Identification and characterization of genes related to m^(6)A modification in kiwifruit using RNA-seq and ATAC-seq

N6-methyladenosine(m^(6)A)RNA modification is a conserved mechanism that regulates the fate of RNA across eukaryotic organisms.Despite its significance,a comprehensive analysis of m^(6)A-related genes in non-model plants,such as kiwifruit,is lacking.Here,we identified 36 m^(6)A-related genes in the kiwifruit genome according to homology and phylogenetic inference.We performed bioinformatics and evolutionary analyses of the writer,eraser,and reader families of m^(6)A modification.Reanalysis of public RNA-seq data collected from samples under various biotic and abiotic stresses indicated that most m^(6)A-related genes were remarkably expressed under different conditions.Through construction of gene co-expression networks,we found significant correlations between several m^(6)A-related genes and transcription factors(TFs)as well as receptor-like genes during the development and ripening of kiwifruit.Furthermore,we performed ATAC-seq assays on diverse kiwifruit tissues to investigate the regulatory mechanisms of m^(6)A-related genes.We identified 10 common open chromatin regions that were present in at least two tissues,and these regions might serve as potential binding sites for MADS protein,C2H2 protein,and other predicted TFs.Our study offers comprehensive insights into the gene family of m^(6)A-related components in kiwifruit,which will lay foundation for exploring mechanisms of post-transcriptional regulation involved in development and adaptation of kiwifruit.

Evaluation of Resistance of Different Kiwifruit Varieties (Lines) to Canker Disease and Brown Spot Disease

Kiwifruit canker and brown spot are significant diseases affecting kiwis,caused by Pseudomonas syringae patho-genic variations(Pseudomonas syringae pv.Actinidiae(Psa))and Corynesporapolytica(Corynespora cassiicola).At present,the research on canker disease and brown spot disease mainly focuses on the isolation and identification of pathogenic bacteria,drug control,resistance gene mining and functional verification.Practice has proved that breeding disease resistant varieties are an effective method to control canker disease and brown spot disease.However,most existing cultivars lack genes for canker and brown spot resistance.Wild kiwifruit resources in nat-ure exhibit extensive genetic diversity due to prolonged natural selection,containing numerous resistance genes.But,due to insufficient understanding of the resistance of most kiwifruit varieties(lines)to canker disease and brown spot disease,some high-quality resources have not been fully utilized.The incidence of canker and brown spot of 18 kiwifruit cultivars(lines)was measured by inoculating isolated branches and leaves,and their resistance to canker and brown spot was analyzed according to the length,disease index,mean diameter,and systematic clustering.The results were as follows:Among 18 different kiwifruit varieties(lines)for canker disease,there were two highly resistant materials,eight disease-resistant materials,four disease-susceptible materials,and two highly susceptible materials.Moreover,regarding brown spot disease,there were one highly resistant material,five dis-ease-resistant materials,four susceptible materials,and three highly susceptible materials.Furthermore,four resources were resistant to both diseases.The outcomes provided a theoretical basis for breeding kiwifruit against canker and brown spot.

Investigation and Identification of Fungal Diseases of Kiwifruit in Qiandongnan Prefecture

[Objectives]The paper was to investigate and identify the fungal diseases of wild and red heart kiwifruit in Qiandongnan Prefecture.[Methods]The pathogenic fungi were isolated from diseased leaves and fruits of wild and red heart kiwifruit by tissue separation method.DNA sequencing was carried out by using the sequence analysis of ribosomal r DNA-ITS region,and molecular evolutionary trees were built by using MEGA 4.0 software.Finally,the pathogenic fungi were classified and identified by combining morphological observation.[Results]The main fungal diseases were anthracnose caused by Colletotrichum gloeosporioides on wild kiwifruit,fruit anthracnose caused by C.acutatum on red heart kiwifruit,leaf soft rot caused by Fusarium incarnatum on red heart kiwifruit,and brown spot caused by Alternaria alternata on red heart kiwifruit.[Conclusions]The study may provide some theoretical basis for the control of kiwifruit diseases in Qiandongnan Prefecture.

