.75 and 1.24–2.93%, respectively, decreased PH by 5.64–13.08 and 13.62–27.30%, respectively, and increased TGW by 4.89–10.94 and 11.12–21.45%, respectively. Furthermore, the constitutive expression of the Ppd-D1 gene in rice significantly delayed heading date and resulted in reduced plant height, thousand-grain weight, grain width (GW), and total protein content. With reference to 40years of data from Chinese wheat breeding, it was found that the appropriate haplotypes Ppd-1-Hapl-A1, Ppd-1-Hapl-B1, and Ppd-1-Hapl-D1 had all been subjected to directional selection, and that their distribution frequencies had increased from 26.09, 60.00, and 52.00% in landraces to 42.55, 93.62, and 96.23% in wheat cultivars developed in the 2010s. A Ppd-B1 methylation molecular marker was also developed to assist molecular wheat breeding. This research is of significance for fully exploring the function of the Ppd-1 gene and its genetic resource diversity, to effectively use the most appropriate haplotypes and to improve crop yield and sustainability.


INTRODUCTION
Flowering is the central process in plant transformation from vegetative growth to reproductive growth, and photoperiod is one of the key environmental factors that regulates this process. In the photoperiod pathway which controls flowering in higher plants, the plant circadian system is at a key position and plays an important role in regulating the flowering of plants. During the long-term evolution of plants, the circadian system gave plants the ability to adapt to periodic changes in the external environment. The circadian system allows plants to sense changes in the external environment and to complete the growth and development process at the appropriate time (Greenham and McClung, 2015;Wei et al., 2018). The circadian system includes inputs from external signals and internal core oscillator and output channels (Harmer, 2009). Certain biological processes regulated by the circadian clock will also feedback and regulate the core oscillator of the circadian clock, forming a complex feedback regulatory network (Nohales and Kay, 2016).
The molecular mechanism of the circadian clock of the model plant Arabidopsis has been studied in depth. The Arabidopsis circadian clock includes several feedback loops. The central feedback loop is composed of genes encoding the core oscillator members TIMING OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY), and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1; Alabadí et al., 2001). CCA1/LHY also forms an early feedback loop with PRR9/PRR7 of the PSEUDO RESPONSE REGULATOR (PRR) gene family. PRR9, PRR7, and PRR5 exhibit expression peaks in sequence every 2-3 h from dawn to evening. They recruit histone deacetylases to form a transcriptional repression complex, which inhibits the expression of CCA1 and LHY at the transcriptional level (Nakamichi et al., 2010;Wang et al., 2013). The late feedback loop contains the evening complex (EC; Nusinow et al., 2011), composed of MYB transcription factor LUX ARRHYTHMO, nuclear protein EARLY FLOWERING 3 (ELF3), ELF4 (Nusinow et al., 2011), and TOC1. These feedback loops interlock to form the basic structure of the repressilator of the core oscillator of the circadian clock.
The PRR gene family, the core component of the circadian clock, has an important function in crops. The "Green Revolution" gene in wheat, Ppd-1 (TaPRR37), encodes a member of the PRR protein family, is homologous to Arabidopsis PRR7, and includes three photoperiod response loci, namely Ppd-A1, Ppd-B1, and Ppd-D1 (Laurie, 1997;Worland and Snape, 2001;Beales et al., 2007). The deletion of the Ppd-A1 promoter region is related to photoperiod insensitivity. Nishida et al. (2013) found that the promoter region of common wheat "Chihokukomugi" has a 1,085 bp deletion. Wilhelm et al. (2009) studied nearisogenic lines of tetraploid durum wheat with different photoperiod responses and found that the photoperiod insensitivity was related to two