Genetic mechanisms associated with floral initiation and the repressive effect of fruit on flowering in apple (Malus x domestica Borkh)

Many apple cultivars are subject to biennial fluctuations in flowering and fruiting. It is believed that this phenomenon is caused by a repressive effect of developing fruit on the initiation of flowers in the apex of proximal bourse shoots. However, the genetic pathways of floral initiation are incompletely described in apple, and the biological nature of floral repession by fruit is currently unknown. In this study, we characterize the transcriptional landscape of bourse shoot apices in the biennial cultivar, 'Honeycrisp', during the period of floral initiation, in trees bearing a high fruit load and in trees without fruit. Trees with high fruit load produced almost exclusively vegetative growth in the subsequent year, whereas the trees without fruit produced flowers on the majority of the potential flowering nodes. Using RNA-based sequence data, we documented gene expression at high resolution, identifying >11,000 transcripts that had not been previously annotated, and characterized expression profiles associated with vegetative growth and flowering. We also conducted a census of genes related to known flowering genes, organized the phylogenetic and syntenic relationships of these genes, and compared expression among homologs. Several genes closely related to AP1, FT, FUL, LFY, and SPLS were more strongly expressed in apices from non-bearing, floral-determined trees, consistent with their presumed floral-promotive roles. In contast, a homolog of TFL1 exhibited strong and persistent up-regulation only in apices from bearing, vegetative-determined trees, suggesting a roles in floral repression. Additionally, we identified four GIBBERELLIC ACID (GA) 2 OXIDASE genes that were expressed to relatively high levels in apices from bearing trees. These results define the flowering-related transcriptional landscape in apple, and strongly support previous studies implicating both gibberellins and TFL1 as key components in repression of flowering by fruit. 

Methods
S1 File was constructed using the StringTie transcriptome assembler following RNAseq read alignment using HISAT2. 

S2 File was constructed by the Trinity transcriptome assembler using reads that failed to map to the GDDH13 reference genome using HISAT2.

S3 File was constructed by running the FASTA output from the Trinity transcriptome assembler through the python program Trinity_gene_splice_modeler.py provided by the Trinity suite.

S4 File was generated using the Cuffdiff program within the Cufflinks suite.

S5 File was generated using the Cuffdiff program within the Cufflinks suite.

All .png files image files are peptide phylogenies of Arabidopsis genes and their apple homologs genereated using ETE3.