Efficacy of a Kiwifruit Extract (PhenActivTM) on Gastrointestinal Tract Function: A Randomised Double-Blind Placebo-Controlled Study

Objective: Gastrointestinal (GI) discomfort is experienced by millions of people every day. This study aimed to evaluate the effect of PhenActivTM, a novel green kiwifruit extract, on gastrointestinal tract (GIT) function in otherwise healthy adults. Methods: 41 healthy adults with mild GI discomfort were enrolled in this double-blind, randomized, placebo-controlled study. Participants were randomized to either take 3.0 g/day of PhenActivTM or a placebo for 6 weeks. Interviews were conducted at baseline, week 3 and week 6, with participants completing questionnaires regarding GI symptoms. Frequency of bowel movements was self-recorded daily. Results: There were no differences in daily and weekly defecation frequency and stool characteristics in either group. The active and placebo groups significantly improve GSRS scores (p , only the active group had a significant improvement in the IBSSS and PAC-QOL scores (p < 0.05) from baseline. Neither group had changes in sleep quality, quality of life and fatigue, plasma zonulin concentrations or macular pigment optical density scores. The product was well tolerated with no GI disturbances or adverse events being reported. Conclusion: Supplementation of 3.0 g/day of PhenActivTM for 6 weeks did not improve defecation frequency or stool composition in healthy adults, but did improve perceived symptoms of GIT function, including symptoms of functional GIT disorders, IBS and constipation. The product was well tolerated and future trials investigating higher doses with more participants and/or a different population would be beneficial.

Variation of Sugar, Acid and Vitamin C Contents in Fruit Development in Different Types of Kiwifruit

The variation of sugar, acid and vitamin C contents in fruits of red flesh kiwifruit （Actinidia chinensis） ＂Hongyang＂ and green fresh kiwifruit （A. deliciosa） ＇Jinkui＇ were investigated during fruit development. The results showed that the to- tal sugar soluble contents of ＂Hongyang＇ and ＇Jinkui＂ during fruit development ex- isted different variations. With the upward trend of ＇Hongyang＇ fruit development, 95 days after full bloom （DAFB）, the total soluble sugar accumulation was relatively slow, and then rose rapidly until harvest with the maximum content （6.87%）. While ＇Jinkui＇ fruit showed a fluctuant process, rising in 50 DAFB, then declining, then rising rapidly and decreasing slightly right before harvest. The variation of fruit titrat- able acid between them was more consistent, which was increased and then de- creased. The only difference was that the titratable acid content of ＇Jinkui＇ fruit de- creased slowly from the late of fruit development to fruit ripening, similar to the maximum value, but that of ＇Hongyang＇ fruit decreased rapidly in the late. Titrat- able acid contents of them in the maturity were 1.08% and 1.20%, respectively. The trends of sugar acid ratio for ＇Hongyang＇ and ＇Jinkui＇ fruit were quite different. ＇Hongyang＇ fruit increased slowly and then rapidly; ＇Jinkui＇ changed from decreas- ing to increasing, followed by slight decreasing in mature stage. In addition, the two kiwifruit varieties had a similar change trend of Vc content during fruit development, which changed from rapid increasing to declining, followed by slight growing in the harvest. The changing tendency of ＇Jinkui＇ fruit was later than that of ＇Hengyang＇.

Effects of Acetylsalicylic Acid (ASA) and Ethylene Treatments onRipening and Softening of Postharvest Kiwifruit

Kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson cv. Bruno) was used toinvestigate the effects of acetylsalicylic acid (ASA, 1.0 mmol/L, pH 3.5) and ethylene (100 mL/L) treat-ments on changes at endogenous salicylic acid (SA) levels and other senescence-related factors duringfruit ripening and softening at 20 ℃. The level of endogenous SA in ripening fruits declined and a closerelationship was observed between the change at endogenous SA level and the rate of fruit ripening andsoftening. ASA treatment elevated SA level in the fruit, slowed down the increases in lipoxygenase (LOX)and allene oxide synthase (AOS) activities, decreased the O22-. production in the preclimacteric phase andthe early phase of ethylene climacteric rise, maintained the stability of cell membrane, inhibited ethylenebiosynthesis, postponed the onset of the ethylene climacteric, and delayed the process of fruit ripeningand softening. On the contrary, application of ethylene to ripening kiwifruit resulted at a lower SA level, anaccelerated increases in the activities of LOX and AOS and the rate of O22-. production, an elevated relativeelectric conductivity and an advanced onset of ethylene climacteric, and a quicker fruit ripening andsoftening. It is suggested that the effects of ASA on ripening kiwifruit can be attributed to its ability toscavenge O22-. and/or to maintain stability of cell membrane.

