Identification of S-RNase genotype and analysis of its origin and evolutionary patterns in Malus plants

Identification of the S genotype of Malus plants will greatly promote the discovery of new genes,the cultivation and production of apple,the breeding of new varieties,and the origin and evolution of self-incompatibility in Malus plants.In this experiment,88 Malus germplasm resources,such as Aihuahong,Xishuhaitang,and Reguanzi,were used as materials.Seven gene-specific primer combinations were used in the genotype identification.PCR amplification using leaf DNA produced a single S-RNase gene fragment in all materials.The results revealed that 70 of the identified materials obtained a complete S-RNase genotype,while only one S-RNase gene was found in 18 of them.Through homology comparison and analysis,13 S-RNase genotypes were obtained:S_(1)S_(2)(Aihuahong,etc.),S_(1)S_(28)(Xixian Haitang,etc.),S_(1)S_(51)(Hebei Pingdinghaitang),S_(1)S_(3)(Xiangyangcun Daguo,etc.),S_(2)S_(3)(Zhaiyehaitang,etc.),S_(3)S_(51)(Xishan 1),S_(3)S_(28)(Huangselihaerde,etc.),S_(2)S_(28)(Honghaitang,etc.),S_(4)S_(28)(Bo 11),S_(7)S_(28)(Jiuquan Shaguo),S_(10)S_e(Dongchengguan 13),S_(10)S_(21)(Dongxiangjiao)and S_(3)S_(51)(Xiongyue Haitang).Simultaneously,the frequency of the S gene in the tested materials was analyzed.The findings revealed that different S genes had varying frequencies in Malus resources,as well as varying frequencies between intraspecific and interspecific.S_(3) had the highest frequency of 68.18%,followed by S_(1)(42.04%).In addition,the phylogenetic tree and origin evolution analysis revealed that the S gene differentiation was completed prior to the formation of various apple species,that cultivated species also evolved new S genes,and that the S_(50) gene is the oldest S allele in Malus plants.The S_(1),S_(29),and S_(33) genes in apple-cultivated species,on the other hand,may have originated in M.sieversii,M.hupehensis,and M.kansuensis,respectively.In addition to M.sieversii,M.kansuensis and M.sikkimensis may have also played a role in the origin and evolution of some Chinese apples.

Influence of soaking Malus domeri(Bois)Chev.leaves on gut microbiota and metabolites of long-living elderly individuals in Hezhou city,Guangxi,China

To investigate the effects of drinking the soaking of Malus domeri(Bois)Chev.leaves on gut microbiota and metabolites of long-living elderly individuals in Hezhou city,Guangxi,China.It has been reported that longevity is closely related to metabolism and the gut microbiota.The 16S rRNA sequencing and liquid chromatography-mass spectrometry(LC-MS)were used to analysis fecal samples and explore the factors affecting longevity in the region.Interestingly,we discovered,that elderly individuals who had been drinking the soaking of M.domeri(Bois)Chev.leaves for a long time exhibited higher diversity of the gut microbiota than without drinking the soaking,notably.The proportions of Ruminococcaceae and Prevotella were decreased in those who did not drink this soaking.In addition,a total of 106 metabolites were characterized,and the people of long-lived people(>90 years old)and elderly people(<90 years old)who drinking soaking of M.domeri(Bois)Chev.leaves significantly altered the gut microbiota and upregulated levels of haplopine,farnesol,genipic acid,momordicinin,2-hydroxyestrone,hydroxyphenyllactic acid,caffeic acid,sophoraflavanone B,and soyasaponin I.We preliminarily determined that M.domeri(Bois)Chev.leaves consumption may be an important factor affecting longevity in this area.

Malus’ Law Derived from Deterministic Particle Behavior

A polarized beam of energy is usually interpreted as a set of particles, all having the same polarization state. Difference in behavior between the one and the other particle is then explained by a number of counter-intuitive quantum mechanical concepts like probability distribution, superposition, entanglement and quantized spin. Alternatively, I propose that a polarized beam is composed of a set of particles with a cosine distribution of polarization angles within a polarization area. I show that Malus’ law for the intensity of a beam of polarized light can be derived in a straightforward manner from this distribution. I then show that none of the above-mentioned counter-intuitive concepts are necessary to explain particle behavior and that the ontology of particles, passing through a polarizer, can be easily and intuitively understood. I conclude by formulating some questions for follow-up research.

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.