independent deletions (1,027 bp deletion and 1,117 bp deletion, respectively) in the Ppd-A1 gene, which caused abnormal gene expression and activation of FLOWERING LOCUS T (FT) expression. The aforementioned Ppd-A1 promoter deletion variant alleles were named Ppd-A1a.1 (1,085 bp deletion; Nishida et al., 2013),  (1,027 bp deletion), and Ppd-A1a.3 (1,117 bp deletion; Wilhelm et al., 2009), respectively. The photoperiod-insensitive allele Ppd-A1a confers wheat with a photoperiod-insensitive phenotype, which is intermediate between the insensitive phenotypes caused by the alleles Ppd-B1a and Ppd-D1a (Bentley et al., 2011;Shaw et al., 2012). Muterko et al. (2015) reported the allelic variation of the photoperiod-sensitive site Ppd-A1b. According to the difference in the movement speed of the 452 bp fragment in the promoter region, Ppd-A1b can be divided into two allele types: Ppd-A1b.AI and Ppd-A1b.AII.
Studies have shown that the Ppd-B1a photoperiod-insensitive allele is caused by copy number variation, with increased copy number leading to increased gene expression levels, and achieving a photoperiod-insensitive phenotype (Díaz et al., 2012). According to the types of Ppd-B1 copy number, it can be divided into Ppd-B1a (three-copy), Ppd-B1b (one-copy), Ppd-B1c (four-copy), Ppd-B1d (two-copy), and Ppd-B1e (null allele; Díaz et al., 2012;Cane et al., 2013). Würschum et al. (2015) studied the distribution characteristics of Ppd-B1 copy number in 1,110 winter wheat accessions and the effects of copy number on flowering time. The results showed that copy number variation in Ppd-B1 facilitated global adaptation in wheat. Sun et al. (2014) proved that the level of DNA methylation in the promoter region of the Ppd-B1 gene affected gene expression and was associated with photoperiod insensitivity. According to the levels of DNA methylation, it can be divided into two types: Ppd-B1 methylation haplotype a and methylation haplotype b (Sun et al., 2014). It is worth noting that DNA hypermethylation at Ppd-B1a is accompanied by higher copy numbers, either of which effects might be factors affecting the development of the Ppd-B1a allele (Sun et al., 2014). Beales et al. (2007) showed that wheat accessions carrying the photoperiod-insensitive allele Ppd-D1a all contained a 2 kb deletion upstream of the coding region. The deletion caused abnormal expression of the Ppd-D1 gene, leading to the expression of FT under short-day conditions. According to the promoter 2 kb deletion and other allelic variants (including TE insertion of the first intron and 5 bp deletion of the seventh exon), Ppd-D1 can be divided into four allele types: Ppd-D1a, , of which only Ppd-D1a contains a 2 kb deletion (Beales et al., 2007;Guo et al., 2010;Cane et al., 2013). In addition to regulating the photoperiod response of wheat, Ppd-1 is also a key regulator of inflorescence architecture and paired spikelet development (Boden et al., 2015).
Although there has been considerable research into the factors underlying the formation of Ppd-1 photoperiod-insensitive alleles, studies on the development of molecular markers for Ppd-1 genetic and epigenetic variation, the relationships between Ppd-1 haplotypes and yield-related traits, grain characteristics and quality traits on a genome-wide scale, and the effects of selection on Ppd-1 haplotypes in wheat breeding programs are incomplete. In the current study, we conducted a systematic functional analysis of Ppd-1, using genome-wide association analysis and studies on transgenics, and explored the relationship between Ppd-1 alleles and yield-and quality-related traits of wheat. Using data from 40 years of wheat breeding in China, the inadvertent effects of selection for increased yield and Frontiers in Plant Science | www.frontiersin.org improved quality on Ppd-1 haplotype were systematically explored. As a consequence, this study provides a theoretical basis for revealing the function of Ppd-1 and identifying its application to wheat breeding.

