Target enrichment sequencing coupled with GWAS identifies MdPRX10 as a candidate gene in the control of budbreak in apple.

The timing of floral budbreak in apple has a significant effect on fruit production and quality. Budbreak occurs as a result of a complex molecular mechanism that relies on accurate integration of external environmental cues, principally temperature. In the pursuit of understanding this mechanism, especially with respect to aiding adaptation to climate change, a QTL at the top of linkage group (LG) 9 has been identified by many studies on budbreak, but the genes underlying it remain elusive. Here, together with a dessert apple core collection of 239 cultivars, we used a targeted capture sequencing approach to increase SNP resolution in apple orthologues of known or suspected A. thaliana flowering time-related genes, as well as approximately 200 genes within the LG9 QTL interval. This increased the 275 223 SNP Axiom® Apple 480 K array dataset by an additional 40 857 markers. Robust GWAS analyses identified MdPRX10, a peroxidase superfamily gene, as a strong candidate that demonstrated a dormancy-related expression pattern and down-regulation in response to chilling. In-silico analyses also predicted the residue change resulting from the SNP allele associated with late budbreak could alter protein conformation and likely function. Late budbreak cultivars homozygous for this SNP allele also showed significantly up-regulated expression of C-REPEAT BINDING FACTOR (CBF) genes, which are involved in cold tolerance and perception, compared to reference cultivars, such as Gala. Taken together, these results indicate a role for MdPRX10 in budbreak, potentially via redox-mediated signaling and CBF gene regulation. Moving forward, this provides a focus for developing our understanding of the effects of temperature on flowering time and how redox processes may influence integration of external cues in dormancy pathways.

The Identification of Small RNAs Differentially Expressed in Apple Buds Reveals a Potential Role of the Mir159-MYB Regulatory Module during Dormancy.

Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.

Unraveling the role of MADS transcription factor complexes in apple tree dormancy.

A group of MADS transcription factors (TFs) are believed to control temperature-mediated bud dormancy. These TFs, called DORMANCY-ASSOCIATED MADS-BOX (DAM), are encoded by genes similar to SHORT VEGETATIVE PHASE (SVP) from Arabidopsis. MADS proteins form transcriptional complexes whose combinatory composition defines their molecular function. However, how MADS multimeric complexes control the dormancy cycle in trees is unclear. Apple MdDAM and other dormancy-related MADS proteins form complexes with MdSVPa, which is essential for the ability of transcriptional complexes to bind to DNA. Sequential DNA-affinity purification sequencing (seq-DAP-seq) was performed to identify the genome-wide binding sites of apple MADS TF complexes. Target genes associated with the binding sites were identified by combining seq-DAP-seq data with transcriptomics datasets obtained using a glucocorticoid receptor fusion system, and RNA-seq data related to apple dormancy. We describe a gene regulatory network (GRN) formed by MdSVPa-containing complexes, which regulate the dormancy cycle in response to environmental cues and hormonal signaling pathways. Additionally, novel molecular evidence regarding the evolutionary functional segregation between DAM and SVP proteins in the Rosaceae is presented. MdSVPa sequentially forms complexes with the MADS TFs that predominate at each dormancy phase, altering its DNA-binding specificity and, therefore, the transcriptional regulation of its target genes.