To compare the effects of different nitrogen enhanced fertilizer on apple growth and to identify suitable nitrogen enhanced fertilizer and application methods for apple cultivation,this study examined the impact of four different types of nitrogen enhanced fertilizer on apple growth,yield,quality,and soil nitrogen supply capacity.The experiment consisted of seven treatments:no nitrogen fertilizer(CK),regular application of ordinary urea by farmers(U),coated urea mixed with ordinary urea 3∶7(CU1),basal application of coated urea and follow-up application of ordinary urea(CU2),loss-control urea(KSU),stabilizing urea(WDU),and humic acid stabilizing urea(FZU).A field experiment was conducted in Qixia City,Shandong Province,to analyze the effects of different fertilization treatments on the growth,yield,and quality of 6-year-old Golden Crown apple trees,as well as nitrogen utilization and soil nitrogen supply capacity.The results demonstrated that the application of diverse synergistic nitrogen fertilizers could markedly enhance spring and autumn growth,elevate leaf SPAD,and enhance apple yield and quality.Among these,the yield of the FZU treatment exhibited a significant increase of 8.89% in comparison to that of the control.The Vc content of the fruits of the CU1 treatment was found to be significantly increased by 66.73% in comparison to the U treatment.Furthermore,the CU1 treatment demonstrated the most pronounced impact on the improvement of growth in both spring and autumn.Nitrogen enhanced fertilizer had been demonstrated to significantly enhance nitrogen accumulation in new shoots and fruit nitrogen accumulation,nitrogen fertilizer agronomic efficiency,nitrogen fertilizer bias productivity and nitrogen utilization in the middle and late stages of apple fertility.Of these,the FZU treatment had been observed to exert the most pronounced effect.The nitrogen accumulation of new shoots in FZU treatment was significantly increased by 37.25%,15.91% and 37.85% compared with that in U treatment at bud differentiation,fruiting and ripening stages,respectively.The CU2 treatment was found to have the most beneficial effect on nitrogen utilization.Nitrogen enhanced fertilizer treatments demonstrated the capacity to significantly enhance the nitrate and ammonium content of soil.Among these treatments,the FZU treatment exhibited the most pronounced effect.The FZU treatment was observed to enhance soil urease activity during the flowering and bud differentiation stages.Additionally,the WDU and FZU treatments were found to significantly reduce the nitrification of soil ammonium and nitrogen loss.The {\mathrm{NO}}_3^{-}-N of the FZU treatment was concentrated at a depth of 20—60 cm soil,which was consistent with the distribution of apple roots in the soil and reduced the risk of {\mathrm{NO}}_3^{-}-N leaching.A comprehensive analysis demonstrated that the FZU treatment exhibited clear advantages in promoting apple growth,increasing apple yield and quality,and enhancing soil nutrient supply capacity.Consequently,the FZU treatment was identified as the optimal treatment.

To clarify the optimal harvesting period and storage temperature for the Venus apple fruit used for storage.The effects of three harvesting periods combined with three precise storage temperatures on fruit quality,anti-browning and preservation of Venus gold apple during 20 ℃ shelf life after cold storage were studied. The results showed that harvesting period Ⅰ(relatively early harvest)combined with storage at-1.5 ℃ (relatively low temperature)could maintain high fruit firmness,but the soluble solid content (SSC)of fruit was low,the taste and flavor of fruit were poor,and different degrees of browning of skin,heart and flesh appeared during shelf life. When harvested at period Ⅲ (relatively late),stored at 3 ℃ (relatively high temperature)until January 20,the fruit had high SSC,good taste and flavor. However,when stored at 20 ℃ for 24 h and stored on the shelf for 7 days,the fruit flesh was spongy and severely browning. The results showed that compared with-1.5 and 3.0 ℃,0 ℃ maintained higher firmness,SSC,titratable acid (TA)and ascorbic acid contents,and better maintained fruit chlorophyll fluorescence activity,and delayed fruit senescence. For the same storage temperature,the contents of TA and ascorbic acid in the fruits of harvest period Ⅱ were higher than those of harvest period Ⅰ and Ⅲ. The comprehensive analysis showed that the harvest period Ⅱ and Ⅲ combined with 0,3 ℃ could maintain the good merchantability and intrinsic quality of Venus gold apple stored in the medium and short term. For the Venus gold apple with medium and long-term storage,the harvest period Ⅱ combined with 0 ℃ accurate temperature storage could not only effectively inhibit fruit browning,slow fruit softening and senescence,but also maintain good flavor and quality of fruit during shelf life.

