 vernalization.

The genome region associated with the presence and absence of awn
The Tabasco × Zi119 F 2 population showed a clear cut-off segregation of awn and awnless spikes, fitting a 3: 1 (χ 2 = 1.17, p = 0.28). The result demonstrated that the awn trait was dominant over the awnless trait in the F 2 population. The integration of the phenotypes and genotypes for each of the F 2 plants allowed mapping of a genomic region on chromosome 5A for this trait. Further analysis of SNP markers in the mapped genomic region identified 10 crossovers between AX109275049 and AX109911223 ( Figure 4A). Fine mapping of these crossovers showed that the candidate gene should be located between AX109857944 at 692,461,596 bp and AX109911223 at 702,081,022 bp ( Figure 4A). TaesCS5A02G542800 representing ALI-A1 at position 698,528,636 bp is located between the two SNP markers. TZ98 and TZ101 had homozygotes for AX109857944 and heterozygotes for AX109911223 and the two plants had no awn, indicating that the candidate gene was linked with ALI-A1. TZ19, TZ196, and TZ207 had homozygotes for AX109911223 and heterozygotes for AX109857944 and these three plants had no awn, indicating that the candidate gene was linked with ALI-A1 too. Combining the genotypic and phenotypic results from these five critical recombinant plants, ALI-A1 was a candidate gene causing the segregation of awn/awnless traits in the F 2 population. Tabasco had the dominant Ali-A1 allele for awn, Zi119 had the recessive ali-A1 allele for awnless, and ALI-A1 was used for the common allele.

Allelic variation in ALI-A1
Sequencing results showed that the Tabasco and Zi119 ALI-A1 alleles had the same sequences in the coding region but differed in two SNPs in the 254 bp promoter region ( Figure 4B). One was at position-111, where "C" in the Ali-A1 allele was replaced by "T" in the ALI-A1 allele. The other one was at position-219, where "A" in the Ali-A1 allele was deleted in the ali-A1 allele. The transcript level of ALI-A1 gene was determined and no significant difference was observed between the two alleles using RNA samples collected from leaves at the seedling stage (p = 0.666, n = 9; Figure 5A) or young spikes at heading date (p = 0.374, n = 9; Figure 5B).
The same primers were used to determine the copy number of genes. Relative to one copy of ali-A1 in Zi119, Ali-A1 in Tabasco was found to have two copies of Ali-A1 ( Figure 5C). According to the copy number and chromosomal locations of ALI-A1 gene in CS and 10 wheat genome sequences, the Ali-A1 allele was found to have one copy on chromosome 5A in six cultivars ( Figure 5D). However, the Ali-A1 allele was found to have four types, Ali-A1a through Ali-A1d. Ali-A1a has one copy on chromosome 5A ( Figure 5E), Ali-A1b on has one copy ChrUN (unknown chromosome; Figure 5F), Ali-A1c has three copies, one at position on chromosome 5A and another two copies on ChrUN ( Figure 5G), and Ali-A1d has five copies, one on chromosome 5A and another four copies on ChrUN ( Figure 5H). It was likely that Tabasco has two copies of ALI-A1, one on chromosome 5A and the other on ChrUN, which is referred to as Ali-A1e allele.