Plant Material
A total of 188 wheat accessions, derived from the major agroecological wheat regions of China and consisting of 25 landraces and 163 modern cultivars, was used for genomewide association analysis and breeding selection analysis (Supplementary Table S1). In the population of modern cultivars, 9, 26, 75, and 53 accessions were released in the 1980s, 1990s, 2000s, and 2010s, respectively Table S2). The test locations are all located in the northern part of China, with long-day conditions. Each accession was planted in 2 m three-row plots with 30 cm between rows. At maturity, six plants in the middle of each plot were selected for each genotype in order to investigate agronomic traits, including plant height (PH), spike length (SL), spike number (SN), total number of spikelets per spike (TNSS), number of grains per spike (NGS), thousand-grain weight (TGW), heading date (HD), grain length (GL), and grain width (GW). PH was measured from the stem base to the top of the main tiller spike. SL was measured from the internodes to the spike tip (excluding awns). TNSS and NGS were measured from the main tiller spike. HD was recorded on 50% spike emergence. TGW, GL, and GW were determined using the Intelligent Test and Analysis System (TOP Cloud-agri Technology, Zhejiang, China) using seeds after harvesting.

Ppd-1 Functional Molecular Markers
Based on the Ppd-B1 differentially methylated region, a target fragment (−1,250 to −665), containing methylation-sensitive restriction endonuclease (MSRE) HpaII or BstUI recognition sites (CCGG or CGCG), was selected to develop MSRE markers. DNA samples were extracted from 7-days-old seedlings during the light period using a phenol-chloroform method (Sharp et al., 1988). When detecting the methylation level of the target material, HpaII or BstUI was first used to digest the genomic DNA, and then, the digested product was amplified with the primers B1-HpaII-F1/R1 (Supplementary Table S3). The methylation type of Ppd-B1 can be distinguished according to the presence or absence of the target fragment after electrophoresis on 1.5% agarose gel. Amplification of the target band indicates that the identified material is the Ppd-B1 hypermethylated type, whereas the absence of an amplification product indicates that the identified material is of the Ppd-B1 hypomethylated type.
The 2 kb deletion in the promoter region of Ppd-D1a allele was amplified using a common forward primer Ppd-D1_F combined with two reverse primers, Ppd-D1_R1 and Ppd-D1_R2. Markers D78 and D520 were used to detect the insertion of TE in the first intron. Marker D5 was used to detect the 5 bp deletion in the seventh exon. This assay used nested PCR with two pairs of primers, D5-1F/D5-1R and D5-2F/D5-2R. Primers exon8_F1/exon8_R1 were used to detect the 16 bp insertion in the eighth exon (Beales et al., 2007;Guo et al., 2010). All Ppd-1 functional molecular markers are listed in Supplementary Table S3.

Bisulfite Genomic Sequencing
Hexaploid wheats "Am3, " "Laizhou953, " "Chinese Spring, " "Lumai14, " and "Yanzhan1" were used for bisulfite genomic sequencing. Bisulfite conversion of genomic DNA was achieved using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, CA, United States). The PCR products were purified and cloned into the pEASY-T1 Cloning Vector (TransGen, Beijing, China). At least 8-10 individual clones were sequenced, and three biologically independent replicates were carried out on each genotype to determine the methylation status of the target genomic regions. The sequencing data were analyzed by Kismeth software (Gruntman et al., 2008). The primer used for bisulfite genomic sequencing was referred to Sun et al. (2014;Supplementary

Reverse Transcription PCR and Quantitative Real-Time PCR
"Chinese Spring" was used to analyze the expression of Ppd-A1, Ppd-B1, and Ppd-D1 in various organs during wheat development. The materials were placed in a vernalization incubator (16 h light/8 h dark, 5°C) for 15 days and then transferred to a controlled environment room under LD conditions (16 h light/8 h dark, 24°C). "Chinese Spring" tissue samples included the shoot, leaf, leaf sheath, tiller base, flag leaf, pulvinus, young ear, and grain, which were collected at the seedling stage, three-leaf, tillering, flag leaf, full boot, ear emergence, anthesis, and milk grain stages, respectively. Four plants were mixed at each time point, and three biologically independent replications were performed for each tissue sample.
Frontiers in Plant Science | www.frontiersin.org Total RNA was extracted using RNAiso Plus (Takara, Ohtsu, Shiga, Japan). DNA was removed by digestion with DNase I (Fermentas, Ontario, Canada), and first-strand cDNA was synthesized using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen, CA, United States). The cDNA was diluted 5-fold for reverse transcription PCR (RT-PCR). The amplified products were detected following gel electrophoresis on 2% agarose. The cDNA was diluted 10-fold for quantitative real-time PCR (qPCR). Reactions included 10 μl 2 × TB Green Premix Ex Taq II Mix, 0.8 μl forward primer, 0.8 μl reverse primer, and 2 μl cDNA template in a total volume of 20 μl. Reaction conditions were [95°C 30 s; (95°C 5 s, 60°C 34 s) × 40 cycles], followed by a melting curve with 0.2°C steps between 60 and 95°C. qPCR was conducted using SYBR ® Premix Ex Taq™ (Takara) on an ABI PRISM 7500 bio-analyzer (Applied Biosystems, Foster City, CA, United States). Fluorescence threshold is set in the exponential phase, and the Ct value (the cycle value at which each sample reached the fluorescence threshold) was extracted for each sample. Expression levels of Ppd-A1, Ppd-B1, and Ppd-D1 genes were standardized against that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and quantitative data were normalized using the 2 -ΔΔCt method (Livak and Schmittgen, 2001). All primers used in this study were designed using Primer Premier 5.0 software (Supplementary Table S4).