In order to investigate the effect of 1-methylcyclopropene(1-MCP)on the physiology,storage quality and electronic nose characteristic of Starking delicious apple.Fruits were treated with 1-MCP and then stored at ambient temperature(20±1)℃.Respiration rate,ethylene production rate,fruit intrinsic quality,color difference and the electronic nose response was analyzed during storage.The results showed that the postharvest respiration rate and ethylene release rate of Starking delicious apple increased at first,then decreased after peaking at 10 d and 15 d,respectively.At the same time,fruit firmness and titratable acid(TA)content decreased,soluble solid content(SSC)increased then decreased.Fruit chromaticity varied,a^*,b^*,C^* and ΔE^* values increased.1-MCP treatment inhibited fruit respiration rate and ethylene release rate,delayed the decline of fruit firmness,SSC and TA content,inhibited increasing of a^*,b^*,C^* and ΔE^*.1-MCP treatment significantly reduced the production of sulfides and terpene compounds(W1W),nitrogen oxides(W5S),organic sulfides and aromatic compounds(W2W),methyl aromatic compounds(W1S),alcohols,aldehydes and ketones aromatic compounds(W2S).Linear discriminant analysis(LDA)combined with electronic nose could distinguish control and 1-MCP fruit at different storage periods.Loading analysis showed that W1W,W5S,W2W,W1S and W2S sensors played key roles in differentiating control and 1-MCP treatment fruit during storage.During ambient temperature storage,1-MCP treatment delayed fruit softening and kept a lower solid-acid ratio,effectively maintained fruit flavor and color of Starking delicious apple.However,1-MCP decreased the response value of sensitive sensors in electronic nose,and inhibited the production of fruit volatile substances.According to correlation analysis,electronic nose sensor response values were significantly correlated with the internal quality and color difference,which may provide a basis for the rapid and nondestructive detection of Starking delicious apple.

To study the effect of soil water and fertilizer management mode on orchard in dryland area,and to provide support and basis for optimizing water and fertilizer management and realizing the best matching of water and fertilizer in apple orchard.Put into use the three main modes that include integrated water-fertilizer and plastic mulching(T1),ridge film rainwater harvesting + straw mulching in ditch(T2),use fertilizer gun to integrate the fertilizer and water(T3),and conventional tillage fertilization(T4)were used as the comparison where in apple orchards of Gansu Province Longdong area.Measure and analysis the contents that the soil organic matter and mineral nutrition,chlorophyll and nutrient content in leaves and fruit quality in apple orchard.In different water and fertilizer management modes of apple orchards in dryland area.The contents of soil organic matter,total nitrogen,alkali-hydrolyzable nitrogen,total phosphorus,available phosphorus and available potassium showed a decreasing trend with the increase of soil depth from 0 to 60 cm,while the decreasing range gradually decreased.The contents of organic matter and mineral nutrients in shallow soil(0-20 cm)were significantly higher than those in middle and deep soil(20-60 cm). The content of soil total K was higher in different modes and layers.The results showed that the soil nutrients were enriched in shallow layer and the soil potassium was rich.In young fruit stage,fruit expansion stage and fruit maturity stage of apple,the decrease rates of the soil total nitrogen,total phosphorus and total potassium were significantly lower than those of available nutrient that the alkali-hydrolyzed nitrogen,available phosphorus and available potassium.The absorption and utilization efficiency of the soil mineral nutrients were different with different water and fertilizer patterns,while the nutrient consumption degree was also different,especially in shallow soil. In 0-20 cm soil depth,the content of soil organic matter in T4 treatment which the young fruit stage and fruit expansion stage was significantly higher than that in other treatments,while it was significantly lower at the fruit maturity stage.This showed that the clear tillage management(CK)had a low capacity of soil fertilizer conservation and nutrient sustainable supply.Under different water and fertilizer management modes,the chlorophyll contents which the young fruit stage and fruit expansion stage varied greatly,and the chlorophyll a,b and total chlorophyll content of T3 were significantly higher than those at T1 and T4 at young fruit stage. However, there was no significant difference with T2.There was no significant difference that the mineral nutrient content in leaves between different modes and different periods.The contents of total nitrogen,phosphorus on a downward trend with the growth and development of apple.While the potassium content in leaves showed an upward-downward trend.The contents of the nitrogen,phosphorus and potassium in leaves were the lowest at fruit maturity stage.The soluble sugar,total sugar and solid acid ratio of T2 were significantly higher than those of other treatments. The content of VC in T2 was significantly higher than T4,but not significantly different from T1 and T3.The application of "ridge film rainwater harvesting + straw mulching in ditch" technology can significantly improve water and fertilizer utilization efficiency and fruit quality in apple orchard in dry farming area.

In order to explore the effect of RNA extraction and virus detection of different parts of apple organs,apple plants infected by three viruses were used as experimental materials.Adsorption column method was used to extract total RNA from flowers(pistil,anther,filament,petals,calyx,receptacle,and flower stalk),leaves(petiole,mesophyll,and veins),fruits(fruit stalk,peel,flesh,score,episperm,and endosperm)and branches(phloem),a total of 17 tissues of four organs.The extraction effects of different sampling parts were compared,the detection effects of three viruses in different parts of organs were analyzed by regular RT-PCR,and the relative content of viruses in different parts of organs were analyzed by Real-time fluorescence quantitative PCR.The results showed that the effects of RNA extraction from different organs of apple were different,with the highest concentration in leaves,followed by flowers and phloem of branches,and the lowest concentration in fruits.Anther,filament and petiole were best extracted from flowers and endosperm and petiole were best extracted from fruits and leaves,respectively.Three apple viruses could be detected in 16 tissues except endosperm.Quantitative analysis showed that the relative content of the three apple viruses was the highest in flowers.Furthermore,the relative contents of viruses in pistil,episperm and petiole were correspondingly higher than that in other tissue parts of flower,fruit and leaf,respectively.In addition,the content of the three viruses in different tissue parts of fruits and leaves showed similar trends,but there were certain differences in the distribution of different tissue parts of flowers.

