Human selection has reshaped crop genomes. Here we report an apple genome variation map generated through genome sequencing of 117 diverse accessions. A comprehensive model of apple speciation and domestication along the Silk Road is proposed based on evidence from diverse genomic analyses. Cultivated apples likely originate from Malus sieversii in Kazakhstan, followed by intensive introgressions from M. sylvestris. M. sieversii in Xinjiang of China turns out to be an “ancient” isolated ecotype not directly contributing to apple domestication. We have identified selective sweeps underlying quantitative trait loci/genes of important fruit quality traits including fruit texture and flavor, and provide evidences supporting a model of apple fruit size evolution comprising two major events with one occurring prior to domestication and the other during domestication. This study outlines the genetic basis of apple domestication and evolution, and provides valuable information for facilitating marker-assisted breeding and apple improvement.

Skin color is an important factor in pear breeding programs. The degree of red coloration is determined by the content and composition of anthocyanins. In plants, many MYB transcriptional factors are involved in regulating anthocyanin biosynthesis. In this study, a R2R3-MYB transcription factor gene, PyMYB10, was isolated from Asian pear (Pyrus pyrifolia) cv. 'Aoguan'. Sequence analysis suggested that the PyMYB10 gene was an ortholog of MdMYB10 gene, which regulates anthocyanin biosynthesis in red fleshed apple (Malus x domestica) cv. 'Red Field'. PyMYB10 was identified at the genomic level and had three exons, with its upstream sequence containing core sequences of cis-acting regulatory elements involved in light responsiveness. Fruit bagging showed that light could induce expression of PyMYB10 and anthocyanin biosynthesis. Quantitative real-time PCR revealed that PyMYB10 was predominantly expressed in pear skins, buds, and young leaves, and the level of transcription in buds was higher than in skin and young leaves. In ripening fruits, the transcription of PyMYB10 in the skin was positively correlated with genes in the anthocyanin pathway and with anthocyanin biosynthesis. In addition, the transcription of PyMYB10 and genes of anthocyanin biosynthesis were more abundant in red-skinned pear cultivars compared to blushed cultivars. Transgenic Arabidopsis plants overexpressing PyMYB10 exhibited ectopic pigmentation in immature seeds. The study suggested that PyMYB10 plays a role in regulating anthocyanin biosynthesis and the overexpression of PyMYB10 was sufficient to induce anthocyanin accumulation.

Background: Pear is one of the most important fruit crops worldwide. Anthocyanins and procyanidins (PAs) are important secondary metabolites that affect the appearance and nutritive quality of pear. However, few studies have focused on the molecular mechanism underlying anthocyanin and PA accumulation in pear. Results: We conducted metabolome and transcriptome analyses to identify candidate genes involved in anthocyanin and PA accumulation in young fruits of the pear cultivar 'Clapp Favorite' (CF) and its red mutation cultivar 'Red Clapp Favorite' (RCF). Gene-metabolite correlation analyses revealed a 'core set' of 20 genes that were strongly correlated with 10 anthocyanin and seven PA metabolites. Of these, PcGSTF12 was confirmed to be involved in anthocyanin and PA accumulation by complementation of the tt19-7 Arabidopsis mutant. Interestingly, PcGSTF12 was found to be responsible for the accumulation of procyanidin A3, but not petunidin 3, 5-diglucoside, opposite to the function of AtGSTs in Arabidopsis. Transformation with PcGSTF12 greatly promoted or repressed genes involved in anthocyanin and PA biosynthesis, regulation, and transport. Electrophoretic mobility shift and luciferase reporter assays confirmed positive regulation of PcGSTF12 by PcMYB114. Conclusion: These findings identify a core set of genes for anthocyanin and PA accumulation in pear. Of these, PcGSTF12, was confirmed to be involved in anthocyanin and PA accumulation. Our results also identified an important anthocyanin and PA regulation node comprising two core genes, PcGSTF12 and PcMYB114. These results provide novel insights into anthocyanin and PA accumulation in pear and represent a valuable data set to guide future functional studies and pear breeding.

A mutant tree with blushed coloring pattern in fruit skin was found and identified as a bud sport of Malus domestica Borkh. cultivar 'Ralls'. This study was aimed at elucidating the genetic basis of this pattern by a comparison of 'Ralls' (striped red) with this bud sport. These two varieties were grown either under normal sunlight (control group) or the fruits were bagged during development (bagged group) and the bags were removed 1 month before harvesting. Anthocyanin content increased during fruit ripening and was higher in the blushed sport than in 'Ralls', by 57% in the control group and by 73% in the bagged group after bag removal. Correspondingly, in both the varieties transcription of six anthocyanin biosynthetic genes was coordinately up-regulated, concomitant with an increase in levels of transcripts of the regulatory gene, MdMYB1, and in general these levels were higher in the blushed sport than in ''Ralls''. However, there were no significant differences between the two varieties in the nucleotide sequences of the genomic region or coding sequence of MdMYB1; nor in the sequences of cDNAs of the other known transcription factors MdbHLH3 and MdbHLH33. Interestingly, DNA methylation in the promoter region of MdMYB1 was inversely correlated with transcript levels of MdMYB1 and was significantly lower in the blushed sport. Thus, we conclude that DNA methylation plays a role in regulating MdMYB1 expression, which in turn regulates anthocyanin biosynthesis and thereby influences the color pattern of the apples.

