Malus baccata is one of four wild apple species that can hybridize with the cultivated apple species (Malus domestica). It is widely used in high-latitude apple-producing areas as a rootstock and breeding resource because of its disease resistance, and cold tolerance. A lack of a reference genome has limited the application of M. baccata for apple breeding. We present a draft reference genome for M. baccata. The assembled sequence consisting of 665 Mb, with a scaffold N50 value of 452 kb, included transposable elements (413 Mb) and 46,114 high-quality protein-coding genes. According to a genetic map derived from 390 sibling lines, 72% of the assembly and 85% of the putative genes were anchored to 17 linkage groups. Many of the M. baccata genes under positive selection pressure were associated with plant-pathogen interaction pathways. We identified 2,345 Transcription factor-encoding genes in 58 families in the M. baccata genome. Genes related to disease defense and cold tolerance were also identified. A total of 462 putative nucleotide-binding site (NBS)-leucine-rich-repeat (LRR) genes, 177 Receptor-like kinase (RLK) and 51 receptor-like proteins (RLP) genes were identified in this genome assembly. The M. baccata genome contained 3978 cold-regulated genes, and 50% of these gene promoter containing DREB motif which can be induced by CBF gene. We herein present the first M. baccata genome assembly, which may be useful for exploring genetic variations in diverse apple germplasm, and for facilitating marker-assisted breeding of new apple cultivars exhibiting resistance to disease and cold stress.

Shoot branching is regulated by phytohormones, including cytokinin (CK), strigolactone (SL), and auxin in axillary buds. The correlative importance of these phytohormones in the outgrowth of apple axillary buds remains unclear. In this study, the outgrowth dynamics of axillary buds of a more-branching mutant (MB) and its wild-type (WT) of Malus spectabilis were assessed using exogenous chemical treatments, transcriptome analysis, paraffin section, and reverse transcription-quantitative PCR analysis (RT-qPCR). High contents of CK and abscisic acid coincided in MB axillary buds. Exogenous CK promoted axillary bud outgrowth in the WT but not in MB, whereas exogenous gibberellic had no significant effect on bud outgrowth in the WT. Functional analysis of transcriptome data and RT-qPCR analysis of gene transcripts revealed that MB branching were associated with CK signaling, auxin transport, and SL signaling. Transcription of the SL-related genes MsMAX1, MsD14, and MsMAX2 in the axillary buds of MB was generally upregulated during bud outgrowth, whereas MsBRC1/2 were generally downregulated both in WT and MB. Exogenous SL inhibited outgrowth of axillary buds in the WT and the apple varieties T337, M26, and Nagafu 2, whereas axillary buds of the MB were insensitive to SL treatment. Treatment with N-1-naphthylphalamic acid (NPA; an auxin transport inhibitor) inhibited bud outgrowth in plants of the WT and MB. The transcript abundance of MsPIN1 was generally decreased in response to NPA and SL treatments, and increased in CK and decapitation treatments, whereas no consistent pattern was observed for MsD14 and MsMAX2. Collectively, the present results suggest that in apple auxin transport from the axillary bud to the stem may be essential for the outgrowth of axillary buds, and at least, is involved in the process of bud outgrowth.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and co-expression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

Shoot branching is an important factor that influences the architecture of apple trees and cytokinin is known to promote axillary bud outgrowth. The cultivar ‘Fuji’, which is grown on ~75% of the apple-producing area in China, exhibits poor natural branching. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes BRANCHED1/2 (BRC1/2) are involved in integrating diverse factors that function locally to inhibit shoot branching; however, the molecular mechanism underlying the cytokinin-mediated promotion of branching that involves the repression of BRC1/2 remains unclear. In this study, we found that apple WUSCHEL2 (MdWUS2), which interacts with the co-repressor TOPLESS-RELATED9 (MdTPR9), is activated by cytokinin and regulates branching by inhibiting the activity of MdTCP12 (a BRC2 homolog). Overexpressing MdWUS2 in Arabidopsis or Nicotiana benthamiana resulted in enhanced branching. Overexpression of MdTCP12 inhibited axillary bud outgrowth in Arabidopsis, indicating that it contributes to the regulation of branching. In addition, we found that MdWUS2 interacted with MdTCP12 in vivo and in vitro and suppressed the ability of MdTCP12 to activate the transcription of its target gene, HOMEOBOX PROTEIN 53b (MdHB53b). Our results therefore suggest that MdWUS2 is involved in the cytokinin-mediated inhibition of MdTCP12 that controls bud outgrowth, and hence provide new insights into the regulation of shoot branching by cytokinin.

Insufficient production of flower buds is an intractable problem in ‘Fuji’ apple orchards. Although cytokinin (CK) promotes flower bud formation in apple trees, little is known about the mechanisms regulating this phenomenon. In the present study, high-throughput RNA sequencing (RNA-Seq) of ‘Nagafu No. 2’ buds was conducted to characterize the transcriptional response to 6-BA treatment during key period of floral transition. A weighted gene co-expression network analysis (WGCNA) of the differentially expressed genes identified hormone signal transduction pathways, totaling 84 genes were highly correlated with the expression pattern of flowering-time genes. The up-regulation of CK signal components and a gibberellin (GA) signal repressor were found to contribute to the promotion of floral transition. In relative comparison to non-treated buds, a series of sugar metabolism- and signal- related genes were associated with relatively high levels of sucrose, fructose, and glucose during floral induction in the 6-BA treated buds. Several transcription factors (i.e. SPLs, SOC1, FD, and COL) that are involved in GA, aging, and photoperiod-regulated flowering pathways were also upregulated by the 6-BA treatment. In addition, potential transcription factors integrating CK signaling to trigger floral induction in apple were also assessed; including PHYTO-CHROME-INTERACTING FACTOR (PIF1,3), WUSCHEL-related homeobox (WOX3,13), and CK response regulators (ARR2). The present study provides insight into the response of flowering and development-related pathways and transcription factors to 6-BA during the period of floral transition in apple. It extends our knowledge of the fundamental mechanisms associated with CK-regulated floral transition in apple trees.