MdWRKY75e enhances resistance to Alternaria alternata in Malus domestica

The Alternaria alternata apple pathotype adversely affects apple(Malus domestica Borkh.)cultivation.However,the molecular mechanisms underlying enhanced resistance to this pathogen in apple remain poorly understood.We have previously reported that MdWRKY75 expression is upregulated by A.alternata infection in‘Sushuai’apples.In this study,we discovered that overexpression of MdWRKY75e increased the resistance of transgenic apple lines to A.alternata infection,whereas silencing this gene enhanced susceptibility to A.alternata infection.Furthermore,we found that MdWRKY75e directly binds to the MdLAC7 promoter to regulate the biosynthesis of laccase and increase the biosynthesis of lignin during A.alternata infection.Moreover,the thickening of the cell wall enhanced the mechanical defense capabilities of apple.In addition,we found that jasmonic acid remarkably induced MdWRKY75e expression,and its levels in transgenic apple lines were elevated.These results indicate that MdWRKY75e confers resistance to the A.alternata apple pathotype mainly via the jasmonic acid pathway and that pathogenesis-related genes and antioxidant-related enzyme activity are involved in the disease resistance of MdWRKY75e transgenic plants.In conclusion,our fi ndings provide insights into the importance of MdWRKY75e for resistance to A.alternata infection in apples.

Genome-wide identification of the radiation sensitivity protein-23(RAD23)family members in apple(Malus×domestica Borkh.)and expression analysis of their stress responsiveness

Radiation sensitivity proteins-23 （RAD23） are DNA repair factors participate in the ubiquitin/proteasome system （UPS）. Although the genome-wide analysis of RAD23 family members has been conducted in some species, little is known about RAD23 genes in apple （Malusxdomestica Borkh.）. We analyzed this gene family in M. domestica in terms of genomic locations, protein and promoter structures, and expressions in response to stresses. Various members showed a ubiqui- tous pattern of expression in all selected apple parts. Their expressions were altered under chilling, heat, and hydrogen peroxide treatments, as well as abscisic acid （ABA） treatment and water deficiency, suggesting their possible roles in plant stress responses. These results provide essential information about RAD23 genes in apple and will contribute to further functional studies.

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.

Physical mapping of three fruit ripening genes: Endopolygalacturonase, ACC oxidase and ACC synthasefrom apple (Malus x domestica) in an apple rootstock A106 (Malus sieboldii)

The apple rootstock, A106 (Malus sieboldii), had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell. Karyotypes were prepared from root-tip cells with 2n= 34 chromosomes. Seven out of 82 karyotypes (8.5%) showed one pair of satellites at the end of the short arm of chromosome 3. C-bands were shown on 6 pairs of chromosomes 2, 4,6, 8, 14, and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica: endopolygalacturonase (EPG,0. 6 kb ) , ACC oxidase (1.2 kb), and ACC synthase (2 kb) were hybridized in situ to metaphase chromosomes of A106. Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11. For the ACC oxidase gene, hybridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively proximal to the centromere of chromosome 1 in 81% of the spreads, and on the long arm of chromosome 13 in 50% of the spreads. Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads, chromosomes 9 and 10 in 76% of the spreads, and chromosome 17 in 56% of the spreads.

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.

Genetic Diversity of Volatile Components in Xinjiang Wild Apple (Malus sieversii)

To evaluate genetic relationships using qualitative and/or quantitative differentiation of volatile components in Xinjiang Wild Apple （Malus sieversii （Lebed.） Roem.） and to acquire basic data for the conservation and utilization of the species, aroma components in ripe fruit of M. sieversii obtained from 30 seedlings at Mohe, Gongliu County, Xinjiang Autonomic Region, China, and in ripe fruit of 4 M. purnila cultivars （＇Rails＇, ＇Delicious＇, ＇Golden Delicious＇, and ＇Fuji＇） were analyzed using head space-solid phase microextraction and gas chromatography-mass spectrometry. The results indicated that the values of similarity coefficient concerning volatile types between the two species were in accordance with the evolution of M. pumila cultivars （forms）, and that M. sieversii seedlings showed considerable genetic variations in these aspects： the total content of volatile components, the classes and contents of each compound classes, the segregation ratio, and content of main components. The results showed significant difference among seedlings and wide genetic diversity within the populations. Comparison of the volatile components in M. sieversii with those in M. pumila cultivars showed that the common compounds whose number were larger than five with the contents over 0.04 mg/L simultaneously between M. sieversii and M. pumila cultivars belonged to esters, alcohols, aldehydes or ketones. This suggests fundamental identity in main volatile components of M. sieversii and M. pumila cultivars. The results above sustained the conclusion ＂M. sieversii is probably the ancestor ofM. purnila＂. However, there were 48 compounds present in M. puraila that were not detected in M. sieversii, including 6 character impact components （i.e., propyl acetate, （Z）-3-hexenal, 2-methyl-l-butanol acetate, pentyl acetate, 3-furanmethanol, and benzene acetaldehyde）. This suggested that in the domestication of M. pumila, introgression of other apple species, except for M. sieversii, by interspecies hybridization was possible. There were 177 compounds in total belonging to 11 classes detected in 30 M. sieversii seedlings, including esters, alcohols, ketones, aldehydes, acids, benzene ramifications, terpenes, heterocycles, hydrocarbon derivates, acetals, and lactones. Among them, acetals and lactones were not detected in M. pumila cultivars, 90 compounds were unique to M. sieversii, and 7 components （l-butanol, ethyl hutanoate, 1-hexanol, ethyl hexanoate, 3-octen-1-ol, ethyl octanoate, and damascenone） belonged to character impact odors. Thus, the potential of M. sieversii in ＂utilization conservation＂ is enormous as a rate germplasm on genetic improvement of M. pumila cultivars.

