Understanding the different physiological responses of Malus species under salt stress in the seedling stages will be useful in breeding salt-tolerant dwarfing apple rootstocks. Seedlings of Malus Zumi (Mats.) Rehd. (M. zumi), Malus sieversii (Led.) Roem. (M. sieversii), and Malus baccata (L.) Borkh. (M. baccata) were treated with solution of 0, 0.20%, 0.40%, and 0.60% salinity. Physiological parameters of their leaves and roots were measured at 0 d, 4 d, 8 d and 12 d after salinity treatments. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA), solution protein (SP), and proline (PRO) initially increased and then decreased. The activities and contents of these parameters were higher in the 0.40% and 0.60% NaCl treatments than in the 0.20% treatment and in the 0% control. M. zumi was the most resistant to salt stress, showing the lowest content of MDA in the leaves and roots, which increased slightly under salt stress. M. baccata had the highest increase in both the content and proportion of MDA. High enzyme activity was shown to play an important role in the salt resistance of M. zumi. Moreover, it can be speculated that there are other substances that also play a major role. We found that osmotic regulation played a key role in response to salt stress for M. baccata even though it was sensitive to salt stress. For M. sieversii, both the osmotic regulation and enzymatic antioxidants were observed to play a major role in mitigating salt stress.

We used 256 apple germplasm resources for a thorough examination of the genetic diversity associated with 26 phenotypic traits (i.e., genetic diversity analysis, cluster analysis, correlation analysis, principal component analysis, and membership function). The average coefficient of variation for 12 morphological traits was 66.39% (21.10–201.5%). The coefficient of variation was highest and lowest for the fruit arris and the width of the eye basin, respectively. Additionally, the diversity index ranged from 0.54 to 1.33. Moreover, the coefficient of variation for 14 numerical traits varied from 5.37% to 50%. The titratable acid content had the highest coefficient of variation, with a diversity index ranging from 2.01 to 2.08 (average of 2.045). A cluster analysis categorized 256 germplasms into four groups, among which Group I included germplasms with large fruits and the best comprehensive performance. Of the top 10 principal components revealed by the principal component analysis, principal component 1 was mainly related to fruit size and flavor. The top 10 germplasms were selected on the basis of comprehensive scores using the membership function method. Furthermore, a stepwise regression analysis identified 15 key traits for identifying apple germplasms, including the vegetative growth day, fruit weight, and the firmness of the fruit without skin. These results can serve as the foundation for future analyses of the phenotypic diversity of apple germplasms, while also providing a theoretical basis for screening, characterizing, and further improving excellent apple germplasms.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs that play pivotal roles in various biological processes. However, circRNAs’ roles in different tissues of apple are currently unknown. A total of 6495 unique circRNAs were identified from roots, phloem, leaves, flowers and fruits; 65.99% of them were intergenic circRNAs. Similar to other plants, tissue-specific expression was also observed for apple circRNAs; only 175 (2.69%) circRNAs were prevalently expressed in all five different tissues, while 1256, 1064, 912, 904 and 1080 circRNAs were expressed only in roots, phloem, leaves, flowers and fruit, respectively. The hosting-genes of circRNAs showed significant differences enriched in COG, GO terms or KEGG pathways in five tissues, suggesting the special functions of circRNAs in different tissues. Potential binding interactions between circRNAs and miRNAs were investigated using TargetFinder; 2989 interactions between 647 circRNAs and 192 miRNA were predicated in the present study. It also predicted that Chr00:18744403|18744580-mdm-miR160 might play an important role in the formation of flowers or in regulating the coloration of flowers, Chr10:6857496|6858910–mdm-miR168 might be involved in response to drought stress in roots, and Chr03:1226434|1277176 may absorb mdm-miR482a-3p and play a major role in disease resistance. Two circRNAs were experimentally analyzed by qRT-PCR with divergent primers, the expression levels were consistent with RNA-seq, which indicates that the RNA-seq datasets were reliable.

