Studies on Mode of Expression of Apple （Malus x domestica Borkh.） Bud Dormancy under Tropical and Temperate Climatic Conditions

A study was conducted to describe the progression of bud dormancy in 1-year-old apple （Malus x domestica Borkh） shoots grown at two contrasting climatic conditions （Belgium, temperate and Ethiopia, tropics）. The experiment was carried out on ＂Golden＂ and ＂Gala＂ cultivars for two consecutive years （2010/2011 and 2011/2012）. Moreover, a validation experiment was conducted on ＂MM106＂ apple rootstock during 2010/2011 only in Ethiopia. Variations in inverse of time to 50% budburst were interpreted in terms of evolution of growth capacity of the buds. Despite differences observed with chilling accumulation later in winter or early in spring, depending on environments, depth of endodormancy intensity during winter can be summarized as follows： buds from pruned shoots were less endodormant than terminal buds of the intact shoots and terminal buds were more endodormant than the dormancy intensity of upper buds of the disbudded shoots, suggesting proximal buds can grow more readily than does terminal ones. Our results provide evidence for the existence of a considerably strong paradormancy inhibition by distal shoot parts and buds, which was more pronounced in Ethiopia than in Belgium, highlighting the importance of designing and applying appropriate pruning and dormancy avoidance strategies in mild-winter climates. Finally, as still there is knowledge gap on bud dormancy progression and its control mechanism especially under mild climates, our study highlights the need for further in-depth research using biological and biochemical tests.

东北山定子(Malus baccata(L.) Borkh)野生居群表型遗传多样性分析及生态地理分布研究

东北山定子（Malus baccata（L.）Borkh）野生居群集中分布区位于49°11＇-50°40＇N,119°42＇-120°16＇E之间,海拔520-670 m,呈带状分布于大兴安岭西南麓与呼伦贝尔平原交接的河床半湿地,面积约230 km^2,处于原始野生状态。调查423份自然变异类型表明：树龄最大400年生以上,30-50年生为主体占37%;树姿以直立型与开张型为主,垂枝型占14%;株高与冠径均呈正态分布,矮生型占19.8%;果实颜色由黄经红到紫色变异,浓红型占43.8%,黄色与紫色类型分别占4.5%、3.2%;果实酸、酸甜、甜酸共占总群体的72.9%;自然授粉前提下,平均每花序坐果3个的占47.7%,坐果5个的占26.5%,3.5%的成龄树没有果实;叶形与果形有扁圆、圆、卵圆、长椭圆等,但叶形与果形之间无遗传相关性。群体遗传分析参考国内外研究进展,结合分布区的GPS、河流走向、地貌特征、树龄分布、采访记录等推测：东北山定子是山定子组（Malus baccatus Jiang）的地理亚种,集中分布于大兴安岭西南麓的山谷间,海拔800m左右,种子经河水、鸟类等作用,延伊敏河、根河、海拉尔河、雅鲁河等流域自然迁徙扩散至海拉尔的黑羊站、蒙古屯、哈克镇,满洲里黑山头口岸的弘吉拉布等地,在近河的山谷地带逐渐适应繁衍形成二级集中分布区。

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

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

山定子(Malus baccata Borkh.)根系蛋白质提取及双向电泳技术体系的建立

为开展山定子（Malus baccata Borkh.）根系蛋白质组学研究,以当年生15片真叶的山定子实生苗白色幼嫩根系为试材,采用TCA-丙酮沉淀法、酚抽提法和Trizol抽提法3种方法提取其总蛋白质,并对样品上样量、IPG胶条的pH值范围和等电聚焦参数进行优化,建立适用于山定子根系的双向电泳技术体系。结果表明：采用Trizol抽提法提取的蛋白质产率最高,可达3.15mg·g^-1 FW,且其SDS-PAGE图谱明显优于其他两种方法 ;选用800μg的上样量,17 cm pH值4～7的IPG胶条和改进后的等电聚焦程序所得到的二维电泳图谱蛋白点最多,最清晰,分辨率最高。

Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function.However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed.Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium(MS basal salts, 30 g Lsucrose, 8 g Lagar, 5 mg L6-benzylaminopurine(6-BA), 2 mg Lthidiazuron(TDZ), and 1 mg L1-naphthlcetic acid(NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime(150 mg L) and kanamycin(15 mg L). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.

