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.

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。

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。

关于Malus定律表述的研究

计算了各种检偏器出射最大光强 Ｉｍａｘ 和通过检偏器前的光强 Ｉ０ 的关系，强调了两者的不同，并指出某些教材中在 Ｍａｌｕｓ 定律叙述中的缺陷．

Bonus-Malus模型在银行贷款机制中的应用

将Bonus-Malus模型应用到银行贷款业务,通过调整银行的Bonus-Malus贷款利率来减少贷款欺诈行为。主要是建立借款人的银行个人信用体系,通过借款人上阶段的还款利率和表现决定其下阶段还款利率,这样就提供了一种与完全审计机制不同的奖惩机制。在一些简单假设下可证明Bonus-Malus利率将会消除所有欺诈行为,而非仅仅减少欺诈行为。

Physiological and transcriptomic analyses of roots from Malus sieversii under drought stress

Water deficit is one of the main limiting factors for apple growth and production.Root architecture plays an important role in drought tolerance of plants.However,little is known about the molecular basis of root system in apple trees under drought.In this study,we compared root morphology of two widely used rootstocks of apple(R3 and Malus sieversii)under drought.Our results suggested that M.sieversii is more tolerant to drought than R3,since M.sieversii had a higher ratio of root to shoot as well as root hydraulic conductivity under long-term drought conditions.We then performed whole-genome transcriptomic analysis to figure out the molecular basis of root responses in M.sieversii under drought.It was found that genes involved in transcription regulation,signaling or biosynthesis of hormones,and oxidative stress were differentially expressed under drought.Consistent with the gene expression profile,roots of M.sieversii had higher activities of peroxidase(POD)and superoxide dismutase(SOD)under drought,as well as higher content of abscisic acid(ABA)and lower content of auxin.Taken together,our results revealed the physiological and transcriptomic analyses of M.sieversii roots in response to drought.

The Growth-Promoting Mechanism of Brevibacillus laterosporus AMCC100017 on Apple Rootstock Malus robusta

Plant growth-promoting rhizobacteria(PGPR) located in rhizobacteria soil are beneficial to plant growth and development. A PGPR strain AMCC100017 of Brevibacillus laterosporus synthesizes the plant hormone IAA in a tryptophan-dependent manner. In this study, the AMCC100017 strain was used to treat Malus robusta, an excellent natural rootstock for apple production, and assess its ability to promote growth. The fresh weight, dry weight, plant height, and lateral root growth of M. robusta were significantly increased with treatment. The presence of the AMCC100017 strain increased IAA content in M. robusta and promoted root secretion of tryptophan. Colonization of the strain in the roots allowed continuous synthesis of IAA and promoted plant growth. In addition, the photosynthetic efficiency in leaves increased after microbial treatment, and the utilization of nitrogen, zinc, iron, copper and magnesium in leaves was increased, which was conducive to photosynthesis.Interestingly, the activities of CAT and SOD, as well as the contents of ROS in plants colonized by AMCC100017 were increased compared to control plants, but the activities of POD and MDA contents were decreased. AMCC100017 strain affected the antioxidant capacity and stress resistance of plants. AMCC100017 strain increased the content of soluble protein and soluble sugar in plants, improved plant metabolic activity and stress resistance. Therefore, AMCC100017 not only increased IAA content and photosynthetic efficiency to promote M. robusta growth, but also affected plant through multiple metabolic pathways.

Analysis of genetic diversity and structure across a wide range of germplasm reveals genetic relationships among seventeen species of Malus Mill,native to China

China is a center of diversity for Malus Mill,with 27 native species including 21 wild species and six domesticated species.We applied a set of 19 simple sequence repeat markers to genotype 798 accessions of 17 species(12 wild species and five cultivated species)of Malus originating from 14 provinces in China.A total of 500 alleles were detected.Diversity statistics indicated a high level of genetic variation as quantified by the average values of the effective allele number(N_(e)).expected heterozygosity(H_(e)),and Shannon's Information Index(I)(10.309,0.886,and 2.545,respectively).Malus sieversii(MSR;H_(e)=0.814,I=2.041,N_(e)=6.054),M.baccata(MBB;H_(e)=0.848,/=2.350,N_(e)=8.652),M.toringoides(MTH;He=0.663,I=1.355,N_(e)=3.332),and M.hupehensis(MHR;H_(e)=0.539,I=0.912,N_(e)=0.579)showed a higher level of genetic diversity in this study than the previous studies.MSR and MBB contributed to the origin and evolution of some accessions of M.domestica subsp.chinensis(MDC).However,other accessions of MDC showed a closer genetic distance with MBB and cultivated species,especially M.robusta(MRB),M.asiatica(MAN),and M.prunifolia(MPB).Not all accessions of MDC were descended from MSR in Xinjiang Uygur Autonomous Region of China.This research provides novel insights into the genetic relationships of Malus native to China,which will be useful for genetic association studies,germplasm conservation,and breeding programs.

