Genetic map based quantitative trait locus (QTL) analysis is an important method for studying important horticultural traits in apple. To facilitate molecular breeding studies of fruit quality traits in apple, we aim to construct a high density map which was efficient for QTL mapping and possible to search for candidate genes directly in mapped QTLs regions. A total of 1733 F1 seedlings derived from ‘Jonathan’ × ‘Golden Delicious’ was used for the map constructionand QTL analysis. The SNP markers were developed by restriction site-associated DNA sequencing (RADseq). Phenotyping data of fruit quality traits were calculated in 2008-2011. Once QTLs were mapped, candidate genes were searched for in the corresponding regions of the apple genome sequence underlying the QTLs. Then some of the candidate genes were validated using real-time PCR. A high-density genetic map with 3441 SNP markers from 297 individuals was generated. Of the 3441 markers, 2017 were mapped to ‘Jonathan’ with a length of 1343.4 cM and the average distance between markers was 0.67 cM, 1932 were mapped to ‘Golden Delicious’ with a length of 1516.0 cM and the average distance between markers was 0.78 cM. Twelve significant QTLs linked to the control of fruit weight, fruit firmness, sugar content and fruit acidity were mapped to seven linkage groups. Based on gene annotation, 80, 64 and 17 genes related to fruit weight, fruit firmness and fruit acidity, respectively, were analyzed.Among the 17 candidate genes associated with control of fruit acidity, changes in the expression of MDP0000582174 (MdMYB4) were in agreement with the pattern of changes in malic acid content in apple during ripening, and the relative expression of MDP0000239624 (MdME) was significantly correlated withfruit acidity. We demonstrated the construction of a dense SNP genetic map in apple using next generation sequencing and that the increased resolution enabled the detection of narrow interval QTLs linked to the three fruit quality traits assessed. The candidate genes MDP0000582174 and MDP0000239624 were found to be related to fruit acidity regulation. We conclude that application of RADseq for genetic map construction improved the precision of QTL detection and should be utilized in future studies on the regulatory mechanisms of important fruit traits in apple.

Adventitious root formation is essential for the vegetative propagation of perennial woody plants. During the juvenile-to-adult phase change mediated by the microRNA156 (miR156), the adventitious rooting ability decreases dramatically in many species, including apple rootstocks. However, the mechanism underlying how miR156 affects adventitious root formation is unclear. In the present study, we showed that in the presence of the synthetic auxin indole-3-butyric acid (IBA), semi-lignified leafy cuttings from juvenile phase (Mx-J) and rejuvenated (Mx-R) Malus xiaojinensis trees exhibited significantly higher expression of miR156, PIN-FORMED1 (PIN1), PIN10, and rootless concerning crown and seminal roots-like (RTCS-like) genes, thus resulting in higher adventitious rooting ability than those from adult phase (Mx-A) trees. However, the expression of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE26 (SPL26) and some auxin response factor (ARF) gene family members were substantially higher in Mx-A than in Mx-R cuttings. The expression of NbRTCS-like but not NbPINs and NbARFs varied with miR156 expression in tobacco (Nicotiana benthamiana) plants transformed with 35S:MdMIR156a6 or 35S:MIM156 constructs. Overexpressing the miR156-resistant MxrSPL genes in tobacco confirmed the involvement of MxSPL20, MxSPL21&22 and MxSPL26 in adventitious root formation. Together, high expression of miR156 was necessary for auxin-induced adventitious root formation via MxSPL26, but independent of MxPINs and MxARFs expression in M. xiaojinensis leafy cuttings.

Nitrogen is critical for plant growth and development. With the increase of nitrogen fertilizer application, nitrogen use efficiency decreases, resulting in wasted resources. In apple (Malus domestica) rootstocks, the potential molecular mechanism for improving nitrogen uptake efficiency to alleviate low-nitrogen stress remains unclear. We utilized multi-omics approaches to investigate the mechanism of nitrogen uptake in two apple rootstocks with different responses to nitrogen stress, Malus hupehensis and Malus sieversii. Under low-nitrogen stress, Malus sieversii showed higher efficiency in nitrogen uptake. Multi-omics analysis revealed substantial differences in the expression of genes involved in flavonoid and lignin synthesis pathways between the two materials, which were related to the corresponding metabolites. We discovered that basic helix–loop–helix 130 (bHLH130) transcription factor was highly negatively associated with the flavonoid biosynthetic pathway. bHLH130 may directly bind to the chalcone synthase gene (CHS) promoter and inhibit its expression. Overexpressing CHS increased flavonoid accumulation and nitrogen uptake. Inhibiting bHLH130 increased flavonoid biosynthesis while decreasing lignin accumulation, thus improving nitrogen uptake efficiency. These findings revealed the molecular mechanism by which bHLH130 regulates flavonoid and lignin biosyntheses in apple rootstocks under low-nitrogen stress.

Deciphering the mechanism of malate accumulation in apple (Malus domestica Borkh.) fruits can help to improve their flavor quality and enhance their benefits for human health. Here, we analyzed malate content as a quantitative trait that is determined mainly by genetic effects. In a previous study, we identified an R2R3−MYB transcription factor named MdMYB44 that was a candidate gene in qtl08.1 (quantitative trait locus mapped to chromosome 8) of fruit malate content. In the present study, we established that MdMYB44 negatively regulates fruit malate accumulation by repressing the promoter activity of the malate-associated genes Ma1 (Al-Activated Malate Transporter 9), Ma10 (P-type ATPase 10), MdVHA-A3 (V-type ATPase A3), and MdVHA-D2 (V-type ATPase D2). Two single-nucleotide polymorphisms (SNPs) in the MdMYB44 promoter, SNP A/G and SNP T/−, were experimentally shown to associate with fruit malate content. The TATA-box in the MdMYB44 promoter in the presence of SNP A enhances the basal activity of the MdMYB44 promoter. The binding of a basic-helix–loop–helix transcription factor MdbHLH49 to the MdMYB44 promoter was enhanced by the presence of SNP T, leading to increased MdMYB44 transcript levels and reduced malate accumulation. Furthermore, MdbHLH49 interacts with MdMYB44 and enhances MdMYB44 activity. The two SNPs could be used in combination to select for sour or non-sour apples, providing a valuable tool for the selection of fruit acidity by the apple breeding industry. This research is important for understanding the complex molecular mechanisms of fruit malate accumulation and accelerating the development of germplasm innovation in apple species and cultivars.