Genome-Wide Associations and Population Structure Analysis
The 55 K single nucleotide polymorphism (SNP) genotyping assay was filtered in PLINK1.9 "--maf 0.01 --geno 0.2 --mind 0.2. " A total of 24,904 unique markers was analyzed for genomewide associations using the mixed linear model (PCA + K) method by TASSEL v5.2.72 (Bradbury et al., 2007), which took the population structure and relative kinship into account. A linkage disequilibrium heatmap was constructed using the package LDBlockShow v1.40 (Dong et al., 2020). Q-Q plots were generated using R package "rMVP" (Yin et al., 2021). Data for different phenotypic traits were represented by boxplots using the best linear unbiased estimate (BLUE) mean under each environment and were conducted using the R package "lme4" (Douglas et al., 2015).

Vector Construction, Plant Transformation, and Trait Measurements
The full-length open reading frame of Ppd-D1 was amplified and inserted into the binary vector pCAMBIA1301 (Sun et al., 2021), using the homologous recombination method. The pUbi:Ppd-D1 construct was transferred into the japonica rice cultivar Zhonghua 17 (ZH17) by Agrobacterium-mediated transformation. Phenotypic measurements of the positive transgenic plants were performed using three independent transgenic lines (10-20 individuals per line) in Ludong University (121.35°E, 37.51°N). Ludong University is located in the northern part of China, with long-day conditions. Yield-related traits assessed included heading date (HD), PH, panicle length, number of grains per main panicle, TGW, and tiller number. Grainrelated traits, including grain length (GL), grain width (GW), grain length/width ratio, and grain area, were determined using the Intelligent Test and Analysis System (TOP Cloud-agri Technology, Zhejiang, China). Grain quality traits, namely total protein content, total starch content, total amylose content, and total lipid content, were determined, with the total protein content and total lipid content of each grain sample being measured as described previously (Bradford, 1976;de Castro and Priego-Capote, 2010), whereas the total starch and amylose contents were determined with specific kits in accordance with the manufacturer's instructions (Megazyme, County Wicklow, Ireland).

Statistical Analysis
All statistical tests were performed using SPSS Statistics 18.0 (IBM, Armonk, NY, United States). Tukey's multiple comparison test was used to determine statistical differences identified by one-way ANOVA. Significance was accepted at p < 0.05 (*) and p < 0.01 (**) levels.
The expression patterns of the Ppd-1 genes were analyzed in various organs of "Chinese Spring" during wheat development under LD conditions (Figure 2). Overall, Ppd-A1, Ppd-B1, and Ppd-D1 exhibited similar expression characteristics. They all showed relatively high expression levels during the tillering, flag leaf, ear emergence, and anthesis stages, whereas the expression level of Ppd-1 was relatively low at the seedling, full boot, and milk grain stages. Moreover, Ppd-1 expression in the flag leaf was higher than that in the young ear and the grain. It is worth noting that the expression level of Ppd-A1  (Wilhelm et al., 2009;Muterko et al., 2015). Ppd-B1 is divided into two haplotypes, Hapl-B1 and Hapl-B2, according to the copy number (Díaz et al., 2012) and DNA methylation level (Sun et al., 2014). The alleles corresponding to Hapl-B1 are Ppd-B1a, Ppd-B1c, and Ppd-B1d, and the allele corresponding to Hapl-B2 is Ppd-B1b (Cane et al., 2013). Ppd-D1 is divided into two haplotypes Hapl-D1 (Ppd-D1a) and Hapl-D2 (Ppd-D1b, Ppd-D1c, and Ppd-D1d) according to whether the promoter region has a 2 kb fragment deletion (Beales et al., 2007;Guo et al., 2010;Cane et al., 2013). ADMIXTURE software was used to analyze the natural population structure. The results showed that the cross-validation error (CV error) value was lowest when k (number of subpopulation) = 3, indicating that the population material could be divided into three subgroups ( Figure 3A). In order to investigate the relationship between Ppd-1 haplotype and yield-related traits, we performed an association analysis of each haplotype with nine yield traits (PH; SL; SN; TNSS; NGS; TGW; HD; GL; and GW  Table S2).
Based on the Ppd-1 genotyping data, combined with the Wheat 55 K SNP array of the natural population, a genomewide association analysis was performed. Association analysis showed the homeolog-specific functions of Ppd-1. For Ppd-A1, there were weak associations between Ppd-A1 and spike length (three environments; Figure 3B). However, Ppd-B1 was significantly associated with plant height (seven environments) and heading date (four environments), and weakly associated with thousand-grain weight (four environments) and grain width (four environments; Figure 3C). Association analysis showed that Ppd-D1 had the strongest effect. Similar to Ppd-B1, Ppd-D1 was strongly associated with plant height and heading date in all environments and moderately associated with thousand-grain weight and grain width (six environments; Figure 3D).