In order to determine the effects of V.villosa Roth cultivation in apple orchard on soil microbial community structure,the high-throughput sequencing technology was used to analyze the microbial structure of V.villosa Roth cultivation soil in seven areas of Shandong Province and different phenological period of the same orchard,taking natural grass treatment as comparison.The correlation between microbial community structure and physicochemical properties of grass cultivation soil was analyzed.Results showed that,in seven sampled sites of Shandong Province,the relative abundances of Metarhizium in V.villosa Roth cultivation treatment were all higher than those in the control,and the difference was biggest in Dongying Area.The relative abundances of Fusariumin V.villosa Roth cultivation treatment of six sites were lower than those of the control,except for Zoucheng.The relative abundances of Gibberella in V.villosa Roth cultivation treatment of six sites were lower than those in the control,except for Rongcheng.In Zoucheng and Longkou,the relative abundances of Alternaria were lower than those of the control.Analysis of microbial community structure in different apple phenological stages of V.villosa Roth cultivation in the same orchard showed that,the relative abundance of Aspergillus in flowering stage soil was much lower than that of natural grass.The relative abundances of Fusarium and Aspergillus in fruit setting stage in V.villosa Roth cultivation treatment were lower than those of natural grass.The relative abundance of Bacillus in V.villosa Roth treatment was higher in fruit expansion stage.The relative abundance of Bradyrhizobium was higher in mature stage,while Gibberella,Cladosporium and Bipolaris were much lower than those of natural grass.Effects of V.villosa Roth cultivation in apple orchard on soil microbial community structure differed in different region and phenological stage.However,the relative abundance of beneficial bacteria in soil was increased,while that of pathogenic fungi was decreased,which was helpful to improve orchard soil micro-ecological environment and promote the healthy cultivation of fruit trees.

Discuss the difference and change rule of texture characteristics of Huahong and Huayue apples after different shelf temperature combined with 1-MCP treatment during the shelf life,in order to determine the appropriate shelf temperature and time for apples.Using Huahong and Huayue as the test materials,the physical property analyzer texture multi-faceted analysis(TPA)and puncture test were used to determine the pulp elasticity,gummy stickiness, chewability, pulp hardness, rupture work, rupture force, rupture displacement, yield work, yield force and yield displacement.Factor analysis was applied to these indicators,and the original indicators were reduced in dimensionality,so that a comprehensive index was used to evaluate 1-MCP(1.0 μL/L)treatment,it was placed at shelf temperatures of 5,10,15,20 ℃ to maintain the texture properties of Huahong and Huayue apples.The shelf life of Huahong and Huayue fruit texture characters could be prolonged by 1-MCP treatment,especially for Huahong fruit.Under the same treatment temperature,the fruit decline of the two varieties treated with 1-MCP was significantly less than that of the untreated group,which could be reduced by about 10%.Correlation analysis showed that there was a very significant positive correlation between the texture indexes of apple fruit,but there were some differences in the degree of tightness.After factor analysis and based on the principle that the eigenvalue was greater than 1,two main factors were extracted,and the cumulative variance contribution rate was 91.194%.According to the representative indexes of each main factor,they were named puncture factor F₁ and TPA factor F₂,and the variance contribution rates were 79.870% and 11.324%,respectively.According to the score of the main factor,the most suitable shelf temperature of untreated Huahong fruit was 5 ℃.The temperature of the treated fruit was 15 ℃.The optimum shelf temperature of Huayue fruit without treatment was 10 ℃,and the suitable shelf temperature of 1-MCP treated fruit was 5,10 ℃.The CK group of Huahong fruit could be stored for 8 d at 5 ℃,and the shelf temperature of the treatment group could be extended to 16 d at 15 ℃;the CK group and the treatment group of Huayue fruit could be stored at 10 ℃,could be stored for 16 d.

In order to clarify the mechanism of pyroligneous acids improving replantation soil,the effect of pyroligneous acids treatment on plant growth was studied by field experiment using apple rootstock-Malus micromalus as experimental material.The main physical and chemical properties and enzyme activities of soil in July and October were analyzed based on Illumina high-throughput sequencing to analyze the changes of microbial diversity in rhizosphere soil.The results showed that compared with the control,the annual increase of plant height,ground diameter and leaf area of Malus angustifolia seedlings were significantly increased after 100-fold pyroligneous acids irrigation.Pyroligneous acids root irrigation treatment significantly increased the contents of main nutrients and enzyme activities in the soil of replanting disease.The contents of soil organic matter,total nitrogen,alkali hydrolyzable nitrogen,available phosphorus and available potassium were 1.31,1.38,1.20,1.60 and 1.65 times of CK1,respectively in July,and 1.12,1.03,1.58,1.40 and 1.25 times of CK2 in October,respectively.Sucrase and urease activities increased significantly in July and October.Pyroligneous acids increased the microbial diversity of rhizosphere soil in summer and autumn.Under CK and pyroligneous acids treatments in July and October,the top five dominant gates of rhizosphere bacteria were Proteobacteria,Acidsobacteria,Gemmatimonadetes,Chloroflexi and Rokubacteria;at the genus level,they were mainly uncultured_bacterium_c_Subgroup_6, uncultured_bacterium_f_Gemmatimonadaceae, uncultured_bacterium_o_Rokubacteriales, RB41 and MND1. The top five dominant gates of rhizosphere fungi were Ascomycota,Basidiomycota, Mortierellomycota,Chytridiomycota and Glomeromycota.At the genus level,they were mainly Cladosporium,Mortierella,Ilyonectria,Guehomyces and Fusarium.According to the correlation network map,the beneficial bacteria RB41 and uncultured_bacterium_f_Gemmatimonadaceae in the rhizosphere and the pathogenic fungi Fusarium and Ilyonectria were negatively correlated with other populations.Pyroligneous acids root irrigation can increase soil nutrients and enzyme activity,increase soil microbial diversity,improve soil microbial community structure,promote plant growth,enhance plant resistance and reduce the harm of replanting disease.