Dynamic Variation in Sugar,Acid,and ASA Contents of ‘Ganmi 6' Kiwifruit(Actinidia eriantha Benth) Fruits

[Objective] The variation of sugar, acid and AsA contents in fruits of ‘Ganmi 6＇ kiwifruit （Actinidia eriantha Benth） were investigated during fruit develop- ment. [Method] Kiwi fruits were randomly taken as materials every 15 days since 20 days after full bloom （DAFB） to 170 DAFB until in mature stage. [Result] The results showed that during fruit development of ‘Ganmi 6＇, the total sugar soluble contents had a rising trend with relatively stable at 95 DAFB, then rose until har- vest with the maximum content （10.35%）. The titratable acid content showed a trend of increasing, then declining, then increase to the harvest content （1.10%）. From the sugar acid ratio, we can knew it decreased in 95 DAFB, then up to the max （9.38）. The changes of AsA contents showed double ‘S＇ shape, decreased af- ter the first increased rapidly, slightly increased and then decreased in the early harvest. [Conclusion] It provided a theoretical basis for scientific cultivation methods to explore the nutrients regulation.

Analysis on SSR Information of EST Resources of Kiwifruit(Actinidia ssp.)

Objctive The aim was to make better use of the EST-SSR resources of kiwifruit for further molecular biological studies and new EST-SSR marker development by screening and mining the SSR repeats in the EST database of kiwifruit （Actinidia spp. ） [ Method] 56 400 of EST sequences were randomly selected from EST（ Expressed Sequence Tag）sequences of kiwifruit in the database of NCBI. EST sequences were analyzed and the SSR（Microsatellite） was screened by using the SSRHunter software. [ Result] 7 939 SSRs were identified from the ran- domly selected kiwifruit EST resources, among which there were 5 131 （64.63%） dinucleotide, 1 237 （ 15.58% ） trinucleotide, 284 （ 3.58% ） tetra nucleotide, 397 （5.00%） pentanucleotide and 890 （ 11.21% ） hexanucleotide SSRs. Among the dinucleotide sequences, AG/CT repeat motif was accounted for 4 654（90.70% ）. The frequency of SSRs was approximately 1/2.48 kb, which could exist to 1 SSR in 7 unigenes. [ Conclusion] The dinucleotide repeats appeared to be the most abundant SSRs, followed by the trinucleotide and hexanucleotide repeats. Among them the repeat motif such as AG/CT was predominant in each type of SSRs.

Pre-harvest spraying of oxalic acid improves postharvest quality associated with increase in ascorbic acid and regulation of ethanol fermentation in kiwifruit cv. Bruno during storage

The kiwifruit trees(Actinidia deliciosa cv.Bruno)were sprayed with 5 mmol L-1 oxalic acid(OA)or water(as control)at 130,137 or 144 d after full-blossom,and then the fruit were harvested at commercial maturity and stored at room temperature(20±1)℃ for 13 d.The effect of pre-harvest spraying of OA on postharvest quality of kiwifruit was evaluated during storage.The OA spraying slowed the increase in soluble solids content(SSC)and decrease in titratable acid(TA),as well as increased contents of ascorbic acid(AsA)and total-AsA accompanied with higher AsA/DHA ratio in kiwifruit during storage.Moreover,the OA spraying significantly reduced the contents of acetaldehyde and ethanol in kiwifruit,along with significant decrease in activities of enzymes involved in ethanol fermentation metabolism during the later period of storage,which was beneficial to control off-flavor associated with over accumulation of ethanol during postharvest.It was suggested that pre-harvest spraying of OA might maintain the postharvest quality of kiwifruit in relation to delay in fruit ripening,AsA maintenance and regulation of ethanol fermentation.