Genome-wide identification of droughtresponsive microRNAs in two sets of Malus from interspecific hybrid progenies

Drought stress can negatively impact apple fruit quality and yield.Apple microRNAs(miRNAs)participate in apple tree and fruit development,as well as in biotic stress tolerance;however,it is largely unknown whether these molecules are involved in the drought response.To identify drought-responsive miRNAs in Malus,we first examined the drought stress tolerance of ten F1 progenies of R3(M.×domestica)×M.sieversii.We performed Illumina sequencing on pooled total RNA from both drought-tolerant and drought-sensitive plants.The sequencing results identified a total of 206 known miRNAs and 253 candidate novel miRNAs from drought-tolerant plants and drought-sensitive plants under control or drought conditions.We identified 67 miRNAs that were differentially expressed in drought-tolerant plants compared with drought-sensitive plants under drought conditions.Under drought stress,61 and 35 miRNAs were differentially expressed in drought-tolerant and drought-sensitive plants,respectively.We determined the expression levels of seven out of eight miRNAs by stem-loop qPCR analysis.We also predicted the target genes of all differentially expressed miRNAs and identified the expression of some genes.Gene Ontology analyses indicated that the target genes were mainly involved in stimulus response and cellular and metabolic processes.Finally,we confirmed roles of two miRNAs in apple response to mannitol.Our results reveal candidate miRNAs and their associated mRNAs that could be targeted for improving drought tolerance in Malus species,thus providing a foundation for understanding the molecular networks involved in the response of apple trees to drought stress.

Change of Mineral Elements and Amino Acids in <i>Malus hupehensis var. pingyiensis</i>Leaves under Cd Treatment

The objective of this paper is to research the effects of CdCl2 treatment on mineral elements and amino acids in leaves of Malus hupehensis var. pingyiensis. The seedlings of Malus hupehensis var. pingyiensis with 6 leaf were cultured in 1/2 Hoagland nutrient solutions of different CdCl2 treatments (0, 0.5, 5 and 10 mg·L-1), respectively. The mineral elements and amino acids of the leaves in Malus hupehensis var. pingyiensis were measured in the day 30. Compared with the control (0 mg·L-1 CdCl2), the treatments significantly decreased the contents of Mg, Fe and Zn in the tested leaves and obviously increased the contents of Cd in the experimental leaves. As to Ca and Mn, low concentration Cd treatment (0.5 mg·L-1 CdCl2) promoted their absorption, however, high concentration Cd treatments (5 and 10 mg·L-1 CdCl2) inhibited their absorption. The metabolism pathway and content of amino acids in the Malus hupehensis var. pingyiensis leaves under Cd treatment were modified, the content of amino acids in the glycolate pathway became larger than that in control, the content of amino acids in the pyruvic acid synthesis pathway and tyrosine and phenylalanine became smaller than that in control, the content of other amino acids also had made a certain degree change. The results provided the important basis for safety production and quality evaluation of leaves in Malus hupehensis var. pingyiensis.

The mitigation effects of exogenous dopamine on low nitrogen stress in Malus hupehensis

Dopamine plays numerous physiological roles in plants.We explored its role in the regulation of growth,nutrient absorption,and response to nitrogen(N)deficiency in Malus hupehensis Rehd.Under low N condition,plant growth slowed,and the net photosynthetic rates,chlorophyll contents,and maximal quantum yield of PSII(Fv/Fm)decreased significantly.However,the application of 100μmol L−1 exogenous dopamine significantly reduced the inhibition of low N stress on plant growth.In addition to modifying root system architecture under low N supply,exogenous dopamine also changed the uptake,transport,and distribution of N,P,and K.Furthermore,exogenous dopamine enhances the tolerance to low nitrogen stress by increasing the activity of enzymes(nitrate reductase,nitrite reductase,glutamic acid synthase and glutamine synthetase)involved in N metabolism.We also found that exogenous dopamine promoted the expression of ethylene signaling genes(ERF1,ERF2,EIL1,ERS2,ETR1,and EIN4)under low N stress.Therefore,we hypothesized that ethylene might be involved in dopamine response to low N stress in M.hupehensis.Our results suggest that exogenous dopamine can mitigate low N stress by regulating the absorption of mineral nutrients,possibly through the regulation of the ethylene signaling pathway.