Gene STAYGREEN(SGR)plays an important role in the process of plant chlorophyll degradation and metabolism.To explore the biological function of gene SGR2,MdSGR2 gene sequence was cloned from the peel of Granny Smith by RT-PCR.The homology,physicochemical properties,protein structure and cis-acting elements of the promoter were predicted and analyzed by bioinformatics software.The plant overexpression vector was constructed by double enzyme digestion.The results showed that the complete open reading frame(ORF)cDNA of gene MdSGR2 was 840 bp.Encoding 279 amino acids in total,it belonged to the Staygreen superfamily.Physicochemical analysis showed that the total molecular weight of the protein was 31.27 ku,and the isoelectric point pI was 8.52,indicating that the protein was an unstable hydrophilic protein.Homology analysis showed that MdSGR2 encoded amino acid sequence was of the highest homology with Pyrus ussuriensis,reaching 84.12%.The protein structure analysis showed that the secondary structure and tertiary structure of MdSGR2 was determined structurally to be 40.55% α-helix and 44.44% random coil.Promoter analysis showed that the promoter region of the gene contained cryogenic cis-acting elements,photo-responsive elements and induced cis-acting elements such as abscisic acid,salicylic acid and methyl jasmonate response elements.These results suggest that gene MdSGR2 may be regulated by many factors such as environment and hormones.The plant overexpression vector pCAMBIA2301-MdSGR2 was successfully constructed.

To explore the effects of spraying CaCl₂ at different concentrations on physiological characteristics of apple seedlings under salt stress, two-year-old T337 seedlings were used as test materials, and the pot experiment was used to set 100 mmol/L NaCl(T1) and 100 mmol/L NaCl + four different concentrations of CaCl₂ (5.0(T2), 7.5(T3), 10.0(T4), 12.5 mmol/L(T5)) treatment, watering with deionized water was used as the control(CK).Determine the chlorophyll content, photosynthetic and fluorescence parameters, antioxidant enzyme activity and osmotic adjustment substance content of each treatment group, and carry out correlation and principal component analysis. The results showed that with prolonged stress time, the catalase(CAT), peroxidase(POD), superoxide dismutase(SOD) of T337 leaves first increased and then decreased, malondialdehyde(MDA), intercellular CO₂ concentration(Ci), proline(Pro) and relative conductivity(REC) showed a rising trend, initial fluorescence(F0), PSⅡ maximum light energy conversion rate(Fv/Fm) stomatal conductance(Gs) and the transpiration rate(Tr) showed a downward trend;compared with CK and T1, the variation amplitudes of T337 leaves after treatment with exogenous CaCl₂ were all reduced, and there was a significant concentration effect. Among them, the relative chlorophyll content(SPAD), Pn, Tr, POD, F0 and SOD decreased the least under T4(10mmol/L) treatment, which was significantly higher than other treatment groups;MDA, REC and Ci increased the least, which was significantly higher than CK and lower than others processing group. Correlation analysis of 14 indicators showed that Pn was positively correlated with CAT, Tr, Gs, F0, Fv/Fm, ETR, SPAD, SOD and POD, and negatively correlated with Pro, MDA, Ci, and REC. Principal component analysis was performed on 14 related indicators after salt stress treatment, and 2 principal components with eigenvalues greater than 1 were extracted, and their eigenvalues were 11.190, 2.295, respectively;The first and second principal component variance contribution rates were 79.930%, 16.394%, respectively, the cumulative variance contribution rate reached 96.324%, which meets the analysis requirements. According to the ranking of principal component scores, the mitigation ability of exogenous CaCl₂ to T337 salt stress was T4>T3>T2>T5 from high to low. Therefore, 10 mmol/L CaCl₂ could improve the photosynthetic fluorescence characteristics of T337 rootstock leaves under salt stress, improve the antioxidant enzyme system and osmotic adjustment ability, and enhance the stability and permeability of biofilms to alleviate salt stress.

