Materials and Methods

In our previous study, we found a C/T polymorphism in exon 4 that is responsible for the amino acid change at Leu117/Phe117 in TaVRN-A1 between the Jagger allele and the 2174 allele. A PCR marker for this polymorphism showed an association with developmental variation in the Jagger × 2174 RIL population tested in the field (Chen et al. 2009). Primers used for the SNP in exon 4 are vrn-A1F4F 5′-CAACTTGTTTGGGACTAAAGGC-3′ and vrn-A1F4R 5′-CTGCAACTCCTTGAGATTCAAAG-3′. PCR was performed for 40 cycles (90 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s per cycle) followed by a 10-min final extension at 72 °C. PCR products were digested with DpnII and run on a 1 % agarose gel.

In this study, we found that there was another SNP in exon 7, and we developed the PCR marker for the SNP in exon 7 to screen wheat germplasm available in our laboratory. The hexaploid wheat cDNA sequences in GenBank for each of three homoeologous VRN1 genes were divided into three groups, and genomic DNA sequences for each group were derived from the wheat genome sequence database (cerealsdblished.uk.net/). Specific primers for each homoeologous VRN1 were designed. Chinese Spring nullitetrasomic lines, N5AT5D, N5BT5D or N5DT5B missing each of three homoeologous group five chromosomes were used to determine specificity of the primers. Primers vrnA1F7B (5′-GTGGAGAAGCAGAAGGCGCATG-3′) and vrn-A1R7 (5′-CCGACAGAACTGCATAGAGACC-3′) were designed to detect the SNP encoding A180/V180 between the Jagger vrn-A1a allele and 2174 vrn-A1b allele.

The two primers amplified a 221 bp fragment using an annealing temperature of 55 °C and extension time for 1 min. The PCR products digested with restriction enzyme SphI were run on a 1 % agarose gel, showing polymorphic bands between the vrn-A1a allele (199 bp) and vrn-A1b allele (221 bp).

Results and Discussion

The vrn-A1 Gene Controlling Vernalization Requirement Duration in Winter Wheat Cultivars

Jagger required 3 weeks to reach the maximum vernalization effect on flowering, whereas 2174 required 6 weeks to reach the maximum vernalization effect on flowering under the same condition (Li et al. 2013). We generated a BC1F2 population using RIL23 to backcross with 2174. When vernalized for 3 weeks, heading date of 90 F2 plants showed a clear segregation. On average, 24 plants homozygous for the Jagger vrn-A1a allele headed at 110 days after planting, 20 plants homozygous for the 2174 vrn-A1b allele headed at 138 days, and 46 plants heterozygous at VRN-A1 headed at 118 days (Fig. 13.1a). The 70 plants either homozygous or heterozygous for the Jagger vrn-A1a allele for early heading showed a significant difference from the 20 plants homozygous for the 2174 vrn-A1b allele for late heading (p < 0.001). The observed segregation ratio between the earlier heading and later heading groups was not significantly different from a 3:1 ratio (X2 = 0.37, df = 1, p = 0.54) and fit a one-gene model.

A Critical Point Mutation in vrn-A1 at the Protein Level

We used the positional cloning strategy to prove that quantitative vernalization requirement in winter wheat is controlled by vrn-A1 at the protein level (Li et al. 2013). There were 29 SNPs in vrn-A1 between the Jagger allele (11,922 bp, JQ915055) and the 2174 allele (11,921 bp, JQ915056), including 464 bp from the start codon for translation, the complete gene from the start codon and the stop codon, 41 bp after the stop codon. These SNPs were confirmed by sequencing two

Fig. 13.1 Allelic variation and genetic effects of vrn-A1. (a) The segregation of heading date in plants carrying different vrn-A1 alleles in a BC1F2 population. vrn-A1a for the Jagger allele and vrn-A1b for the 2174 allele. (b) PCR marker for the SNP in exon 4 of vrn-A1 using primers vrnA1F4F and vrn-A1F4R. (c) PCR marker for the SNP in exon 7 of vrn-A1 using primers vrn-A1F7F and vrn-A1F7R

independent PCR products or digesting PCR products using restriction enzymes wherever appropriate. No difference was observed between the Jagger vrn-A1a allele and 2174 vrn-A1b allele in the previously identified regulatory sites in the promoter or intron one that accounted for allelic variation between the winter vrnA1 allele and the spring Vrn-A1 allele.

There were two SNPs in exon 4 and exon 7, both of which resulted in an altered amino acid in the conserved domain between the Jagger and 2174 alleles. An amino acid Ala180 encoded in exon 7 in the Jagger vrn-A1a allele controlling less vernalization or early flowering was mutated to Val180 in the 2174 vrn-A1b allele. The Ala180/ Val180 substitution accounted for the differential interactions of vrn-A1a and vrnA1b with TaHOX1 in pull-down assays and protein immune-precipitation analyses (Li et al. 2013).

 
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