Discussion
Zi119 is a spring wheat cultivar that has no vernalization for flowering about 50-70 days (depending on experiment seasons in the greenhouse) as presented in this study. In wheat production, however, Zi119 is planted in October and harvested in next May, spanning approximate 7 months in field in Zone III; where a winter season is clear (Zhang et al., 2008). Zi119 wheat was found to have the Vrn-D1 allele for early flowering, when this cultivar was crossed with strong winter cultivar Tabasco and the resulting F 2 population was tested under constant warm temperature and long day conditions. In the field, the spring wheat cultivar is not accelerated but delayed for flowering by low temperature, whereas a winter cultivar is accelerated in the developmental transition for flowering (Li et al., 2013). The Vrn-D1 allele could make spring wheat or semi-winter cultivar to flower in a difference of a few months in the greenhouse but a few days in the field.
Frontiers in Plant Science 08 frontiersin.org The Vrn1 gene was initially cloned from diploid wheat T. monococcum (Yan et al., 2003). The availability of the gene sequence allowed isolation of three homoeologous genes, VRN-A1, VRN-B1, and VRN-D1 in common wheat and identification of allelic variations in each of them. Compared with VRN-A1 that has multiple alleles (Yan et al., 2003(Yan et al.,, 2004aPidal et al., 2009;Golovnina et al., 2010;Li et al., 2013;Shcherban et al., 2015), VRN-B1 and VRN-D1 are observed to have fewer haplotypes. Vrn-D1 in spring wheat has a large deletion in intron one in hexaploid wheat (Fu et al., 2005). The effect intensity of the three VRN-1 genes was ranked as Vrn-A1 > Vrn-B1 > Vrn-D1, based on analyses on Triple Dirk isogenic lines (Loukoianov et al., 2005), Chinese wheat cultivars (Zhang et al., 2008), and a biparental population (Li et al., 2015). Due to the greatest effect of the Vrn-A1a allele, it has been incorporated into spring wheat cultivars in Canadian breeding programs and spring-sown wheat cultivars in China to provide frost avoidance in short-season environments or frozen damage in spring season (Iqbal et al., 2007;Zhang et al., 2008). This study provided experimental evidence for the desirable selection of Vrn-D1 on ideal heading date in wheat Zone III. The genetic corporation of Vrn-D1 with two genes on chromosome arms 2AS and 2DS could be used to make wheat cultivars with different developmental durations at joining state, heading date, and physiological maturity (Chen et al., 2009(Chen et al.,, 2010, which are adapted to gradually changed environments from southern to Northern regions in Zone III. Genotypes and phenotypes of 10 recombinant plants that have crossovers at the ALI-A1 locus. (A) Physical locations of 10 crossovers that occurred at the ALI-A1 locus. Markers mapped chromosome arm 5AL that are indicated in different colors are arranged based on the sequences of Chinese Spring (CS). ALI-A1 is annotated TaesCS5A02G542800. "A" is the Tabasco allele, "B" for the Zi119 allele, and "H" for the heterozygote. "X" indicates a crossover detected between two neighboring markers. Phenotype of each recombinant plant was of awn or awnless. (B) Comparison of promoter sequences between the Tabasco allele and the Zi119 allele.
Frontiers in Plant Science 09 frontiersin.org Awn Length inhibitor 1 (i.e., TraesCS5A02G542800) was believed to be a strong candidate for the dominant Tipped1 or B1 as an awn suppressor (DeWitt et al., 2020;Huang et al., 2020;Niu et al., 2020;Wang et al., 2020;Würschum et al., 2020). This gene was cloned by analyses of association maps and biparental populations, and polymorphisms were observed not in the ALI1 coding region but a nearby region (a 30-bp deletion at 4 kb downstream) that was predictive of regulatory elements (DeWitt et al., 2020). It could be a 25-bp deletion upstream of the Ali-1 allele that was linked with the awnless trait (Huang et al., 2020), but this deletion is also found in T. urartu, raising a doubt about the causal. It could be one of five SNPs at positions from-1,630 to-709 in the promoter region of ALI1 that is responsible for awn elongation (Wang et al., 2020). In addition, SNPs at the ALI1 locus could be a gene that has a dosage effect on awn length based on segregation for long, intermediate and short awn phenotypes two F 8 recombinant inbred lines (RILs; Niu et al., 2020). Collectively, it is not yet clear how allelic variation in ALI-1 caused differences between the awnless and awn traits or between the long awn and short awn traits (Würschum et al., 2020). ALI-1 is located within a genomic region, where it is surrounded by >100 kb of insertion or deletion of transposable or repetitive elements that may hinder assembly of genome sequences, resulting in unknown chromosome. Among the 10 sequenced  192,