Large-scale amplicon sequencing of the SP3D gene responsible for fruit-yield heterosis in tomato

Hybrid tomato plants being heterozygous for certain induced loss-of-function mutations of the SP3D gene displayed fruit-yield heterosis. In order to investigate the possible occurrence of spontaneous mutations of that kind, the SP3D amplicon of 262 tomato seed bank accessions was deep sequenced at about 2000-fold coverage. Compared to the SL2.50 genome assembly, twenty-two novel SNPs in total were identified in the 652 bp upstream, 3817 bp coding and 613 bp downstream regions of the gene. Neither novel exonic mutations in the 262 accessions of this study nor possible loss-of-function mutations amongst the known exonic variants were identified in the SP3D gene of Solanum lycopersicum indicating that, very likely, no such spontaneous mutations occur in cultivated tomato. In wild tomato, however, there are missense variations in the coding region of SP3D, which might result in disabled SP3D proteins. This difference between cultivated and wild tomatoes might have reproductive-biological reasons, while in cultivated tomato missense SP3D mutations might have been eliminated during breeding because they could result in extremely late flowering phenotype. In the promoter region of the SP3D gene, two SNPs, of which one was novel, were identified in the − 62 and − 61 positions upstream of the ATG start codon. These positions were genotyped in the 262 accessions and, comparing our results with Ensembl Plants data, it could be concluded that the dinucleotide is a conserved CT in Solanum lycopersicum varieties, while it is either CA or TA in other Solanum species. This dinucleotide is in a predicted binding site of BELL homeodomain transcription factor(s), regulating meristem, fruit and inflorescence development, thus it might be an element of the regulatory cascade in which the SP3D gene is involved to regulate flowering in tomato. We presented a hypothesis that the SP3D and Single Flower Truss (SFT) genes might not be the same, as it is currently considered, and that SFT might be equivalent to the gene Solyc03g071710 encoding a putative Self-pruning interacting protein. This would logically explain both the absence of natural loss-of-function mutations in the SP3D gene and the absence of DNA lesions in the SP3D gene of sft mutant tomato.