Dong Mei Chen

A dense SNP genetic map constructed using restriction site-associated DNA sequencing enables detection of QTLs controlling apple fruit quality

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.

Differences in Gene Expression and Regulation during Ontogenetic Phase Change in Apple Seedlings

A woody perennial plant has to undergo a gradual and continuous process of transition from juvenility to reproductive maturity. To better understand the underlying mechanism of ontogenesis, our aim in this study was to identify differentially expressed genes between juvenile phase and adult phase not only among different seedlings but also among different hybrid crosses. Two reciprocal subtracted cDNA libraries of juvenile versus adult phases were constructed using suppression subtractive hybridization (SSH) in an apple (Malus domestica Borkh.) hybrid seedling (Jonathan × Golden Delicious). The expression uniformity of genes between the two ontogenetic phases was reconfirmed by real-time PCR (RT-qPCR) in three seedlings from the same population and also in three seedlings from different hybrid populations. Some potential post-transcriptional regulated genes were confirmed by semi-quantitative PCR. Eighty-five expressed sequence tags (ESTs) were up-regulated in the juvenile phase, and 103 ESTs were up-regulated in the adult phase. The transcription of genes associated with lipid metabolism, chloroplast protein metabolism and secondary metabolism was up-regulated in the adult phase; however, the expression of some phytohormone responsive genes was up-regulated during the juvenile phase. Several ontogenetic differential genes, such as rbcS, differed in sequences or elements in mRNA 3′ untranslated region (3′ UTR). In summary, the ontogenetic variations are probably under both transcriptional and post transcriptional regulation. The expression of redox related genes differed between juvenile and adult phase, indicating that redox homeostasis may play a role in vegetative phase change and floral transition.

Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

BackgroundAlthough protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms.MethodsPhosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry.ResultsA total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases.ConclusionsProtein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations.

Inheritance and Molecular Marker of Resistance to Bot Canker in Malus domestica

Abstract Apple bot canker [ Botryosphaeria dothidea (Moug.) Ces. et de Not.] is distributed worldwide, resulting in a serious crop loss every year in apple ( Malus domestica Borkh.) production. The resistance of each seedling derived from a hybrid population (JonathanxGolden Delicious) was evaluated by disease index either from natural infection in the field or from inoculation with five isolates of B. dothidea, Ls1, Lw023, Lw048, Mx1, and Zz26. The inheritance of the resistance to bot canker was analyzed via frequency distribution analysis, and microsatellite and AFLP markers linked to the resistance loci were screened. From the binary frequency distribution patterns, it was found that the segregation ratio of resistant/susceptible genotypes infected by pathogen isolates Lw023 and Ls1 was 1:15; and that by Zz26 and Mx1 was 15:1. The variation of resistance was involved in the segregation of two to four alleles of major genes, the resistance was recessive when infected by Lw023 and Ls1, but was dominant when infected with Mx1 and Zz26. A microsatellite maker, CH02a04-450, and two AFLP markers, E-AG/M-GAC-280 and E-AGG/M-CTT-110, were identified, and their map distances to the resistance loci were 5.1, 5.1 and 6.2 cM, respectively. The three markers are located in different linkage groups, while CH02a04-450 is on linkage group 2 or 7. E-AG/M-GAC-280 was successfully converted into SCAR159. Finally, CH02a04-450 and SCAR159 were re-examined in inoculated segregation population and presented a good reliability on predicting phenotypes of resistance.