Discovery of Allelic Variation in the Blueberry TFL1 Gene By Ecotilling

TERMINAL FLOWER1 (TFL1) has been found to repress the transition from vegetative to reproductive growth in diverse plant species. Natural mutations of TFL1 orthologs cause repetitive flowering in rose and woodland strawberry and a determinate growth habit in soybean, tomato, and cowpea. We investigated allelic variation in blueberry TFL1 among accessions in the USDA Vaccinium germplasm collection. The VcTFL1 gene sequence was identified from the draft genomic sequence of diploid V. corymbosum line W85-20. VcTFL1 is a member of a PEBP gene family and it could be distinguished from its family members by sequence comparison with PEBP family genes from other plants. To examine allelic variation, DNA was isolated from 160 Vaccinium accessions, most of which were autotetraploid V. corymbosum genotypes. For each accession, the four exons of VcTFL1 were amplified separately by PCR. Exons that differed in sequence from the W85-20 control were identified by high-resolution melting analysis. Single nucleotide polymorphisms (SNPs) were confirmed by sequencing. The analysis of tetraploid accessions with variant TFL1 alleles could distinguish simplex, duplex, and triplex haplotypes. A total of 18 SNPs were confirmed in VcTFL1 coding sequences among the 160 accessions. Three of these SNPs caused a change in an amino acid. The effect of these amino acid changes on protein function was examined with multiple bioinformatic tools. An alanine to valine change in exon 4 was predicted by these analyses to be deleterious to VcTFL1 function. The diploid V. corymbosum accession DE596 is heterozygous for this missense mutation. Exon 4 is critical to TFL1 function and is the site of missense mutations in soybean and cowpea that alter growth habit. The utility of V. corymbosum accession DE596 for developing an ornamental blueberry with novel flowering or architecture will be examined.