Effects of Exogenous Manganese (Mn) on Mineral Elements, Polyamines and Antioxidants in Apple Rootstock Malus robusta Rehd.

Manganese(Mn)is one of the essential microelements in all organisms.However,high level of Mn is deleterious to plants.In this study,the effects of exogenous manganese application on mineral element,polyamine(PA)and antioxidant accumulation,as well as polyamine metabolic and antioxidant enzyme activities,were investigated in Malus robusta Rehd.,a widely grown apple rootstock.High level of Mn treatments decreased endogenous Mg,Na,K and Ca contents,but increased Zn content,in a Mn-concentration-dependent manner.Polyamine metabolic assays revealed that,except the content of perchloric acid insoluble bound(PIS-bound)spermine,which increased significantly,the contents of putrescine(Put),spermidine(Spd)and spermine(Spm)all decreased progressively,accompanied with the decreased activities of arginine decarboxylase(ADC,EC 4.1.1.19)and ornithine decarboxylase(ODC,EC 4.1.1.17),and the increased activities of diamine oxidase(DAO,EC 1.4.3.6)and polyamine oxidase(PAO,EC 1.5.3.3).Further antioxidant capacity analyses demonstrated that contents of anthocyanin,non-protein thiols(NPT)and soluble sugar,and the activities of guaiacol peroxidase(POD,EC 1.11.1.7),catalase(CAT,EC 1.11.1.6)and superoxide dismutase(SOD,EC 1.15.1.1),also increased upon different concentrations of Mn treatments.Our results suggest that endogenous ion homeostasis is affected by high level of Mn application,and polyamine and antioxidant metabolism is involved in the responses of M.robusta Rehd.plants to high level of Mn stress.

Analysis of Leaf Volatiles of Crabapple (<i>Malus</i>sp.) Individuals in Different Aphids’ Resistance

The aim of this experiment was to analyze the leaf volatiles of crabapple (Malus sp.) individuals at different aphid’s resistance, to ascertain the particular ingredients which has lure or aversion effects on aphid, and to provide reference for finding out a simple method to control effectively aphids. Volatiles of leaves from twenty-one different crabapple individuals were evaluated with the method of head space-solid phase micro-extraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS). Volatiles profiles of them were then compared. There are one hundred eighty-six kinds of volatiles were detected with varied contents found in different individuals. And all plants contain eight kinds of common components: 3-Hexen-1-ol, acetate, (Z)-, 4-Hexen-1-ol, (Z)-, n-Decanal, n-Tetradecane, .alpha.-Farnesene, Diethyl Phthalate, Oxime-, methoxy-phenyl- and Dibenzofuran-, wherein the relative content of higher have 3-Hexen-1-ol, acetate, (Z)- and 4-Hexen-1-ol, (Z)-. Specific volatile substances in high resistance plants contain 3-Hexenal, (Z)- and 2-Hexenal, (E)-. Leaf volatiles differ in twenty-one crabapple individuals. High resistance plants specific volatile substances contains 3-Hexenal, (Z)-, 2-Hexenal, (E)- and other small molecular volatile substances, and no-resistance plants all have .beta.-Farnesene.

Tissue distribution and changes in dopamine during development and stress responses in Malus germplasm

Dopamine is a catecholamine and an anti-oxidant which functions in responses to stress and it interacts with plant hormones to mediate plant development.At present,there are few studies on the functions of dopamine in apple.This study developed a method for dopamine determination which was used to analyze dopamine in Malus germplasm,in order to clarify the tissue distribution,developmental changes,diurnal variations,and stress responses in apple trees.First,the proposed method was verified.The linear range of quantification was robust from 0.1 to 20 ng mL^(–1).The instrumental,inter-day precision,and sample repeatability relative standard deviations were 1.024,5.607,and 7.237%,respectively.The spiked recovery was greater than 100%,indicating the feasibility of the method and its suitability for the rapid analysis of dopamine in Malus.Next,the dopamine content was measured in 322 Malus tissues.The results showed that the dopamine level in Malus was low and the average dopamine content in leaf was higher than in peel and flesh.The dopamine had a skewed distribution that deviated to the right in cultivars and wild accessions.Finally,the tissue specificity,developmental changes,diurnal changes,and responses to stress were analyzed.In cultivar ‘Pinova’(Malus domestica),the dopamine concentration was the highest in leaf buds and lowest in flesh.The dopamine contents in leaf and flesh decreased with the growth and development of cultivar ‘Liangxiang’(Malus domestica).The dopamine content of apple leaves was higher after either drought or salinity stress as compared to the control.In this study,a dopamine detection method for apple was established based on HPLC-MS and shown to be a robust approach.This study provides a framework for future research on elucidating the tissue distribution,developmental changes,diurnal variation,and stress responses of dopamine in apple trees.