Malus domestica and Solanum lycopersicum Mixtures for the Synthesis of Graphene Silver and Zinc Oxide Nanocomposites

This study reports on the novel and simple green method involving the use of apple (Malus domestica) and tomato (Solanum lycopersicum) extracts in the synthesis of electroactive layers of silver nanoparticles|graphene oxide (AgNPs|GO) and zinc oxide nanoparticles|graphene oxide (ZnONPs|GO). The surface morphology of the green synthesized nanocomposites was studied using High-Resolution Transmission Electron Microscopy (HRTEM), High-Resolution Scanning Electron Microscopy (HRSEM) while the elemental analysis was studied using Fourier Transform Infrared Spectroscopy (FTIR), Raman spectroscopy and X-Ray diffraction (XRD) and their optical properties were further characterised using Ultraviolet Spectroscopy (UV-vis). The electrochemical studies of these nanocomposites were achieved using cyclic voltammetry (CV) where an increase in electron conductivity of the AgNPs|GO and ZnONPs|GO nanocomposite was observed. Comparatively, the silver nanoparticulate-based platforms were observed to have superior electrochemical properties as opposed to the zinc oxide-based platform. The observed electrochemical activities of the synthesized nanocomposites are a good indication of their suitability as electroactive platforms towards the development of electrochemical sensors. Electrochemical sensors are popular in the Electrochemistry field because they may be developed using different methods in order to suit their intended analytes. As such, the synthesis of a variety of electrochemical platforms provides researchers with a vast range of options to select from for the detection of analytes.

Resistance index and browning mechanism of apple peel under high temperature stress

Apples are one of the most important economic crops worldwide.Because of global warming and an aggravation of environmental,abnormally high temperatures occur frequently in fruit-growing season and seriously affect normal fruit growth and reduce fruit quality and yield.We took five-year-old Ruixue’(Qinfu 1×Pink Lady;CNA20151469.1) fruits as test materials,and the ambient temperature during fruit development was monitored.The results showed that during the fruit-growing season,especially during the rapid growth stage (July to August),the maximum daily temperature exceeded 30℃ and lasted for more than 40 days.To determine the effects of high temperature stress on the apple fruit resistance,we treated expanding,veraison,and maturity-period fruits at different temperatures.It was found that the fruits of the expanding period showed strong resistance to high temperature stress,whereas during veraison and maturity,fruit resistance to high temperature stress decreased,and the fruit peel browning phenotype appeared.Meanwhile,the content of malonaldehyde (MDA),hydrogen peroxide (H_(2)O_(2)),and superoxide anion (O._(2)^(-)) in the peel gradually increased with increasing temperature.The content of total phenols,flavanol,and flavonoids in the peel decreased substantially at 45℃.Moreover,it was found that polyphenol oxidase gene (MdPPO1) was most sensitive to high temperature stress in apple.Furthermore,transient and stable MdPPO1 overexpression significantly promoted peel browning.The transgenic materials were more sensitive to high temperatures,and browning was more severe compared to non-genetically modified organism (WT).Stable MdPPO1 knockout calli obtained via clustered regularly interspersed short palindromic repeats (CRISPR/Cas9) gene knockout technology reduced the browning phenotype,and the resultant fruits were not sensitive to the effects of high temperature stress.Thus,MdPPO1 expression may be a key factor of high temperature-related changes observed in the browning phenotype that provides a scientific theoretical basis for the selection of high temperature-resistant varieties and apple cultivation and management in the future.

苹果MdWRKY23基因提高植株抗寒性的功能分析

为探究MdWRKY23基因对苹果抗寒能力的影响及其抗寒机制,以苹果(Malus domestica)为试验材料,通过基因克隆得到全长为1452 bp的WRKY家族基因MdWRKY23,该基因编码483个氨基酸。亚细胞定位研究发现,MdWRKY23蛋白定位于细胞核。实时荧光定量PCR结果表明,MdWRKY23基因在植物根、成熟叶和新叶中表达量较高,在根尖表达量较低。低温处理时,MdWRKY23基因在新叶和根中表达量均呈先升后降趋势。与对照植株相比,MdWRKY23基因过表达植株具有更强的低温耐受力,且转基因株系中低温胁迫相关基因表达显著上调。研究表明MdWRKY23基因可显著提高植株抗寒性。

