Large-scale amplicon sequencing of the SP3D gene responsible for fruit-yield heterosis in tomato Ferenc Marincs a,b, ⁎, Tibor Nagy a,1 , Krisztina Miró a , Zsuzsanna Kollár c , Endre Barta a , Péter Kaló a a Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, 2100 Gödöllő, Szent-Györgyi Albert u. 4, Hungary b Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 2462 Martonvásár, Brunszvik u. 2, Hungary c Center for Plant Diversity, 2766 Tápiószele, Külsőmező 15, Hungary abstract article info Article history: Received 18 May 2016 Received in revised form 3 November 2016 Accepted 17 December 2016 Available online 27 December 2016 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 up- stream, 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 eliminat- ed 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 vari- eties, 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 pre- sented a hypothesis that the SP3D and Single Flower Truss (SFT) genes might not be the same, as it is currently con- sidered, 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. © 2016 Elsevier B.V. All rights reserved. Keywords: Amplicon sequencing Fruit-yield Gene variation SNP Solanum lycopersicum SP3D gene The phenomenon called heterosis or hybrid vigour is the superior performance of hybrid progenies compared to their genetically aver- age/inferior parents. Heterosis is generally considered to be under multi-gene control affecting multiple physiological and phenotypic traits. In the long history of heterosis research, which goes back to Dar- win (Jiang et al., 2013), a number of hypotheses were born about its ge- netic background and emerging genomic, transcriptomic and epigenetic studies have also contributed to understand this phenomenon (for a re- cent review see Fu et al., 2015). In crop species, heterosis is one of the key research topics, since it influences agronomically important traits such as vegetative growth, yield and stress-resistance. For obvious economic reasons, fruit yield is one of the most impor- tant agronomic traits in cultivated varieties of tomato (Solanum lycopersicum). Therefore, the discovery that F1 hybrid tomato plants being heterozygous for a single gene produced up to 60% higher fruit yield (Krieger et al., 2010) had a great significance and was considered as an evidence for the single gene overdominance hypothesis of heter- osis (Birchler et al., 2003). The hybrid plants were obtained by crossing wild-type and mutant M82 lines, where every mutant line carried a sin- gle loss-of-function allele of the SP3D gene (Lifschitz et al., 2006), which belongs to the self-pruning (SP) gene family and is an orthologue of the Arabidopsis FLOWERING LOCUS T (FT) gene (Carmel-Goren et al., 2003). The mutant lines were generated by induced mutagenesis using differ- ent agents (Menda et al., 2004) and were selected for their extreme late- flowering phenotype (Lifschitz et al., 2006). Sequencing of the SP3D Plant Gene 9 (2017) 45–49 ⁎ Corresponding author at: Agricultural Biotechnology Institute, NARIC, Szent-Györgyi Albert u. 4., 2100 Gödöllő, Hungary. E-mail addresses: marincs.ferenc@abc.naik.hu (F. Marincs), black007@abc.hu (T. Nagy), miro.krisztina@abc.naik.hu (K. Miró), zskollar@mail.nodik.hu (Z. Kollár), barta.endre@abc.naik.hu (E. Barta), kalo.peter@abc.naik.hu (P. Kaló). 1 Present address: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom. http://dx.doi.org/10.1016/j.plgene.2016.12.002 2352-4073/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Plant Gene journal homepage: www.elsevier.com/locate/plantgene