To understand the effects of SH1 and Y-1 dwarfing interstocks on the growth and leaf photosynthesis of Fuji apple saplings, and provide theoretical basis for the selection and utilization of dwarfing interstocks in production, the Fuji Nagafu 2 apple trees grafted on SH1 and Y-1 interstocks were used to study the effects of two kinds of interstocks on the growth, branch composition and leaf photosynthetic indexes of young Fuji trees. The results showed that the annual growth trend of Fuji on SH1 and Y-1 interstocks was basically the same, but the growth of spring and autumn shoots and the length of new shoots of Y-1 were significantly lower than that of SH1, and the ratio of short branches was significantly higher than that of SH1, and the fruit color index, hardness, soluble solids and solid acid ratio significantly increased. The diurnal and annual dynamic trends of net photosynthetic rate(Pn) of Fuji Nagafu 2 were different between SH1 and Y-1 interstocks. The Pn of leaf in SH1 in the early growth period was higher than in Y-1, but the Pn of leaf in Y-1 in the later growth period was significantly higher than in SH1, analysis of light response parameters and chlorophyll fluorescence parameters showed that the light saturation point(LSP) of Y-1 in the early growth period was lower than SH1, the compensation point(LCP) was higher than SH1, non photochemical quenching coefficient (NPQ) was significantly higher than SH1, and the primary light energy conversion efficiency (Fv/Fm), photosynthetic electron transfer quantum efficiency (ΦPSⅡ) and apparent electron transfer rate (ETR) of Y-1 were significantly lower than that of SH1 in the early growth stage, and the LSP, PSⅡ, Etr, the chlorophyll content and net photosynthetic rate of Y-1 were significantly higher than those of SH1 at the later growth stage. Different intertstocks had great effects on the growth and leaf photosynthetic characteristics of grafted varieties. Some characteristics of Fuji Nagafu 2 grafted onto Y-1 were better than that of SH1, such as dwarfing, early flowering, easy shaping. In the early growth period, the leaf maturity of Fuji Nagafu 2 in Y-1 lags behind in SH1, so the photosynthetic efficiency of the leaves was low. In the late growth period, the photosynthetic pigment accumulation and Fv/Fm of leaves in Y-1 were higher or significantly higher than that of SH1, and it could effectively control the growth of autumn shoots, which was beneficial to the accumulation of nutrients and the formation of flower buds.

To explore the botanical agents to prevent and control apple ring spot disease,the inhibitory of the four common of Allium plant extracts,on the hypha growth of Botryosphaeria berengriana were tested.On this basis,the metabolites of the four Allium plants,Chinese leek,garlic,onions and welsh onion,were analyzed by metabolomics methods,and the suppressive effects of the main components of the four Allium plants on the mycelial growth of B.berengriana and the incidence of the apple ring disease on the apple fruits were also determined.The results showed that in the whole experiment period the inhibition of four Allium plant extracts(1×)on the growth of mycelia of B.berengriana was as high as 100%.78 metabolites were detected in the four Allium plants.Among them,the three main components,pyroglutamic acid,phosphoric acid and citric acid,inhibted the growth of mycelia of B.berengriana by 29.10% to 100.00%,and inhibited the incidence of ring disease on apple fruit caused by B.berengriana by 44.38% to 91.45%.The results of this study laid a theoretical foundation for further understanding and developing the antimicrobial components in Allium,and provided a potential way for the prevention and treatment of apple ring disease.

This study aims to explore the genetic diversity of Malus at the level of chloroplast gene and the phylogenetic relationship of haplotypes of different species in different regions, so as to provide a basis for its origin and evolution process, protection and utilization. Based on chloroplast intergenic regions trnH-psbA, trnS-trnG spacer+intron, trnT-5'trnL and 5'trnL-trnF, the sequences of 722 accessions of Malus from twelve provinces were analyzed. After four regions merged, the length of four merge regions of chloroplast DNA was 4 120 bp, and the haplotypes of chloroplast DNA fragments were 100, the nucleotide diversity and haplotype diversity of the merged regions were 0.009 52 and 0.879 respectively. The region with highest nucleotide diversity and haplotype diversity was trnH-psbA (Hd=0.808,Pi=0.034 09). Tajima's test showed that the value of four regions merged was -1.503 16, and it was not significant at the P>0.10 level and followed the neutral theory of molecular evolution. AMOVA showed that genetic variation mainly existed among populations and within intraspecific populations.The results of haplotype distribution and network analysis showed that the missing haplotypes in the center of adjacent networks are closed into a ring, and different species of Malus have different evolutionary routes. There were complex relationships among species and germplasms of same species in the process of origin and evolution in different regions. Relatively old haplotypes H_6 and H_15 experienced population expansion, and there were many derived branches. The genetic diversity of Malus at chloroplast gene level was high, and the genetic variation mainly occurs among populations and within intraspecific populations. The genetic evolution of chloroplast genes was dominated by mutation or random drift within populations. The main factors of genetic variation of Malus were intraspecific differences and intrapopulation differences caused by geographical isolation.

The fruit softening of precocious apple cultivar is an important factor for promotion of apple production. It is important to elucidate the mechanism of fruit softening to prolong the fruit storage period and maintain the fruit quality. In this experiment, Mato 1 and XU2-5 were used as the materials to analyze the changes of fruit hardness, ethylene release rate, related enzyme activity and related gene expression during fruit softening. The main results showed that, along with the ripening of fruits, the fruit hardness decreased and the ethylene release rate increased. The ethylene release rate of Mato 1 was significantly higher than that of XU2-5. The relative expression pattern of MdACS6 gene was basically the same in the two varieties, of which the relative expression level was low in early time, and gradually increased after that. With the ripening of fruits, the relative expression amount of MdPG1 gene and the change of enzyme activity in Mato 1 were consistent, which reached the highest at 100 d after flowering, while XU2-5 showed the consistency between relative expression level of MdPG1 gene and the change of enzyme activity in 80-90 d after flowering, of which the relative expression of MdPG1 was the highest at 90 d after flowering, and the enzyme activity was the highest at 100 d after flowering. In 80-85 d after flowering, the relative expression of MdPE gene and PE enzyme activity in the two varieties (lines) presented an increasing trend, and the enzyme activity reached to a peak value. In 90-100 d after flowering, the relative expression of MdPE gene decreased in the two varieties (lines), while the PE enzyme activity increased gradually. The LOX enzyme activity in the two varieties (lines) decreased first and then increased, and the lowest point appeared at 85 d after flowering. The relative expression of MdLOX gene in XU2-5 increased first and then decreased, and the peak appeared at 85 d after flowering, while the relative expression of MdLOX gene in Mato 1 increased gradually, and the highest value appeared at 100 d after flowering.In summary,taking measures to regulate gene expression may be effective in regulating fruit softening.