Growth and physiological responses of four kiwifruit genotypes to salt stress and resistance evaluation

In this study,four genotypes(Acva-1,Acva-2,Acva-3 and ZM-2) of Actinidia germplasm resources were grown in different NaCl concentrations(0,0.4,0.8 and 1.2 g L–1).The growth,physiological and biochemical indicators were measured,and a graded scale was developed as the salt damage index(SDI) according to different damage symptoms in leaves.The results showed SDI increased gradually,and average number and length of new shoot decreased significantly.Three antioxidant enzymes(superoxide dismutase,peroxidase and catalase) and two osmotic adjustment substances(soluble sugar and proline) showed different changes in old and new leaves of four genotypes.SPAD values exhibited a decreased trend in the whole except in the new leaves of Acva-2.Malonaldehyde contents increased and root activity decreased with the increasing salt concentrations.Principal component analysis was used to assess the salt tolerance,and the results showed Acva-3,from Actinidia valvata Dunn.,had the strongest tolerance to salt,and could be a potential resistant resource to the salt-tolerance dedicated rootstock breeding of kiwifruit.

Increasing dietary fiber intake in terms of kiwifruit improves constipation in Chinese patients

AIM： To investigate if increased dietary fiber, in terms of kiwifruit, is effective in Chinese constipated patients. METHODS： 33 constipated patients and 20 healthy volunteers were recruited for a 4-wk treatment of kiwi fruit twice daily. Response during wk 1-4 was defined as an increase in complete spontaneous bowl, motion （CSBM）≥ 1/wk. Secondary efficacy included response during wk 1-4, individual symptoms and scores of bowel habits and constipation. Responses were compared with the baseline run-in period. Colonic transit time and anorectal manometry were performed before and after treatment. RESULTS： Responder rate was 54.5% in the constipated group. The mean CSBM increased after treatment （2.2±2.6 vs 4.4± 4.6, P = 0.013）. There was also improvement in the scores for bothersomeness of constipation （P = 0.02）, and satisfaction of bowel habit （P = 0.001）, and decreased in days of laxative used （P = 0.003）. There was also improvement in transit time （P = 0.003） and rectal sensation （P 〈 0.05）. However, there was no change in the bowel symptoms or anorectal physiology in the healthy subjects. CONCLUSION： Increasing dietary fiber intake is effective in relieving chronic constipation in Chinese population.

Reducing nitrogen fertilization of intensive kiwifruit orchards decreases nitrate accumulation in soil without compromising crop production

Excessive nitrogen （N） fertilization of high value horticultural crops is a common problem that not only increases the cost to farmers, but also negatively affects crop growth and the environment. A three-year field experiment was conducted in an intensive kiwifruit orchard in Shaanxi Province, China to compare the effects of reduced N fertilization applied as urea （U）, and controlled release urea （CRU） on the N nutrition of kiwi vines, fruit yield and quality, and nitrate-N accumulation in the soil profile. The three treatments included a conventional N application rate （CF-U, 900 kg N ha-1 yr-1 as urea）, two reduced N fertilization treatments where the amount of N fertilizer applied as U and CRU was reduced by 25％ in 2013 and 2014, and by 45％ in 2015. The 25 and 45％ reduced N treatments had no adverse effects on the N concentrations in leaves and pruning branches and the fruit yield and quality of kiwi vines. However, they significantly enhanced the partial factor productivity of applied N （PFPN） and the economic benefits, and reduced nitrate accumulation in the 0-200 cm soil profile. The same benefits of reduced N fertilization were observed for both the U and CRU treatments, but the CRU treatment had the added benefit of decreasing the loss of nitrate through leaching. We concluded that the current level of N fertilization in kiwi orchards is very excessive, and reducing the N fertilizer rate by 25-45％ could not only guarantee fruit yield, but also reduce N accumulation and loss.