新疆野生苹果(Malus sieversii)的总多酚、总黄酮提取物的抗氧化活性研究

以采集于新疆不同地区的20个野生苹果品种为研究对象,福林酚法测总多酚,分光光度计法测定总黄酮,DPPH、ABTS、还原能力法测定总抗氧化能力,相关系数法分析各个指标之间的相关性。结果显示,不同品种的野生苹果的总多酚、总黄酮及抗氧化活性之间具有显著差异,其中,HDM-23的总多酚含量最高(4714.33μmol没食子酸/100g鲜果),XY-31的总黄酮含量最高(2452.84mg芦丁/100g鲜果)。总多酚、总黄酮及抗氧化活性之间呈极显著(p<0.01)正相关,表明总多酚与总黄酮是野生苹果抗氧化活性的主要贡献物质。

Genetic Structure of Malus sieversii Population from Xinjiang,China,Revealed by SSR Markers

One hundred and nine Malus sieversii accessions from four geographical populations located at Kuerdening in Mohe town, Gongliu County, Jiaowutuohai, in Xinyuan County, Daxigou in Houcheng County of Ily State, and Baerluke Mountain in Yumin County of Tacheng State, Xinjiang Uygur Autonomous Region of China were studied by SSR markers. The purpose of the study was to determine the genetic structure and diversity in these eco-geographical populations with eight pair SSR primers of apple. The results indicated that： an average of 16 bands was detected in the four populations. The percentage of polymorphic bands in Gongliu population （89.06%） was the highest in the four populations. The average Nei＇s gene diversity index was 0.257 for all the loci. Totally, 128 polymorphic loci were detected and the percentage of polymorphic loci （P） was 100%, 88.28%, 84.83%, 87.50%, and 87.12%, respectively, at the species level and Gongliu, Xinyuan, Huocheng, and Yumin population levels. The Nei＇s gene diversity index （H = 0.2619） and Shannon＇s information index （1 = 0.4082） in the species level were higher than in the population level. The Nei＇s gene diversity index and Shannon＇s information index in the four populations were Gongliu 〉 Huocheng 〉 Xinyuan 〉 Yumin. Gongliu population and Xinyuan population were the highest in genetic identity and the closest in genetic distance. Gene flow between the populations was 7.265 based on genetic differentiation coefficient （GST = 0.064）. The UPGMA cluster analysis indicated that the genetic relationships between the Gongliu and Xinyuan population were the closest, and the Yumin population were the farthest with the other three populations. The UPGMA cluster analysis indicated that the four geographical populations located in Gongliu, Xinyuan, Huocheng, and Yumin were relatively independent populations. Concurrently, there was also mild gene exchange between the populations. On the basis of the study of population genetic structure and the highest genetic diversity, Gongliu population should be given a high priority consideration in Malus sieversii population＇s in situ germplasm conservation.

新疆野苹果(Malus sieversii)超低温保存及其植株再生

以新疆野苹果(Malus sieversii(Lebed.)M.Roem.)无菌试管苗为试材,对其离体茎尖玻璃化超低温保存的影响因素进行研究。结果表明,新疆野苹果茎尖在含有5%二甲基亚砜(DMSO)的0.4mol/L蔗糖培养基上预培养3 d,60%玻璃化溶液(PVS2)中室温装载30m in,PVS20℃下处理40m in,经液氮保存至少24h后,转入继代培养基上再培养,成活率和再生率分别为93.3%和86.7%。再生植株生长和分化正常;同时对再生植株进行SSR标记检测,未发现超低温保存前后的DNA谱带存在差异。