miR395a-MdWRKY26调控苹果植物耐盐的分子机制

【目的】为了揭示miR 395 a-MdWRKY 26表达模式在盐处理下调控苹果耐盐性的分子机制,并且为苹果耐盐新品种的选育提供基础。【方法】以‘嘎啦’野生型苹果苗和通过稳定遗传转化方法得到的miR 395 a-OE、miR 395 a-RNAi、MdWRKY 26-OE、MdWRKY 26-RNAi转基因苹果苗为试验材料,对其进行200 mM的NaCl处理,观察表型并且用RT-qPCR检测盐处理下的表达量,用紫外分光光度计法测量丙二醛含量。【结果】结果表明,200 mM的NaCl处理后,与对照相比,miR 395 a-OE和MdWRKY 26-RNAi转基因植株受到的伤害程度较高,对盐更敏感;而MdWRKY 26-OE和miR 395 a-RNAi转基因植株受到的损害程度较轻,表明植株MdWRKY 26-OE和miR 395 a-RNAi转基因植株具有耐盐性。【结论】通过耐盐性分析,得到miR 395 a基因负调控苹果耐盐性,而MdWRKY 26基因发挥正调控作用。揭示了miR 395 a-MdWRKY 26调控苹果耐盐性的分子机理,可以减弱植物在盐处理下受到的伤害。

苹果不同品种果实香气物质研究

【目的】研究不同品种苹果果实的香气成分。【方法】利用热脱附-气相色谱-质谱联用技术(TCT-GC-MS)分别测定了苹果不同品种未成熟无香气和成熟有香气果实的挥发性物质,并对不同品种果实的香气成分进行了探讨。【结果】未成熟苹果无香气果实的挥发性物质以己醛、2-己烯醛等为主;成熟苹果有香气果实的挥发性物质以酯类和某些醇类物质为主。富士苹果果实主要香气成分为丁酸乙酯、1-丁醇、乙酸3-甲基丁酯、乙酸乙酯和2-甲基丁酸乙酯。乙酸丁酯、乙酸3-甲基丁酯、乙酸丙酯、乙酸乙酯、1-丙醇、1-丁醇、2-甲基丁醇和2-甲基丁酸乙酯为新红星苹果果实的主要香气成分。乔纳金苹果主要香气物质有1-丙醇、乙酸丙酯、乙酸丁酯、2-甲基丁醇、1-丁醇和乙酸3-甲基丁酯。丁酸乙酯、乙酸丁酯、乙酸乙酯和2-甲基丁酸乙酯为王林苹果果实的主要风味物质。【结论】苹果不同品种成熟果实的挥发性物质与未成熟果实不同,在成熟果实中大量增加的挥发性物质为果实的主要香气物质。

苹果加工品种果实中的酚类物质与褐变研究

【目的】研究加工苹果品种的酚类物质组成及其与果实褐变的关系,为选择加工品种和加工工艺提供依据。【方法】利用Folin-C法和高效液相色谱法等测定分析了10个苹果加工品种(4个酿酒品种、6个制汁品种)的果实酚类物质组成、果实褐变度及多酚氧化酶活性。【结果】果实中酚类物质的含量和比例因品种类型而异,苦涩酿酒苹果总酚含量高于甜或酸苹果。原花青素、绿原酸、表儿茶素及儿茶素是苹果果肉中含量较高的酚类物质,苹果特征酚类物质根皮苷在苦绯甘品种中大量存在。果实的褐变度与总酚、原花青素、儿茶素和根皮苷相关性较高,绿原酸与果实的褐变度相关性较低。【结论】酚类物质含量与苹果加工品种类型有关,酿酒品种的总酚含量高于制汁品种。果实中黄烷-3-醇含量对褐变度影响较大。

应用AFLP分子标记鉴定苹果品种

在建立苹果品种AFLP分析体系的基础上 ,从 6 8对引物中筛选出了 4对多态性高、分辨能力强的引物 (即P32M4 6、P4 4M53、P59M4 1和P79M6 1)。分析了P32M4 6引物绘制的我国 2 5个苹果重要品种的AFLP指纹图谱的遗传多样性 ,确定了各供试品种的差异带 ,区分了供试的 2 5个苹果品种。对AFLP分析的关键步骤进行了讨论 ,并对生产上应用AFLP技术检测苹果品种真实性和纯度作了展望。