Kiwifruit(Actinidia chinensis)R1R2R3-MYB transcription factor AcMYB3R enhances drought and salinity tolerance in Arabidopsis thaliana

Kiwifruit is an important fruit crop that is highly sensitive to environmental stresses,such as drought,heat,cold,water logging and phytopathogens.Therefore it is indispensable to identify stress-responsive candidate genes in kiwifruit cultivars for the stress resistance improvement.Here we report the isolation and characterization of a novel kiwifruit R1R2R3-MYB homolog(AcMYB3R)whose expression was induced by drought,salinity and cold stress.In vitro assays showed that AcMYB3R is a nuclear protein with transcriptional activation activity by binding to the cis-element of the kiwifruit orthologue of G2/M phase-specific gene KNOLLE.The Arabidopsis transgenic plants overexpressing AcMYB3R showed drastically enhanced tolerance to drought and salt stress.The expressions of stress-responsive genes such as RD29A,RD29B,COR15A and RD22 were prominently up-regulated by ectopic expression of Ac MYB3R.Our study provides a valuable piece of information for functional genomics studies of kiwifruit and molecular breeding in improving stress tolerance for crop production.

1-MCP Regulates Ethanol Fermentation and GABA Shunt Pathway Involved in Kiwifruit Quality During Postharvest Storage

Kiwifruit(Actinidia deliciosa)‘Bruno’is prone to accumulate ethanol rapidly after respiratory climacteric during storage at ambient conditions without stresses,which causes quality deterioration of the fruit associated with alcohol off-flavor.For maintaining the postharvest quality of kiwifruit‘Bruno’,the effects of 1.0μL•L^−11-methylcyclopropene(1-MCP)treatment on regulating the ethanol fermentation andγ-aminobutyric acid(GABA)shunt pathway associated with control of alcohol off-flavor were investigated during storage at room temperature(24±1)°C for 27 days.The results showed that 1-MCP treatment significantly reduced the respiration rate,ethylene production,decay rate,ascorbic acid(AsA)loss,and delayed the decline in the firmness and titratable acidity(TA),and the increase in total soluble solid(TSS)in kiwifruit.Furthermore,1-MCP treatment effectively inhibited the increases in contents of acetaldehyde,ethanol,and GABA along with the suppressed activities of key enzymes involved in ethanol fermentation and GABA shunt pathway,such as pyruvate decarboxylase(PDC),alcohol dehydrogenase(ADH),glutamate decarboxylase(GAD),and GABA-transaminase(GABA-T)in kiwifruit during storage.In conclusion,1-MCP treatment efficiently regulated the ethanol fermentation and GABA shunt pathway by delaying the ripening process to avoid the alcohol off-flavor development,thereby contributed to maintaining the quality of the kiwifruit.

Alginate oligosaccharide improves resistance to postharvest decay and quality in kiwifruit(Actinidia deliciosa cv. Bruno)

Kiwifruit is an extremely perishable fruit;postharvest disease and senescence during storage can reduce the fruit quality,resulting in economic loss.Considerable research effort has focused on identifying safe and cost-effective ways to preserve fresh kiwifruit.To this end,the present study investigated the effects of alginate oligosaccharide(AOS)soaking treatment on postharvest quality and disease in the‘Bruno’variety of kiwifruit.The involved physiological mechanisms were further explored.The results showed that AOS did not inhibit the growth of Botrytis cinerea in vitro,the causal agent of gray mold in kiwifruit,but reduced the incidence of gray mold and diameter of lesions of kiwifruit during storage.Kiwifruit treated with 50 mg·L-1 AOS showed a higher degree of firmness and lower soluble solid content than control fruit treated with distilled water.Moreover,AOS treatment inhibited the activity of polygalacturonase and pectinesterase,while enhancing the activity of polyphenoloxidase,l-phenylalanine ammonia lyase andβ-1,3-glucanase related to pathogen defense,and also improved total antioxidant capacity determined by the DPPH,FRAP,and ABTS methods in kiwifruit.These results indicate that 50 mg·L-1 AOS can confer disease resistance in kiwifruit during storage.