In order to effectively discover the apple fruit russet gene, based on SLAF-seq technology, the genetic map construction and QTL analysis for fruit russet gene were conducted using a population derived from a cross between Miyazaki Spur and Sakata Tsugaru. The results showed that 522 122 315 reads (110.32 Gb) of the sequencing data from seedlings was obtained, in which the average Q30 sequencing was 95.07%, and the average GC content and its parents was 40.11%. Then, 20 440 SLAF markers and 7 309 729 SNPs were developed. Based on these markers, seventeen linkage groups were constructed, of which 4 075 markers presented in the genetic map, and marker integrity was 99.92%. The total diagram spacing was 2 235.23 cM, and the average diagram spacing was 0.55 cM. A total of 9 QTLs related to fruit russet were detected, which were distributed on Chr3, Chr9, Chr11, and Chr15 chromosomes, with a marker distance of 0-4.9 cM and a contribution rate of 18.0%-85.5%. Among them, Qru-9, Qru-15 and Qru-3, located on Chromosome 9, 3, and 15, respectively, might be novel QTLs for fruit russeting of apple. A genetic map based on SLAF-seq method was constructed in apple progeny Miyazaki Spur crossing Sakata Tsugaru and nine QTLs related to apple russeting were obtained. These results provided the important information for further exploration of russeting QTL in apple.

As the basic component of plant microtubules,tubulins are of great significance to the formation of cytoskeleton. To investigate the effect of one member of the β-tubulin gene family with the transcript number of MDP0000749824.1 on the stem growth,the coding sequence of this gene was cloned from the apple young shoot tips of standard apple variety Yanfu 3 and the columnar variety Telemon,respectively. This sequence was 1 332 bp in length which encoded a peptide with 443 amino acids. The results of DNAMAN alignment showed that the coding sequence of the gene was identical between Yanfu 3 and Telemon.Using DNA of Yanfu 3 and Telemon as templates,1 661 bp nucleotide sequences was obtained by PCR amplification with promoter specific primers. There were six bases differences between them. Promoter sequence analysis revealed that some hormone responsive elements of gibberellin,abscisic acid,methyl jasmonate and salicylic acid existed in it. Cam35S-MDP0000749824-GFP recombinant vector was constructed. Subcellular localization analysis with tobacco leaves indicated that the protein encoded by MDP0000749824.1 was on the cell membrane and microtubules. qRT-PCR detected that the transcript level of this gene in apical stems of columnar apple Telemon was significantly lower than that of standard apple Yanfu 3. It was speculated that the gene could affect the stem elongation be cause its expression level was regulated by hormones.

In order to select antifreeze negative gene in apple, lay a foundation for cultivating new varieties of antifreeze apples,4 open reading frame CBF genes were cloned from apple by the method of information biology,named as MdCBFL1-MdCBFL4. These genes(MdCBFL1-MdCBFL4) were shown to encode proteins that share a high similarity with plant cold stress-related CBF proteins,which contained a AP2 domain and two classic sequences that conserved PKRPAGRTKFRETRHP nuclear location signal area and DSAWR conservative sequence. Phylogenetic analysis revealed that the isolated apple genes could be classified into the Arabidopsis A-1 subgroup, it was speculated that it was similar to the function of Arabidopsis A-1 transcription factor, and the expression of MdCBFL1-MdCBFL4 proteins was induced by stress of drought, low temperature and salt stress.The expression of MdCBFL1-MdCBFL4 gene in apple was detected by semi-quantitative RT-PCR in high salt(200 mmol/L NaCl),drought and low temperature.The results showed that MdCBFL1 gene was response to the low temperature,drought and salt stress. MdCBFL2-MdCBFL4 gene was induced by low temperature and high salt. This provides useful clues to further elucidate the function and mechanism of apple MdCBF gene in stress response.