A new species of Malus in China,Malus shizongensis Liu sp.nov

Based on morphological,molecular biological,and molecular systematic studies,we describe here a new species of Malus from Yunnan,China.We compared the morphology of this new species,Malus shizongensis Liu sp.nov,with three Malus species,including M.hupehensis,M.baccata,and M.micromalus.Although the appearance of M.shizongensis was similar to these three species,it differed in height,branch color,branch hair,and flower color.To better identify the taxonomy of this new species,genome of M.shizongensis and that of seven Malus species,including M.prunifolia,M.sylvestris,M.sieversi,M.hupehensis,M.baccata,M.robusta,and M.micromalus were analyzed.A phylogenetic tree based on genome analysis indicated that M.shizongensis was close to M.hupehensis.Furthermore,M.shizongensis had its species-specific SNPs,and the number of species-specific SNPs was similar to that of three close species(M.hupehensis,M.baccata,and M.micromalus).Based on the above information,we named this new species as M.shizongensis Liu sp.nov.

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.

Study on Callus Induction and Tissue Culture Regeneration System of Malus toringoides

[Objectives]To study the culture of callus produced by the leaves of the ethnic medicine‘E Se’,the induction of adventitious buds and the regeneration system of tissue culture seedlings.[Methods]Multi-factor orthogonal experiment was used to design and analyze.[Results]The best medium for callus induction was MS+2.0 mg/L 6-BA+0.1 mg/L 2,4-D.By comparing the effects of cytokinin 6-BA,KT and TDZ on induction of adventitious bud differentiation,it was found that the optimum medium for adventitious bud regeneration was MS+0.5 mg/L 6-BA+0.5 mg/L KT+0.15 mg/L IBA.Finally,adding 0.2 mg/L IBA(0.1 or 0.2 mg/L)to MS medium could effectively make regenerated seedlings produce roots,and the efficiency was more than 70%.[Conclusions]It laid a foundation for the large-scale cultivation of the seedlings of the ethnic medicine‘E Se’and the promotion of their artificial cultivation.

Dormancy Characteristics of Malus ‘Snowdrift’ and Effects of Spraying 6-benzyladenine on Its Germination and Flowering in Long-day Treatment

[Objective] The dormancy characteristics of Malus ‘Snowdrift’ in long-day treatment were studied, and 6-BA was used to break the dormancy, with the aim to achieve the purpose of flowering in autumn. [Method] The new shoots of ‘Snowdrift’ in long-day treatment were conducted with hydroponics to investigate their dormancy time. And cytokinin 6-benzyladenine(6-BA)was used to treat the dormant shoots, to investigate the budding, flowering and flower bud differentiation. [Result] The shoots in long-day treatment entered endodormancy after August 7^(th) and the shoots in the natural daylight entered endodormancy before July 18^(th). In long-day treatment, 116 buds, 198 flowers were observed after 6-BA spraying. [Conclusion] The optimum concentration of 6-BA was 300 mg/L. And the flower bud differentiation of ‘Snowdrift’ in long-day treatment was faster than those in natural daylight after 6-BA spraying.

Effect of Acid Treatment on Root Architecture in Seedlings of <i>Malus hupehensis</i>var. <i>pingyiensis</i>

The purpose of this paper is to explore the effects of acid treatment on root morphology and architecture in seedlings of Malus hupehensis var. pingyiensis. The rootstock seedlings were cultured in 1/2 Hoagland nutrient solutions of different pH (pH 4, pH 4.5, pH 5 and pH 6), respectively. The parameters of root architecture were measured in the day 4, 8 and 12 with the professional WinRHIZO 2007. Compared with the control (pH 6), the treatments significantly decreased the fractal dimension, length, diameter, surface area and volume of roots in day 8 and 12, and they kept decreasing followed the increase of the acidity and treatment time. The growth of lateral roots was more susceptible to acid treatment than taproots. In addition, the acid treatment mainly inhibited the growth of rootlets, significantly decreased the proportion of rootlets that changed the composition of roots, and then simplified the space structure of roots.