台湾林檎(Malus doumeri(Bois.)Chev.)地理分布模拟

依据苹果属最新的系统分类学成果,重新鉴定了中国科学院植物研究所等标本馆的标本,选取了243个有价值的分布记录转换成地理坐标数据,用DIVA-GIS绘制了采集标本的地理分布,这种分布式样主要呈西南—华南—华东的横向连续分布和大陆—台湾的间断性分布。用当前生物气候数据(分辨率30 arc s)采用BIO-CLIM模拟的分布与标本记录分布基本情况是吻合的,并发现了一些潜在分布区域。对落在亚洲地区生物气候变量包络内分布点的19个生物气候变量进行了主成分分析,发现雨季降雨量,年温差,最干月降雨量是台湾林檎分布的主导生物气候因子,变种间仅3个主导生物气候因子的生态位有明显间隔,其它因子生态位不规则重叠。比较了用当前气候数据和未来气候数据对台湾林檎模拟的当前、未来分布格局和潜在分布区,结果显示当前和未来的整体分布范围变化较小,只是整个分布区内部各级分布区的范围和分布值有一定变化。依据台湾林檎的生态需求特性和分布区变化的趋势,推测台湾林檎可能起源于云南、广西、老挝和越南交界的区域。

中国新疆野苹果(Malus sieversii(Ldb.)Roem.)种群地理分布格局及其遗传关系研究

利用SSR(简单重复序列)标记技术,对新疆野苹果的5个主要地理居群(128份材料)进行了种内亲缘关系分析。结果表明:(1)5个居群中巩留居群遗传多样性最丰富;(2)群体的AMO-VA分子方差分析得出野苹果居群遗传多样性有92%来自于居群内,8%来自于居群间,说明遗传多样性主要分布在居群内;(3)5个居群遗传距离与基因流呈极显著负相关,与地理距离呈显著正相关,说明野苹果地理分布格局是决定其遗传关系的主要因素;(4)5个居群间的基因流结果推断野苹果种群在历史上曾发生过迁移,根据种群遗传关系以及地理分布格局推断野苹果在冰期后迁移方向从中亚平原由西向东,由南向北迁移,从而形成现代的地理分布。

西府海棠(Malus micromalus) MaMAPK基因的克隆及表达特性

依据高等植物MAPK基因的保守区设计简并引物 ,用RT PCR方法 ,首次从西府海棠幼苗叶片中克隆了促分裂原活化蛋白激酶 (mitogen activatedproteinkinase,MAPK )基因 (MaMAPK )的cDNA全序列。Southern杂交结果表明在西府海棠中存在一个小的MAPK基因家族。 2 0 %PEG处理西府海棠幼苗不同时间后的Northern杂交分析表明 ,该基因在根系和叶片中均有表达 ,随着胁迫时间延长表达量增加 。

新疆野苹果(Malus sieversii(Ledeb.) M.Roem.)植物学性状遗传多样性及相关性分析

为加强野苹果种质资源利用与研究,促进野苹果研究工作,以3年来调查收集的新疆野苹果（赛威士苹果）的129个单株资源为材料,对单果重、果实纵横经、叶片大小等12个数量性状和叶片颜色、叶尖类型等5个质量性状进行遗传多样性及相关性分析。分析结果表明：叶片颜色、叶尖类型、叶姿、叶缘、叶面状态5个质量性状分布频率较集中;单果重、果实纵横经、叶柄长、叶片长宽、可溶性固形物、干周、树高均存在较大变异,变异系数幅度为16%~51%。各性状多样性指数也较大,均值为1.9264,叶片长的多样性指数最小为1.7359;果梗长的多样性指数最大为2.0525。新疆野苹果资源拥有丰富的遗传多样性,在于果实相关性状的多样性指数高,且变异幅度大,表明丰富的遗传多样性是新疆野苹果资源的重要特征。

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。

苹果无融合生殖型砧木“76—2”(Malus Sp.)的利用研究初报

“76—2”苹果砧木具有无融合生殖特性,染色体数量为2n=4x=68。乔木,寿命长,根系发达,侧根及须根量尤多。固地性好,无根蘖,管理方便。耐旱、耐涝、耐瘠薄,并可忍耐-45℃的低温。能在pH8.3—8.6的土壤条件下正常生长。经六年的嫁接鉴定,它对故道地区普遍潜带褪绿叶斑病毒(CLSV)、茎痘病毒SPV)、及茎沟槽病毒(SGV)的金冠品种不发生敏感,嫁接部位愈合良好,平整光滑,无大、小脚现象。植株直立生长,半矮化。其矮化程度、提早结果作用及丰产能力均与M7相近。果实风味浓厚,维生素C、可溶性固形物都显著高于八楞海棠或M7为砧的金冠品种,是有望的实生系半矮化砧。

新疆野苹果Malus sieversii(Ldb.)Roem花粉形态及其起源演化研究

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