To explore the sequence characteristics of β-glucosidase gene VmGluI from Valsa mali var. mali (Vmm)and analyze its expression pattern in the process of pathogen infection,the cDNA sequence of VmGluI was amplified from the strong pathogenic strain LXS080601 by using RT-PCR technology.The phylogenetic relationship,physicochemical properties and secondary structure of the protein coded by VmGluI were analyzed by bioinformatics software.The expression levels of VmGluI in apple tiwgs(Malus domestica Borkh.Fuji) inoculated by Vmm were analyzed by Real-time quantitative PCR.The results showed that the cDNA sequence of VmGluI from Vmm was obtained and its GenBank accession number was KY646110.The open reading frame of 1 689 bp encoded 562 amino acid residues with a predicted molecular weight of 62.7 kDa.VmGluI shared characteristic conserved domains of glycosyl hydrolase family 1. α -helices and random coils were its main elements of secondary structure of VmGluI,and β-turns were less.Multiple sequence alignment and phylogenetic tree analysis showed that the encoded protein had the closest genetic relationship with β-glucosidase genes from Vmm (KUI68239.1) and V.mali var. pyri (KUI56905.1),and the similarities of amino acid sequences were 98.7% and 87.8%,respectively.However,the relationship between VmGluI and other β-glucosidase genes from non-pathogenic fungi was far away.The Real-time quantitative PCR results showed that VmGluI was significantly up-regulated in Fuji tiwgs after inoculation by Vmm.Thus,the authors speculated that VmGluI could paly an important role in the pathogenic process of Vmm.The expression level of VmGluI began to increase significantly in detached twigs at 48 h and the highest expression level was 6.3 times higher than that in the control at 72 h.However,the gene expression level increased significantly at 12 h in living twigs,which was 11.2 times higher than that in the control at 96 h.Although there was a low level of gene expression at 48 h,the amount of gene expression was 16.8 times higher than that in the control at 96 h.The results indicated that expression levels and the time course changes of VmGluI were obvious difference in detached and living twigs inoculated with Vmm.

To extensively understand the sequence feature and evolution of heat shock transcription factors (Hsf)in the genome of apple,fifty MdHsf genes were identified using bioinformatics methods at the whole-genome level of apple,and a series of analysis including sequence characterization,phylogenetic relationship,gene expression and selective pressure of MdHsf genes were further performed.Phylogenetic relationship and sequence characterization analysis showed that,like the model species Arabidopsis and rice,50 MdHsf genes were divided into three subfamilies A,B and C.Additionally,at least two genes were found in the same end clades in the phylogenetic tree,indicating that the lineage-specific amplification had happened during evolutionary processes of apple Hsf gene family.Although the intron numbers and sizes of MdHsf genes were relatively divergent,the conserved motifs and domains of MdHsf proteins were highly conserved because of functional constraints.Based on EST data,72% of the 50 genes (except 14 genes such as MdHsfA2a and MdHsfA3a/b/c)had transcription activities.Selective pressure signatures demonstrated that no positive selection site was identified in the cleaned codon alignments for 36 MdHsf genes based on site-specific model,suggesting that this protein family was controlled by purifying selection.However,branch-site model had identified a total of five positively selected sites in the d and e clade of the phylogenetic tree,i.e.28R,30L,35D,51M and 67V.28R and 30L were included in the Hsf domains,while 35D,51M and 67V were not mapped on the region of Hsf domains,suggesting that purifying selection was the main evolutionary dynamics of functional conservation Hsf domains except for 28R and 30L.In conclusion,various Hsfs existed in apple genome,and the conserved motifs and functional domains were conserved.The majority of them had transcription activity,and the evolution of this family was dominated by purifying selection.

In order to identify the function of MdCaM gene in apple under different abiotic stresses. MdCaM gene was cloned by RT-PCR from apple, and phylogenetic tree was clustered based on amino acid of it. Also, the relative expression levels of MdCaM gene was analyzed by Real-time quantitative PCR (qPCR) method under wound, low-oxygen, high-temperature stress during apple fruit storage. The results showed that Calmodulin gene had high homology in different species, and MdCaM gene had a closer relationship with Arabidopsis thaliana CaM₁. qPCR analysis showed that the transcription level of MdCaM was up-regulated in the early stage, then returned to the normal level after wound and low-oxygen (0 O₂ and 3%O₂) stress, the difference was wound stress peak at 6 h, while low-oxygen (0 O₂) stress peak at 1 h. Also, MdCaM was up-regulated under high-temperature (20℃ and 40℃) stress compared to control,and the expression level was the highest at 12 h under stress,which indicated that MdCaM gene might be involved in the whole process of high-temperature stress-responsive. These results suggested that MdCaM might function as an stress-responsive gene under wound,high-temperature,low-oxygen stresses during apple storage. This research provided a theoretical basis for further study of the function and molecular regulation mechanism of MdCaM.

In order to study the molecular characterization and expression patterns of GA20 oxidase gene in apple dwarfing rootstocks,RT-PCR method was used to clone GA20 oxidase gene (GA20ox1) from cDNA of the apple dwarfing rootstocks No.2 and No.36, and the genes and the amino acid sequences, as well as gene expression on rootstocks and the grafted varieties at various times, was analysed, respectively. Results showed that cDNA of GA20ox1 gene coded a 1 179 bp sequence, and derived a protein with 393 amino acids including termination codon, which the predicted protein molecular weight was 44.3 kDa and theoretical isoelectric point was 5.89.The protein contained conserved domain characteristics of GA20ox1 gene family,and phylogenetic analysis showed that the protein sequence homology with SH40 was at 96.9%. Real-time fluorescent quantitative PCR analysis revealed that the GA20ox1 expression of No.36 rootstock was significantly lower than the control (Malus robusta Rehd.) in June and higher than that in July to August, the expression of the varieties grafted on it was significantly lower than control in July. From July to August, GA20ox1 gene expression levels of No.2 rootstock and the grafted varieties were significantly lower than control.