Resistant Evaluation of Kiwifruit Rootstocks to Root Zone Hypoxia Stress

In this thesis, 10 species of kiwifruit rootstocks were treated with hydroponics hypoxia to study their root zone hypoxia tolerance. The results were as follows: growth of all kiwifruit seedlings was inhibited. The max length of new root, plant height, plant biomass, root activity, relative growth rate of leaves, and content of chlorophyll in leaves under root zone hypoxia stress obviously declined comparing with control. MDA content, relative conductance in the leaves and roots all increased in 10 kinds of kiwifruit seedlings. The sensitivities of 10 kinds’ kiwifruit seedlings to hypoxia stress were obviously different. With the method of subordinate function and cluster analysis, the adversity resistance coefficient of 10 kinds’ kiwifruit seedlings, were comprehensively evaluated in order to appraise their hypoxia-tolerance abilities. According to the results, “Hayward”, “Qinmei”, “Jinxiang”, “Kuoye”, “Huayou” kiwifruit seedlings held higher tolerance to root zone hypoxia stress, while “Hongyang” kiwifruit seedlings were sensitive to root zone hypoxia stress. The others, including “Xixuan”, “Maohua”, “Jinhua”, “Shanli” kiwifruit seedlings kept moderate resistant intensity to root zone hypoxia stress. The kiwifruit seedlings’ resistance order from strong to weak was: “Hayward” > “Qinmei” > “Jinxiang” > “Kuoye” > “Huayou” > “Xixuan” > “Maohua” > “Jinhua” > “Shanli” > “Hongyang”.

Cell Ultrastructure of Kiwifruit (Actinidia chinensis) Shoot Tips During Cryopreservation

The changes in the cell ultrastructure of in vitro cultured shoot tips from dwarf genotype of kiwifruit （Actinidia chinensis Ganmi 5） during cryopreservation were investigated. Shoot tips were preserved in liquid nitrogen using vitrification, and the cell ultrastructure was examined using transmission electron microscopy （TEM）. The regular ultrastructure of the cell wall, cell membrane and nucleus of shoot tips could be damaged during the freezing and thawing associated with preservation using liquid nitrogen. The cell plasmolysis was increased and freezing tolerance was improved after precultufing and dehydrating in a preservation and vitrification solution （PVS2） （30% glycerol （Gly）＋ 15% ethylene glycol （EG）＋ 15% dimethylsulfoxide （DMSO） ＋ 0.4 mol L^-1 sucrose）. The structure of some cells with low degree of injury and reversible damage was similar to that of the control and they could undergo normal cell division and differentiation. Besides, they could recover automatically and regenerate after their reculture.

Factors Influencing Bacterial Canker Disease of Kiwifruit and the Control Countermeasures

Survey was conducted to investigate the occurrence laws of bacterial canker disease of kiwifruits in Liupanshui area, Guizhou Province, and the patho- gens and hazard symptoms of the canker disease were introduced. The results showed that the factors influencing the occurrence of bacterial canker disease of ki- wifruit were identified as inter-species resistance, agronomic traits, meteorological and ecological conditions, cultivation and management techniques and fertilization levels. Techniques for the comprehensive control of kiwi canker disease were stated, and control suggestions were proposed.

Development Status and Countermeasures of Kiwifruit Industry in Sichuan Province

The kiwifruit industry is one of the dominant agricultural industries in Sichuan province. A kiwifruit industrial zone with Cangxi county,Dujiangyan city,Shifang city,Pengzhou city,Pujiang county,Qionglai city and Dayi county as the core; An county,Beichuan county, Ya'an city,Wenchuan county et al. as radiation zone has been developed during the last 30 years. The kiwifruit cultivation area has reached to 2. 97 × 10 4 hm 2 with an annual fresh kiwifruit output of 1. 3 × 10 5 t; and the production value was nearly 110 million RMB in 2011. The production acreage and yield ranked the second over the country. Advice and ideas were proposed based on the circumstance and problems of kiwifruit industry in Sichuan. According to the advantages of developing kiwifruit industry in Sichuan,measures including strengthening scientific and reasonable planning,optimizing industrial layout,speeding up the breeding,adjusting product structure,standardizing demonstration garden, integrating and innovating the key technology,setting up research alliances,and fostering the leading enterprises were put forward.

Highly-efficient Stereo-cultivation Model in Kiwifruit Orchards Interplanting Konjak

The kiwifruit orchards with plants growing for over two years were chosen for interplanting of konjak, which takes advantages of complementation of the two plants in terms of habits, reducing water and soil erosions effectively, and decreasing water evaporation, and konjak diseases. Furthermore, the interplanting doubles planting benefits and constitutes a highly-efficient planting model.