Ppd-1-Hapl-A1, Ppd-1-Hapl-B1, and Ppd-1-Hapl-D1 Were Positively Selected for in Wheat Breeding
To determine whether favorable haplotypes of Ppd-1 were selected for during wheat breeding, we assessed the frequency changes of Ppd-A1, Ppd-B1, and Ppd-D1 haplotypes in the 188 wheat accessions studied which originated over many decades in different regions of China (Supplementary Table S1). Based on phenotypic data exhibited in seven environments, PH declined from landraces to modern cultivars and fell continually in modern cultivars bred from the 1980s to the 2010s. TGW showed the opposite trend, increasing gradually from landraces to the modern cultivars. The heading date of modern cultivars advanced gradually with the increase in breeding years ( Figure 5A). Compared with Ppd-1-Hapl-B2 and Ppd-1-Hapl-D2, Hapl-B1 and Hapl-D1 exhibited shorter PH, greater TGW, and earlier HD, which are all favorable haplotypes (Figures 4B-D).

Ppd-D1 Affects Rice Heading Date and Yield-Related Traits
To evaluate the effect of Ppd-1 on yield-related traits, we introduced the overexpression construct (pUbi:Ppd-D1, OE) into the japonica cultivar ZH17 (Figure 6A). Under field conditions, three representative homozygous lines overexpressing Ppd-D1 (Ppd-D1-OE) were obtained for detailed analysis. Compared with ZH17, as the wild-type control, the Ppd-D1-OE transgenic plants showed significantly elevated Ppd-D1 expression levels ( Figure 6B). The heading date of Ppd-D1-OE transgenic lines was delayed by 5.92-7.42 days (+4.93 to 6.18%), compared with wild-type plants (p < 0.01; Figure 6C). Meanwhile, the Ppd-D1-OE transgenic lines showed reduced plant height (−12.64 to −13.87%) as well as decreased number of grains per main panicle (−9.44 to −10.91%) and thousand-grain weight (−11.08 to −16.24%; p < 0.01; Figures 6D,F,G). However, there was no significant difference in either panicle length or tiller number (p > 0.05; Figures 6E,H). Thus, the constitutive expression of Ppd-D1 delayed the heading date and affected yield-related traits in rice.

Ppd-D1 Affects Grain Size and Quality in Rice
The functions of Ppd-D1 during seed development were also evaluated ( Figure 7A). Compared with ZH17, as the control, the Ppd-D1-OE transgenic lines showed decreased grain width (−3.87 to −4.37%), decreased grain area (−4.38 to −5.06%; Figures 7C,E), and increased ratio of length to width (+1.84 to 2.93%; p < 0.01; Figure 7D). However, there was no significant difference in grain length (p > 0.05; Figure 7B). We also tested the effect of Ppd-D1 on the nutrient content of rice grains, including total protein content, starch content, amylose content, and total lipid content. The Ppd-D1-OE transgenic lines showed decreased total protein content (−7.56 to −8.79%), compared with the control wild-type plants ( Figure 7F). However, there was no significant effect on total starch content, amylose content, and lipid content (p > 0.05; Figures 7G-I). Thus, the constitutive expression of Ppd-D1 affected grain size and seed quality in rice. These findings confirmed the potential application of Ppd-D1 in improving crop grain traits.

DISCUSSION The PRR Family Members of the Circadian Clock Genes Play an Important Role in Crops
The circadian clock is the core part of the photoperiod regulatory system, and PRR is the key component of the circadian clock regulatory network. In addition to the discovery Comparison of the grain length, grain width, the ratio of grain length to width, and grain area between ZH17 and Ppd-D1-OE (for each plant, n = 50). (F-I) Comparison of total protein content, total starch content, total amylose content, and total lipid content between ZH17 and Ppd-D1-OE (for each line, n = 5). Values are presented as mean ± SD. Phenotypic measurements of the transgenic plants were performed using three independent transgenic lines. Tukey's test was performed between control and transgenic plants (**p < 0.01).
of the PRR family in the model plant Arabidopsis thaliana, which regulates the growth and development of plants and its responses to changes in the external environment, including stress, research into the circadian clock of crops has also gradually developed in recent years. TaPRR1 is a core member of the wheat circadian clock. Sun et al. (2020) found that the expression of the TaPRR1 gene was significantly correlated with yield-related traits and exhibited genetic variation and differentiation between landraces and modern cultivars. The wheat microRNA (tae-miR408)-mediated control of TaPRR1 gene transcription is required for the regulation of heading date . Circadian clock member TaPRR73 affected heading date and plant height and promoted rice heading under long-day conditions (Zhang et al., 2016). The function of PRR gene family members in rice has also been reported. Studies have shown that rice OsPRR37 may be involved in the regulation of Hd3a gene expression, thereby regulating the sensitivity of rice to photoperiod (Koo et al., 2013). The rice circadian clock system not only regulates the heading date of rice, but also participates in the tolerance response of rice to salt and cold stress (Xu et al., 2016;Wei et al., 2021). Studies have shown that OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis .

Epigenetic Modification of the Circadian System and Epigenetic Molecular Markers
The network architecture of the circadian system core oscillator is mainly composed of multiple core components through interlocked transcription-translation feedback loops. In addition to strict transcription and post-transcriptional regulation, their expression level and activity are also regulated by epigenetic modification. Studies have found that DNA methylation is an important epigenetic regulator for the Frontiers in Plant Science | www.frontiersin.org precise maintenance of the plant circadian clock. Tian et al. (2021) proposed a new mechanism of DNA methylation controlled by the protein degradation cascade pathway SDC-ZTL-TOC1 to precisely regulate the clock pace, enriching our understanding of the regulation mechanism of the circadian system at the epigenetic level. Zhao et al. (2016) suggested that microRNAs might function in controlling the wheat heading date by mediating circadian clock gene expression, which provides important new information on the mechanism underlying heading date regulation in wheat. Sun et al. (2014) found that DNA methylation occurs in the promoter region of the wheat photoperiod gene Ppd-B1, which affects gene expression level and subsequently wheat photoperiod response. It is worth noting that the hypermethylated region of the Ppd-B1 gene overlaps with the deletion regions upstream of the Ppd-A1 and Ppd-D1 genes, implying that the upstream regulatory regions of Ppd-A1, Ppd-B1, and Ppd-D1 have common key regulatory elements. The methylation level of Ppd-B1 can be determined by bisulfite genomic sequencing, but this method is expensive. In the current paper, an epigenetic marker of Ppd-B1 methylation has been developed, which can identify the Ppd-B1 methylation level through restriction endonuclease digestion combined with PCR. Meanwhile, the highmethylation haplotype of Ppd-B1 was shown to be positively selected in wheat breeding, and the development of molecular markers will be helpful in assisting wheat molecular breeding and genetic improvement.

Research Prospects of the Crop Circadian Clock
In recent years, scientists have used the model plant A. thaliana to make progress in the study of the signal transduction mechanism of circadian clock-mediated plant growth and development, which has greatly promoted the development of this field. The functions of the circadian clock system in crops are diverse and conservative, but the mechanism of the circadian clock components in regulating the growth and development of crops still needs further study. The general impact of the circadian clock system on crops suggests that, by modifying the circadian rhythm, designing the timing of transgene expression and applying agricultural treatments at the most effective time of the day, future food production may be improved (Steed et al., 2021). Analyzing how the circadian clock system regulates the growth and development process of crops will hopefully illuminate the theoretical basis of chronobiology, provide high-quality genetic resources for crop molecular breeding, and increase crop yields (Wei et al., 2018).
Photoperiod-1 (TaPRR37) encodes a member of the PRR protein family and is homologous to Arabidopsis PRR7. AtPRR7 plays a role in clock function and photoperiod response, but the cereals genes may partially separate these functions, allowing mutations of the PRR37 gene to manipulate photoperiod response without affecting clock function (Higgins et al., 2010). Therefore, the Ppd-1 gene functions in the downstream of the circadian clock and is controlled by the output signal of the oscillator. However, in recent years, more and more reports suggested that Ppd-1 is a member of the circadian clock. Steed et al. (2021) indicated that on the basis of its relationship to Arabidopsis, PRR37 is assumed to have potential roles in the oscillator. Cao et al. (2021) described the PRRs, including the wheat Ppd-1 gene, as a major component of the circadian clock. The function of Ppd-1 in the oscillator needs to be further studied and elucidated in the future. As a member of the circadian clock system, Ppd-1 has made a great contribution to the wheat "Green Revolution" (Borlaug, 1983). Making full use of the diversity of Ppd-1 gene resources and developing simple and usable molecular markers are of great significance for the effective use of its tremendous and valuable allelic variation, thereby improving crop yields and quality.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.