In order to develop novel disease-resistant transgenic plant materials against ASPV,a virus resistant RNAi plant expression vector was constructed by the method of homologous recombination.With the Malus rootstock of M26,genetic transformation was conducted and positive transgenic plantlets were obtained successfully.Firstly,a conserved segment of coat protein gene of ASPV (489 bp) was cloned by PCR from cDNA of the virus-infective leaves of Royal gala.Then the fragment was inserted into the vector pENTR/SD/D by TOPO cloning technology to form the intermediate vector of pEN-ASPV.After this fragment was replaced and connected into the destination vector pHellsgate12 by LR reaction,the RNAi plant expression vector Phe-ASPV was obtained.And the engineering bacteria EH-ASPV for plant transformation was obtained after the constructed RNAi vector was transformed into Agrobacterium strain EHA105 by alternate freezing and thawing method.By the Agrobaterium mediated method,transformation was conducted with the leaf discs of the Malus rootstock of M26.After culture and subculture for antibiotics screening,bacteria elimination and rooting,compact kanamycin resistant plantlets formed from buds were obtained.After total RNA extracted from the well rooted plantlets,RT-PCR test was conducted and the result showed that the gene fragment of 489 bp carried on the RNAi structure had been transformed into the material of M26,which was expressed in plants on the level of RNA.Through this study,novel transgenic materials were obtained successfully,which laid foundation for disease-resistance evaluation by pathogen inoculation and virus-resistant molecular breeding in the near future.

In this study,we aimed to identify the differences of tree morphology,accumulation and distribution characteristics of mineral nutrient in root system of Gala apple young tree with different dwarfing interstocks.The results were expected to reveal the relations between the tree dwarfing ability and accumulation,distribution of mineral nutrient in root system.Gala apple saplings (in pots) grafted onto dwarfing interstocks (GM256,Liaozhen No.2,SH1,SH6,SH38,and SH40) with uniform growth vigour were used as the experimental material.After spring shoot growth stopped,plant height and cross-sectional area were measured.The root morphology indexes were analyzed by WINRhizo analysis software.For the measurement of mineral nutrient contents,healthy plants at the same growth stage were removed from the pots,and were dissected and separated into different diameter root system.The contents and accumulations of mineral nutrient were determined.The results showed that plant height and cross-sectional area of Gala apple trees grafted onto dwarfing interstocks were obviously lower than that of the control.The degree of dwarfing of the interstocks followed the order of SH38 > SH1 > SH6 > GM256 > SH40 > Liaozhen No.2.Differences in mineral nutrient contents in different diameter root system of Gala apple young tree among different dwarfing interstocks were obviously different.The mineral nutrient contents in<0.5 mm diameter root system were highest,and the mineral nutrient contents decreased with the increase of root system diameter.The contents of K and Mg of Gala apple root system for SH38 were highest.Root system for SH1 interstocks had the highest Cu,Fe and Zn contents.The accumulations of P,Ca,Fe in all parts of GM256 interstocks root system and that of K,Mg and Zn in>3 mm diameter root system were higher than control and other treatments.The accumulations of K,Mg and Zn in<3 mm diameter root system for Liaozhen No.2 were highest.The mineral nutrient accumulations of SH38,SH1 and SH6 with weak growth vigour were lower than that of Liaozhen No.2,GM256 and CK with strong growth vigour.The optimal interstocks for Gala apple with better dwarfing effect were SH38,SH1 and SH6.In practical application,site condition,soil supplying nutrient capability and nutrient requirement characteristics of different stock-scion combinations should be fully considered,which for realizing the high quality and high efficiency production of apple.

To obtain the transcription factor related to stress conditions in apple rootstock,an apple SBP gene with the full-length cDNA was cloned in Qingzhen 1(apomictic crabapple Pingyitiancha( Malus hupehensis Rehd.)× columnar apple strain CO(Malus × domestica Borkh.) and M26 via homology-based cloning method.The gene cloned was named as MdSBP20 .Bioinformatics analysis to the cDNA showed that the full-length cDNA was 1 362 bp,it's open reading frame possessed 1 362 bp,and encoded 454 amino acids,which had obviously SBP -domain and two zinc finger structures(Zn-1,Zn-2) and the bothway nuclear location signal(NLS).Phylogenetic analysis suggested that MdSBP20 gene had the closer relationship with Pyrus bretschneideri (XM_009339726.1)and Malus domestica (XM_008376435.1).Real-time PCR analysis showed that MdSBP20 gene played a role in response to low temperature,drought,and salt tolerance.It was inferred that QZ1 might has higher resistance than M26 no matter in stress of low temperature,drought,and salt.Based on the results in this experiment, MdSBP20 gene had the significantly effects in apple rootstock response to abiotic stress.

To explore the physiological response of Malus sieversii to salinity stress, Malus sieversii seedlings were cheared with six salinity levels (0,0.2%,0.4%,0.6%,0.8% and 1.0% NaCl) for 4 hours and seven time levels (0,4,8,12,16,20 and 24 hours) in 0.4% NaCl.Contents of proline,MDA,chlorophyll,and soluble sugar in leaves and activity of SOD and POD were mensurated.The results showed that proline content got the peak when NaCl concentration was 0.6% or 12 h in 0.4% NaCl,increasing 63.1% and 27.0% compared to control respectively.Increasing salt concentration and time resulted in higher levels for malondialdehyde content.Chlorophyll content was decreased first,subseguently increased and decreased once more.Among all treatment they were lower than control.Soluble sugar content and the activity of SOD and POD were increased first,then decreased.The results indicated that Malus sieversii could reduce the damage to the plant by accumulating more proline and soluble sugar and increasing the activity of SOD and POD.It was adapted to salinity stress to some extent.