新疆野苹果矿质元素与糖酸组分的遗传多样性

对新疆野苹果巩留和新源两个种下居群的88个实生株系果实的矿质元素、糖酸组分及其遗传多样性进行研究,并与富士、金帅和红星品种进行比较。结果表明,新疆野苹果的6种矿质元素(Ca、Mg、Fe、Cu、Mn、Zn)、3种糖(果糖、葡萄糖、蔗糖)组分及苹果酸含量的变异系数均在23%以上,表现出丰富的遗传多样性。其中钙平均含量是栽培苹果品种的3·1倍,而且可溶性糖的组成与栽培苹果品种表现出明显的差异。

‘红玉’ב金冠’苹果杂交后代果实糖酸组分遗传分析

【目的】探讨糖酸组分在苹果杂交后代中的遗传规律。【方法】以苹果品种‘红玉’与‘金冠’及其杂交后代的实生群体为试材,利用高效液相色谱方法(HPLC),测定‘红玉’与‘金冠’及其杂交后代果实糖酸组分含量。【结果】糖组分含量在‘红玉’与‘金冠’杂交后代中呈正态分布,符合数量性状的遗传特点。在亲本与其杂交后代果实中,果糖、葡萄糖、蔗糖是主要糖组分。亲本的果糖与葡萄糖含量比例要低于杂交后代中2者的平均比例。杂交后代群体果实中果糖、葡萄糖、蔗糖平均质量分数分别为39.86、15.46、23.02 mg·g^(-1),遗传传递力分别为71.59%、65.56%、182.55%;果糖、葡萄糖的遗传主要是加性效应引起的,蔗糖的遗传除存在加性效应外,还存在一定程度的非加性效应。果实中酸的主要成分有苹果酸、枸橼酸、琥珀酸和草酸,草酸、琥珀酸、枸橼酸含量都呈不连续变异,苹果酸呈连续变异,具有典型的数量性状遗传特征。杂交后代群体果实中苹果酸、枸橼酸、琥珀酸、草酸平均质量分数分别为0.55、0.04、0.03、0.02 mg·g^(-1),遗传传递力分别为90.09%、57.62%、84.26%、91.57%。【结论】果糖、葡萄糖、蔗糖、苹果酸是由微效基因控制的数量性状。草酸、枸橼酸、琥珀酸可能是由2对主效基因控制的质量性状,并受微效基因影响;分别控制琥珀酸、枸橼酸含量的2对基因间可能存在互补作用。

苹果果实糖积累特性与品质形成的关系

以富士和国光苹果为研究对象,对其果实发育过程中糖含量及其代谢关键酶活性的变化进行测定分析,以揭示糖分积累代谢特性对果实品质形成的影响.结果表明:(1)富士和国光均为己糖积累型果实,富士果实以积累果糖最多,果糖/葡萄糖(F/G)值为1.56,而国光以积累葡萄糖最多,F/G值仅为0.68;蔗糖在两品种中含量和所占比例均很低,在近成熟期富士高于国光.(2)富士果实蔗糖磷酸合成酶(SPS)和蔗糖合成酶(SS)活性均随果实糖的累积量增加而显著升高,酸性转化酶(AI)活性也渐趋升高,而中性转化酶(NI)活性波动不大,且其糖累积与AI和SPS活性相关性最大,而与NI相关性不大,SS的作用主要表现在发育后期;在国光果实糖积累过程中SPS起主导作用,SS和NI的作用主要表现在发育前期,而AI的作用不大.(3)富士和国光果实淀粉含量变化趋势相同,在淀粉积累高峰之后,富士果实淀粉降解速度更快,其淀粉含量迅速下降且低于国光,此时其相应淀粉酶活性也高于国光.研究发现,富士和国光果实糖积累和淀粉代谢均存在显著差异,从而直接或间接地影响着果实糖代谢过程,进而导致果实品质的显著差异.

