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.

Background: Apple is an economically important fruit crop worldwide. Developing a genetic linkage map is a critical step towards mapping and cloning of genes responsible for important horticultural traits in apple. To facilitate linkage map construction, we surveyed and characterized the distribution and frequency of perfect microsatellites in assembled contig sequences of the apple genome. Results: A total of 28,538 SSRs have been identified in the apple genome, with an overall density of 40.8 SSRs per Mb. Di-nucleotide repeats are the most frequent microsatellites in the apple genome, accounting for 71.9% of all microsatellites. AT/TA repeats are the most frequent in genomic regions, accounting for 38.3% of all the G-SSRs, while AG/GA dimers prevail in transcribed sequences, and account for 59.4% of all EST-SSRs. A total set of 310 SSRs is selected to amplify eight apple genotypes. Of these, 245 (79.0%) are found to be polymorphic among cultivars and wild species tested. AG/GA motifs in genomic regions have detected more alleles and higher PIC values than AT/TA or AC/CA motifs. Moreover, AG/GA repeats are more variable than any other dimers in apple, and should be preferentially selected for studies, such as genetic diversity and linkage map construction. A total of 54 newly developed apple SSRs have been genetically mapped. Interestingly, clustering of markers with distorted segregation is observed on linkage groups 1, 2, 10, 15, and 16. A QTL responsible for malic acid content of apple fruits is detected on linkage group 8, and accounts for ~13.5% of the observed phenotypic variation. Conclusions: This study demonstrates that di-nucleotide repeats are prevalent in the apple genome and that AT/TA and AG/GA repeats are the most frequent in genomic and transcribed sequences of apple, respectively. All SSR motifs identified in this study as well as those newly mapped SSRs will serve as valuable resources for pursuing apple genetic studies, aiding the apple breeding community in marker-assisted breeding, and for performing comparative genomic studies in Rosaceae.

Apple ring rot, caused by Botryosphaeria dothidea, represents a major challenge for apple cultivation worldwide. However, the genetic factors that confer resistance in apples are not well understood. This study utilized time-series transcriptome profiling, combined with weighted gene co-expression network analysis and previously identified quantitative trait loci, leading to the identification of MdNHL8 as a candidate gene linked to resistance. A genome-wide examination of the apple NDR1/HIN1-like gene family revealed 57 members with diverse conserved domains, confirming that MdNHL8 features a distinct RBR-type E3 ligase domain. Subcellular localization experiments showed that MdNHL8 is localized in the nucleus and plasma membrane. Functional assays indicated that overexpression of MdNHL8 enhances resistance to apple ring rot and significantly elevates the expression of MdPR1, whereas silencing this gene does not enhance resistance and exhibits a trend toward increased susceptibility. Furthermore, additional transcriptome sequencing analysis of MdNHL8 transgenic calli highlighted a turquoise module closely linked to MdNHL8-mediated resistance, identifying MdCYCB3, MdEIX2, and MdABC4 as candidate downstream genes with expression patterns tightly correlated with MdNHL8. This research identifies MdNHL8 as a positive regulator in apple defense against B. dothidea and provides a basis for further exploring the regulatory network centered on MdNHL8 in disease resistance, offering insights for future breeding efforts aimed at enhancing resistance to apple ring rot.

Dwarfing rootstocks are widely used in apple production to control tree vigor and improve fruit quality. However, their adoption in ‘Fuji’ orchards remains limited by high production costs and unstable field performance. In this study, five rootstock materials, namely GM256, GM310, MD001, LiaoZhen 2, and Malus hupehensis Rehd (CK), were evaluated for their effects on cold tolerance, tree growth, and fruit quality in ‘Fuji’ apple. Cold tolerance was assessed using electrolyte leakage and related physiological indicators under low-temperature stress. MD001 and GM256 exhibited significantly stronger cold tolerance than the control, as indicated by lower LT₅₀ values and more favorable physiological responses (p < 0.05). Regarding tree growth, GM310 showed the weakest dwarfing effect, while LiaoZhen 2 had a relatively high proportion of long shoots, suggesting excessive vegetative growth. Meanwhile, GM256 produced the highest yield among all combinations. Principal component analysis of fruit-quality traits indicated that GM256 and GM310 performed better overall than the other combinations. In addition, GM256 produced significantly higher soluble solids, vitamin C content, and sugar–acid ratio than the control and most other rootstocks (p < 0.05). Overall, GM256 combined strong cold tolerance, a balanced shoot composition, moderate tree vigor, and high fruit quality, suggesting that it is a promising rootstock for ‘Fuji’ apple cultivation in Shandong Province.