Gene Mapping and Identification of a Missense Mutation in One Copy of VRN-A1 Affects Heading Date Variation in Wheat

Abstract

Heading date (HD) is an important trait for wide adaptability and yield stability in wheat. The Vernalization 1 (VRN1) gene is a key regulatory factor controlling HD in wheat. The identification of allelic variations in VRN1 is crucial for wheat improvement as climate change becomes more of a threat to agriculture. In this study, we identified an EMS-induced late-heading wheat mutant je0155 and crossed it with wide-type (WT) Jing411 to construct an F₂ population of 344 individuals. Through Bulk Segregant Analysis (BSA) of early and late-heading plants, we identified a Quantitative Trait Locus (QTL) for HD on chromosome 5A. Further genetic linkage analysis limited the QTL to a physical region of 0.8 Mb. Cloning and sequencing revealed three copies of VRN-A1 in the WT and mutant lines; one copy contained a missense mutation of C changed to T in exon 4 and another copy contained a mutation in intron 5. Genotype and phenotype analysis of the segregation population validated that the mutations in VRN-A1 contributed to the late HD phenotype in the mutant. Expression analysis of C- or T-type alleles in exon 4 of the WT and mutant lines indicated that this mutation led to lower expression of VRN-A1, which resulted in the late-heading of je0155. This study provides valuable information for the genetic regulation of HD and many important resources for HD refinement in wheat breeding programs.

Keywords: VRN-A1; copy number; genetic mapping; heading date; wheat.

Figure 1

Comparison of phenotype and yield-related traits between WT and mutant: (a) Phenotypic comparison between WT (Jing411) and late heading mutant je0155. Plants were grown under field conditions and the picture was taken when the WT was heading. Bars = 10 cm; (b) Days to heading; (c) Spike length; (d) Thousand kernel weight; (e) Spikelets per spike. Values are averaged from five replicates. ** indicates significant differences by t-test at p < 0.01.

Figure 2

Mapping of the HD gene by BSA and genetic linkage analysis: (a) Distribution of ED⁴ values across all chromosomes. Higher ED⁴ values indicate a higher association effect with HD; (b) QTL analysis of HD on chromosome 5A. The logarithm of odds (LOD) threshold was set to 3.0, and a QTL located on a 1.63 cM of genetic linkage map between markers V3 and V4 was identified.

Figure 3

Copy number and mutation analysis of VRN-A1 in je0155: (a) The ratio of C- and T-type alleles of exon 4 in WT and je0155; (b) Gene structure and sequence of three copies of VRN-A1 with mutations in exon 4 and intron 5 of different copies of VRN-A1 in je0155. The light orange boxes indicate the 5′ or 3′ UTR region, while the orange boxes indicate the exon regions. The red fonts represent the mutation site in J411 and je0155.

Figure 4

Development of PCR sequencing markers and the phenotypic analysis of the F_2:3 population: (a) Sequences of different genotypes of mutations in intron 5 from the F₂ population detected by Sanger sequencing markers. The red box represents different peaks detected by Sanger sequencing in the population; (b) Heading time comparison of F_2:3 lines genotyped by PCR sequencing markers. WT, H, and Mut indicate homozygous WT, heterozygous, and homozygous mutant genotypes, respectively. *** indicates significant differences at 0.001 level by t-test.

Figure 5

Real-time qPCR of vernalization genes in WT and je0155. Values are means ± SD from five biological replicates. Second leaf indicates the second leaf below the spike: (a) Expression of C-type allele of exon 4 of VRN-A1 in the flag leaf, the second leaf, and the young spike of WT and je0155; (b–d) Relative expression of VRN1 (b), VRN2 (c), and VRN3 (d). ** represents a significant difference at 0.01 level by t-test.