BackgroundGenetic 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.MethodsA 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.ResultsA 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1946-x) contains supplementary material, which is available to authorized users.

BackgroundApple 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.ResultsA 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.ConclusionsThis 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.

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Fruit shape is a critical appearance quality in apple. Quantitative trait loci (QTLs) for apple fruit shape index (FSI) traits were previously mapped by our laboratory to linkage group 11 of the maternal parent in a cross population of 'Jonathan' 9 'Golden Delicious' using simple sequence repeat markers. In this study, QTLs for fruit length, diameter, and FSI were identified again using a high-density single nucleotide polymorphism (SNP) genetic linkage map, and candidate genes associated with FSI were screened via whole-genome re-sequencing data for 'Jonathan' and 'Golden Delicious'. Fifteen QTLs, including four for fruit length, one for fruit diameter, and ten for FSI, were identified in three sampling years. Two overlapping year-stable QTL regions related to FSI were anchored on LG 11 of 'Jonathan'. One candidate gene (MDP0000135244) related to FSI in apple and encoding an LysM domain receptor-like kinase protein was predicted and verified in the segregated population. The nonsynonymous SNP (C11.6053728) of MDP0000135244 was present in 23 of 30 individuals with high FSI, demonstrating a close relationship between MDP0000135244 and FSI trait. These results will be useful for the application of marker-assisted selection for FSI trait in apple.

Apple ring rot, which is caused by Botryosphaeria dothidea, is one of the most devastating diseases of apple. However, the lack of a known molecular resistance mechanism limits the development of resistance breeding. Here, the “Golden Delicious” and ‘Fuji ‘Nagafu No. 2″ apple cultivars were crossed, and a population of 194 F1 individuals was generated. The hybrids were divided into 5 categories according to their differences in B. dothidea resistance during three consecutive years. Quantitative proteomic sequencing was performed to analyze the molecular mechanism of the apple response to B. dothidea infection. Hierarchical clustering and weighted gene coexpression network analysis (WGCNA) revealed that photosynthesis was significantly correlated with the resistance of apple to B. dothidea. The level of chlorophyll fluorescence in apple functional leaves increased progressively as the level of disease resistance improved. However, the content of soluble sugar decreased with the improvement of disease resistance. Further research revealed that sorbitol, the primary photosynthetic product, played major roles in apple resistance to B. dothidea. Increasing the content of sorbitol by overexpressing MdS6PDH1 dramatically enhanced apple calli resistance to B. dothidea by activating the expression of salicylic acid (SA) signaling pathway-related genes. However, decreasing the content of sorbitol by silencing MdS6PDH1 showed the opposite phenotype. Furthermore, exogenous sorbitol treatment partially restored the resistance of MdS6PDH1-RNAi lines to B. dothidea. Taken together, these findings reveal that sorbitol is an important metabolite that regulates the resistance of apple to B. dothidea and offer new insights into the mechanism of plant resistance to pathogens.

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Variation with respect to both ploidy level and morphology was characterized for a set of 690 seedlings of the triploid apple variety Jonagold, of which 481 were obtained via in vitro culture of mature embryos, and 209 via conventional germination. Their ploidy level was determined by a combination of flow cytometry and root tip chromosome counting. The assessed morphological traits were leaf length, width and shape, stomatal density, guard cell length and chloroplast width and number. A total of 452 seedlings were aneuploid, 225 diploid, nine triploid and four tetraploid. All four tetraploid seedlings were derived by in vitro culture. When the triploid seedlings were genotyped at the S-locus and at selected microsatellite loci, we found that the Jonagold stigma was compatible with pollen which shared some of the maternal parent S-locus alleles.

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