苹果不同HRM反应体系分析效果评价

高分辨率熔解曲线(high resolution melting,HRM)分析是一种新型的DNA多态性快速筛查技术。本研究以苹果品种"富士"和"舞姿"为试材,用来自苹果基因组的SSR标记CH03d11对不同反应体积、不同DNA浓度以及不同PCR退火程序下的HRM分型效果进行了测试与评估。结果表明:采用5μL反应体积可以获得与10μL或20μL反应体积相同的检测效果。在5μL反应体积中,DNA浓度小到0.25ng/μL时,PCR扩增及随后的HRM分析效果依然良好。另外,研究表明,采用降落PCR扩增模式也能取得良好的HRM多态性检测结果。

苹果果实酸/低酸性状的SSR分析

以91株‘东光’和‘富士’的正反交F1代群体为试材,测定了成熟果实可滴定酸含量和果实不同发育阶段苹果酸含量的变化,从表型上分析了苹果酸的遗传规律。利用140对共显性遗传的SSR引物对高酸和低酸个体群体分离分析,筛选到一个与果实酸/低酸性状连锁的标记SDY085,遗传距离为8.89cM,表型分析和标记分析都表明苹果果实酸/低酸性状由一对主效基因Ma/ma控制,并且Ma对ma为完全显性,而酸个体中苹果酸的连续分布则是加性多基因作用的结果。

威赛克柱型苹果与旭的AFLP多态性研究

以旭和威赛克等 8个柱型苹果品种 (品系 )为试材 ,建立了AFLP银染技术体系。经过引物筛选 ,发现引物PstI GAC/MseI TA和PstI GAC/MseI CG可分别在旭和威赛克之间各扩增出 1条特异片段 ,初步认为这两条特异片段可能与苹果柱型Co基因相关。

几个苹果主栽品种生长季钙含量动态的比较分析

对8个品种苹果(MalusdomesticaBorkh.)钙含量的动态变化进行了研究。结果表明,苹果叶片、新梢钙浓度随着发育而增加,不同品种生长初期差异较小,后期差异显著,特别是新梢后期差异极显著,以新红星最高,达19638.6mg·kg-1,红富士最低,仅8751.3mg·kg-1。果实中钙浓度以幼果期最高,随果实生长钙浓度降低,以果实迅速膨大期下降幅度最大。品种间差异极为显著,幼果期以新红星最高,达506.562mg·kg-1,其次是粉红女士,而红富士的钙浓度只有新红星的66.27%。不同品种果实成熟时的钙浓度由高到低依次为:新红星>珊夏>粉红女士>千秋>嘎拉>红富士>红将军>新世界。通过分析果实钙吸收累积量发现,8个品种在整个生长季都持续摄入钙,以幼果期钙的吸收量较大,占总钙的35%～46%;6月25日测定的果实钙累积量较少;嘎拉、珊夏和新红星3个中早熟品种在7月27日(即较集中的果实迅速膨大期)吸收量约占果实钙吸收总量的30%,而晚熟品种果实膨大期较长,7月27日和8月29日2个测定时期均有钙摄入,红富士分别为22.6%、19.9%,粉红女士分别为26.7%、24.6%,合计累积量都超过了幼果期;各品种进入生理成熟后钙摄入量较其它时期均极显著下降。果梗中钙浓度亦随果实生长发育而增加,品种间变化规律与果实一致。

新疆野苹果与栽培苹果香气成分的比较

以中国新疆伊犁地区巩留县莫合镇的新疆野苹果4个实生株系及国光、红星、富士、金冠等苹果品种的成熟果实为试材,采用顶空固相微萃取和气相色谱—质谱联用技术,分析果实香气成分,旨在为新疆野苹果资源保护与利用提供基本资料。结果表明,①新疆野苹果含醇类等10类89种香气成分,这些化合物单株间在种类和含量上均存在显著差异;新疆野苹果与栽培苹果品种主要香气的种类和成分基本一致;②但乙醛等12种香气成分在新疆野苹果中没有检测到,其中7种为栽培苹果特有的特征香气成分;③缩醛类和内酯类化合物在栽培苹果品种中没有检测到,同时检测到1-丁醇、丁酸乙酯、辛酸乙酯和大马酮等4种化合物为新疆野苹果特有的特征香气成分。