Apples are a major horticultural crop worldwide. Grafting techniques are widely utilized in apple production to keep the varieties pure. Interstocks are frequently used in Northern China to achieve intensive apple dwarfing cultivation. High-throughput sequencing was used to investigate differentially expressed genes in the phloem tissues of two different xenograft systems, M (‘Gala’/‘Mac 9’/Malus baccata (L.) Borkh.) and B (‘Gala’/Malus baccata (L.) Borkh.). The results showed that dwarfing interstocks could significantly reduce the height and diameters of apple trees while have few effects on the growth of annual branches. The interstocks were found to regulate the expression of genes related to hormone metabolism and tree body control (GH3.9, PIN1, CKI1, ARP1, GA2ox1 and GA20ox1), these effects may attribute the dwarf characters for apple trees with interstocks. Besides, the interstocks reduce photosynthesis-related genes (MADH-ME4 and GAPC), promote carbon (C) metabolism gene expression (AATP1, GDH and PFK3), promote the expression of nitrogen (N)-metabolism-related genes (NRT2.7, NADH and GDH) in rootstocks, and improve the expression of genes related to secondary metabolism in scions (DX5, FPS1, TPS21 and SRG1). We also concluded that the interstocks acquired early blooming traits due to promotion of the expression of flowering genes in the scion (MOF1, FTIP7, AGL12 and AGL24). This study is a valuable resource regarding the molecular mechanisms of dwarf interstocks’ influence on various biological processes and transplantation systems in both scions and rootstocks.

Apple sugar and acid are the most important traits of apple fruit. Bud sport cultivars can provide abundant research materials for functional gene studies in apple. In this study, using bud sport materials with a rather different sugar and acid flavor, i.e., “Jonathan” and “Sweet Jonathan”, we profiled the whole genome variations and transcriptional regulatory network during fruit developmental stages using whole genome sequencing and RNA-sequencing. Variation analysis identified 4,198,955 SNPs, 319,494 InDels, and 32,434 SVs between the two cultivars. In total, 4313 differentially expressed genes among all of the d 44,399 genes expressed were identified between the two cultivars during fruit development, and functional analysis revealed stress response and signal transduction related genes were enriched. Using 24,047 genes with a more variable expression value, we constructed 28 co-expression modules by weighted correlation network analysis. Deciphering of 14 co-expression modules associated with sugar or acid accumulation during fruit development revealed the hub genes associated with sugar and acid metabolism, e.g., MdDSP4, MdINVE, and MdSTP7. Furthermore, exploration of the intra network of the co-expression module indicated the close relationship between sugar and acid metabolism or sugar and stress. Motif-based sequence analysis of the 17 differentially expressed ATP-binding cassette transporter genes and Yeast one-hybrid assay identified and confirmed a transcription factor, MdBPC6, regulating the ATP-binding cassette (ABC) transporter genes and potentially participating in the apple fruit development or stress response. Collectively, all of the results demonstrated the use of parallel bud mutation sequencing and identified hub genes, and inferred regulatory relationships providing new information about apple fruit sugar and acid accumulation or stress response.

The breeding of salt-tolerant rootstock relies heavily on the availability of salt-tolerant Malus germplasm resources. The first step in developing salt-tolerant resources is to learn their molecular and metabolic underpinnings. Hydroponic seedlings of both ZM-4 (salt-tolerant resource) and M9T337 (salt-sensitive rootstock) were treated with a solution of 75 mM salinity. ZM-4’s fresh weight increased, then decreased, and then increased again after being treated with NaCl, whereas M9T337′s fresh weight continued to decrease. The results of transcriptome and metabolome after 0 h (CK) and 24 h of NaCl treatment showed that the leaves of ZM-4 had a higher content of flavonoids (phloretinm, naringenin-7-O-glucoside, kaempferol-3-O-galactoside, epiafzelechin, etc.) and the genes (CHI, CYP, FLS, LAR, and ANR) related to the flavonoid synthesis pathway showed up-regulation, suggesting a high antioxidant capacity. In addition to the high polyphenol content (L-phenylalanine, 5-O-p-coumaroyl quinic acid) and the high related gene expression (4CLL9 and SAT), the roots of ZM-4 exhibited a high osmotic adjustment ability. Under normal growing conditions, the roots of ZM-4 contained a higher content of some amino acids (L-proline, tran-4-hydroxy-L-prolin, L-glutamine, etc.) and sugars (D−fructose 6−phosphate, D−glucose 6−phosphate, etc.), and the genes (GLT1, BAM7, INV1, etc.) related to these two pathways were highly expressed. Furthermore, some amino acids (S-(methyl) glutathione, N-methyl-trans-4-hydroxy-L-proline, etc.) and sugars (D-sucrose, maltotriose, etc.) increased and genes (ALD1, BCAT1, AMY1.1, etc.) related to the pathways showed up-regulation under salt stress. This research provided theoretical support for the application of breeding salt-tolerant rootstocks by elucidating the molecular and metabolic mechanisms of salt tolerance during the early stages of salt treatment for ZM-4.