We established a red callus from the leaves of a red-fleshed apple individual, which was a hybrid offspring of the cross between Malus sieversii f. niedzwetzkyana and Malus domestica cv. 'Fuji'. We analyzed callus growth and anthocyanin biosynthesis/metabolism under different combinations of temperature and light conditions. Incubation in darkness resulted in decreased anthocyanin accumulation, while it promoted callus growth. Exposure to light and low temperature (16°C) induced the expression of MYB10 and bHLH3/33, which are responsible for coordinating the regulation of anthocyanin biosynthesis, as well as the expression of other structural genes. Treatments with light and high temperature (32°C) induced MYB16 expression, which repressed anthocyanin biosynthesis. Additionally, low temperature (16°C) inhibited the expression of MYB111. We analyzed the expression patterns of MYB and bHLH transcription factor genes by quantitative real-time polymerase chain reaction. Our data suggest that light-induced regulation of anthocyanin biosynthesis is primarily caused by altered MYB10 transcript levels, while temperature-induced regulation is the result of changes to the expression of bHLH3/33, MYB16, MYB17, MYB111, and other repressors. In conclusion, we investigated the reciprocal effects of light and temperature on anthocyanin biosynthesis in red-fleshed apple calli. Our findings may provide a theoretical basis for breeding redfleshed apple varieties with high anthocyanin contents.

In plants, the vesicle fusion process plays a vital role in pathogen defence. However, the importance of the vesicle fusion process in apple ring rot has not been studied. Here, we isolated and characterised the apple syntaxin gene MdSYP121. Silencing the MdSYP121 gene in transgenic apple calli increased tolerance to Botryosphaeria dothidea infection; this increased tolerance was correlated with salicylic acid (SA) synthesis-related and signalling-related gene transcription. In contrast, overexpressing MdSYP121 in apple calli resulted in the opposite phenotypes. In addition, the results of RNA sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) assays suggested that MdSYP121 plays an important role in responses to oxidation–reduction reactions. Silencing MdSYP121 in apple calli enhanced the expression levels of reactive oxygen species (ROS)-related genes and the activity of ROS-related enzymes. The enhanced defence response status in MdSYP121-RNAi lines suggests that syntaxins are involved in the defence response to B. dothidea. More importantly, we showed that MdSYP121 forms a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with MdSNAP33, and the complex may participate in regulating resistance to B. dothidea. In conclusion, by regulating the interaction of SA pathway and oxidation–reduction process, MdSYP121 can influence the pathogen infection process in apple.

‘Taishanzaoxia’ fruit rapid softening and dehiscence during ripening stage and this process is very sensitive to endogenous ethylene. In this study, we cloned five ethylene signal transcription factors (ZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1 and ZMdERF2) and one functional gene, ZMdPG1, encoding polygalacturonase that could loose the cell connection which associated with fruit firmness decrease and fruit dehiscence to illustrate the reasons for this specific fruit phenotypic and physiological changes. Expression analysis showed that ZMdERF1 and ZMdEIL2 transcription were more abundant in ‘Taishanzaoxia’ softening fruit and dehiscent fruit and their expression was inhibited by an ethylene inhibitor 1-methylcyclopropene. Therefore, ZMdERF1 and ZMdEIL2 expression were responses to endogenous ethylene and associated with fruit softening and dehiscence. ZMdPG1 expression was induced when fruit softening and dehiscence but this induction can be blocked by 1-MCP, indicating that ZMdPG1 was essential for fruit softening and dehiscence and its expression was mediated by the endogenously occurred ethylene. ZMdPG1 overexpression in Arabidopsis led to silique early dehiscence while suppressing ZMdPG1 expression by antisense ZMdPG1 prevented silique naturally opening. The result also suggested that ZMdPG1 related with the connection between cells that contributed to fruit softening and dehiscence. ZMdERF1 was more closely related with ethylene signaling but it was not directly regulated the ZMdPG1, which might be regulated by the synergic pattern of ethylene transcription factors because of both the ZMdERF1 and ZMdERF2 could interact with ZMdEIL2.

Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from ‘Taishanzaoxia’ apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88%) were assigned to the biological process category, 356 unigenes (80%) were classified in the molecular function category, and 381 unigenes (86%) were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in ‘Taishanzaoxia’. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening.