Cloning and Expression of a cDNA Encoding Endopolygalacturonase from Feicheng Peach (Prunus persica)

Polygalacturonase (PG,EC3.2.1.15) is the key cell wall hydrolase in fruit ripening. The identification and characterization of a full length cDNA (pMT18) encoding for PG from Feicheng peach (Prunus persica (L.) Batsch cv. Feicheng) is described. The pMT18 clone is 1188 bp in length, with an open reading frame of 393 amino acids. The homology and phylogenetic analyses indicate a remarkable similarity between peach PG and other ripening related PG. And seven consensus sequences have revealed in peach PG compared to the PG from other plants. However, the profound divergence with other PG and the unique structure features suggest that peach PG probably belongs to a new evolutionary class. In RT PCR analysis, pMT18 related RNA was undetectable in leaves, and was much abundant in ripe fruits. The ripening specific expression pattern of this cDNA will be useful in investigating the roles of PG in fruit ripening and developing a transgenic peach with the improved post harvesting quality in the future.

Molecular cloning,identification,and chromosomal localization of two MADS box genes in peach(Prunus persica)

MADS box proteins play an important role in floral development. To find genes involved in the floral transition of Prunus species, cDNAs for two MADS box genes, PpMADS1 and PpMADSIO, were cloned using degenerate primers and 5＇- and T-RACE based on the sequence database of P. persiea and P. duleis. The full length of PpMADS1 cDNA is 1,071 bp containing an open reading frame （ORF） of 717 bp and coding for a polypeptide of 238 amino acid residues. The full length of PpMADSIO cDNA is 937 bp containing an ORF of 633 bp and coding for a polypeptide of 210 amino acid residues. Sequence comparison revealed that PpMADS1 and PpMADSIO were highly homologous to genes API and PI in Arabidopsis, respectively. Phylogenetic analysis indicated that PpMADS1 belongs to the euAP1 clade of class A, and PpMADSIO is a member of GLO/PI clade of class B. RT-PCR analysis showed that PpMADS1 was expressed in sepal, petal, carpel, and fruit, which was slightly different from the expression pattern ofAPl; PpMADS10 was expressed in petal and stamen, which shared the same expression pattern as PI. Using selective mapping strategy, PpMADSI was assigned onto the Binl：50 on the G1 linkage group between the markers MCO44 and TSA2, and PpMADSIO onto the Bin1：73 on the same linkage group between the markers Lap- 1 and FGA8. Our results provided the basis for further dissection of the two MADS box gene function.

Accumulated chilling hours during endodormancy impact blooming and fruit shape development in peach(Prunus persica L.)

Winter chill is essential for the growth and development of deciduous species. To understand the relationship between accumulated chilling hours during endodormancy and blooming and fruit shape development, we controlled chilling hours and investigated their effects on blooming date and fruit shape of peaches. The results showed that the number of days to full bloom date and the heat requirement for blooming were negatively correlated with accumulated chilling hours. Accumulated chilling hours were significantly negatively correlated with fruit shape index and fruit tip lengths, suggesting that the number of chilling hours affect the fruit shape development. Fewer accumulated chilling hours may be the major reason for longer fruit shape and protruding fruit tips. In conclusion, our results indicate specifically that decreased winter chilling hours can delay the bloom date and may lead to aberrant fruit shape development in peaches. Our study provides preliminary insights into the response of temperate fruit species to global climate change.

Effects of nitrogen content on growth and hydraulic characteristics of peach(Prunus persica L.) seedlings under different soil moisture conditions

A pot experiment was conducted to investigate the effects of nitrogen content [Nl （no fertilizer）, N2 （0.15 g.kg-l）, and N3 （0.3 g.kg 1）] on the growth and the hydraulic characteristics of peach seedlings under different soil moisture conditions （Wl, W2 and W3, in which the soil water content was 45% to 55%, 60% to 70%, and 75% to 80% of the field water capacity, respectively） by using a specialized high pressure flow meter with a root chamber and a coupling, which was connected to plant organs. Leaf area and leaf hydraulic conductivity （KL） increased significantly in the seedlings because of increased soil moisture and N content. KL increased with leaf area. A linear correlation was documented between KL and leaf area. KL was higher in the morning and began to decline sharply after 16：00, at which KL declined after an initial increase. Soil moisture and N content enhanced shoot （Ks） and root （Kr） hydraulic conductivities, thereby improving the low soil moisture condition to a large extent. Ks and Kr of the seedlings were reduced by 32% and 27% respectively in N~, and by 14.7% and 9.4%, respectively in N2, and both in Wb compared with the control treatment. N3 had no significant effect on Ks and Kr under similar conditions. Linear negative correlations were observed between Kr and the excised root diameter as well as between Ks and the shoot stem diameter. The shoot-to-root ratio increased with in- crease in N content. The shoot-to-root ratio in N3 was increased by 14.37%, compared with N1 in W1 as well as by 12% and 4.39% in Wz and W3, respectively. Knowledge of the effects of soil moisture and N fertilizer on hydraulic characteristics and growth is important. Our results provide basic guidelines for the implementation of water-saving irrigation and fertilization management of nursery stock.

Characterization and Expression of Ammonium Transporter in Peach (Prunus persica) and Regulation Analysis in Response to External Ammonium Supply

As the preferred nitrogen(N)source,ammonium(NH_(4)^(+))contributes to plant growth and development and fruit quality.In plants,NH 4+uptake is facilitated by a family of NH_(4)^(+) transporters(AMT).However,the molecular mechanisms and functional characteristics of the AMT genes in peach have not been mentioned yet.In this present study,excess NH_(4)^(+) stress severely hindered shoot growth and root elongation,accompanied with reduced mineral accumulation,decreased leaf chlorophyll concentration,and stunned photosynthetic performance.In addition,we identified 14 putative AMT genes in peach(PpeAMT).Expression analysis showed that PpeAMT genes were differently expressed in peach leaves,stems and roots,and were distinctly regulated by external NH_(4)^(+) supplies.Putative cis-elements involved in abiotic stress adaption,Ca^(2+) response,light and circadian rhythms regulation,and seed development were observed in the promoters of the PpeAMT family genes.Phosphorylation analysis of residues within the C-terminal of PpeAMT proteins revealed many conserved phosphorylation residues in both the AMT1 and AMT2 subfamily members,which could potentially play roles in controlling the NH 4+transport activities.This study provides gene resources to study the biological function of AMT proteins in peach,and reveals molecular basis for NH_(4)^(+) uptake and N nutrition mechanisms of fruit trees.

Novel in silico EST-SSR markers and bioinformatic approaches to detect genetic variation among peach(Prunus persica L.)germplasm

Because there are thousands of peach cultivars,cultivar classification is a critical step before starting a breeding project.Various molecular markers such as simple sequence repeats(SSRs)can be used.In this study,67 polymorphic primers produced 302 bands.Higher values for SI index(1.903)suggested higher genetic variability in the genotype under investigation.Mean values for observed alleles(Na),expected heterozygosity(He),effective alleles(Ne),Nei’s information index(h),and polymorphic information content(PIC)were 4.5,0.83,5.45,0.83,and 0.81,respectively.The dendrogram constructed based on Jaccard’s similarity coefficients outlined four distinct clusters in the entire germplasm.In addition,an analysis of molecular variance(AMOVA)showed that70.68%of the total variation was due to within-population variation,while 29.32%was due to variation among populations.According to this research,all primers were successfully used for the peach accessions.The EST-SSR markers should be useful in peach breeding programs and other research.

桃(Prunus persica(L.) Batsch.)品种核心种质的构建与评价

为构建桃品种核心种质,通过对56份桃（Prunus persica（L.） Batsch.）初级核心种质的形态农艺性状数据（MOR）和SSR等位基因数据的分析,研究了不同聚类取样方法和完全随机取样方法下9种取样比例的遗传多样性指数、保留比例及各频率段等位基因的丢失比例。结果表明：聚类取样的方法优于完全随机取样,并以在80%的取样比例下MOR结合SSR数据聚类取样的效果最好,利用此方案构建的桃品种核心种质共包括45份材料,该核心种质的基因遗传多样性指数最高,保留了初级核心种质100%的形态农艺性状和96.6%的SSR等位基因,在出现频率低于0.05的等位基因中共丢失了2个等位变异,保留了出现频率在0.05-0.10的所有等位基因;利用6个数量性状对所构建的核心种质的代表性检测表明所构建的核心种质很好地代表558份桃原始种质的遗传变异。

采后包装及钙处理对‘豫甜’桃(Prunus persica)果实品质的影响

以'豫甜'桃为试验材料,研究不同包装处理室温贮藏及采后钙处理低温贮藏对其果实品质的影响.结果表明,在室温贮藏条件下,聚乙烯膜包装袋中加入KMnO4(乙烯吸收剂)的贮藏效果明显优于其它2种处理.与对照相比,果实的呼吸速率减小了5 0mg·kg-1·h-1,果实硬度增加了2 6kg·cm-2,Vc含量增加了10μg·g-1,可溶性酸含量增加了0 07g·kg-1,可溶性固形物含量增加了2 1g·kg-1,总糖含量增加了2 8g·kg-1;经ρ为4%的CaCl2溶液处理后,装入加有KMnO4(乙烯吸收剂)的聚乙烯膜包装袋中,在0～5℃的低温下贮藏效果最好,与对照相比:果实的呼吸速率减小了1 0mg·kg-1·h-1,果实硬度增加了2 1kg·cm-2,Vc含量增加了1 3μg·g-1,可溶性酸含量增加了0 17g·kg-1,可溶性固形物含量增加了-0 25g·kg-1,总糖含量增加了0 5g·kg-1;综合比较认为采后钙处理再低温贮藏能较好地降低桃果实的呼吸速率,保持果实的硬度,抑制Vc的降解,较长时间地维持果实的营养成分,保持其品质不大幅度下降.

Effects of Weak Light on the Ultrastructural Variations of Phloem Tissues in Source Leaves of Three-Year-Old Nectarine Trees(Prunus persica L.var. nectarina Ait.)

Leaves from three_year_old solar greenhouse nectarine trees ( Prunus persica L. var. nectarina Ait. “Zao Hong Yan”) were used as materials in this study. It was the first time that the ultrastructural characteristics of phloem tissues of source leaves were observed and compared in normal and weak light intensities using the transmission electron microscopy. Results showed that the average diameters of companion cells (CC) and sieve elements (SE) of all kinds of veins were bigger in normal than that in weak light intensity, indicating that light could influence the cell development and growth. Dense cytoplasm with abundant mitochondria, endoplasmic reticulums, multivesicular bodies, vesicles and plastids were observed in normal light intensity. On the contrary, CC with small vacuolar structures and few mitochondrias, endoplasmic reticulums were shown in weak light. Misalignment of grana thylakoid margins of nectarine leaves also was seen in weak light. The sieve pores of SEs were obstructed in weak light. Chloroplasts with numerous starch grains and few mitochondrias were noticed in the mesophyll cell (MES) surrounding the bundle sheath in weak light. The storage of starch grains appeared to result from an unbalance between photosynthate production and export of photosynthates. This observation provided a strong support to the point that most leaves export the most of assimilates in the light time. Plasmodesmal densities between SE/CC, CC/PP (phloem parenchyma cell), PP/PP and PP/BSC (bundle_sheath cell) decreased in weak light. Plasmodesmata were observed between CC/SE (NS) (nacreous_walled sieve element), PP/BSC in branch veins in normal light intensity, but not in weak light. Thus apoplasmic pathway may be the main mode of transport of assimilates in weak light, however symplasmic pathway may be the main mode of transport of assimilates in normal light intensity. These results demonstrated that the solar greenhouse nectarine trees could be adapted to the weak light via the ultrastructure variation of phloem tissues of the source leaves.

Study on SSR Markers Linked to Flesh Color around the Stone of Prunus persica(L.) Batsch

[Objective] This study aimed to select SSR molecular markers linked to flesh color around the stone of Prunus persica （L.） Batsch. [Method] P. persica （L.） Batsch varieties Chongyanghong and Yanhong were used as parents to construct F1 orthogonal group. A total of 138 FI individuals were selected as experimental materi- als for construction of color around the stone gene pool （B1） and non-color around the stone gene pool （B2） by using bulked segregant analysis （BSA） method, molec- ular markers linked to the flesh color around the stone of P. persica （L.） Batsch were selected with SSR molecular marker technology. [Result] After selection with 256 pairs of SSR primers, three pairs of molecular markers linked to the gene con- trolling flesh color around the stone of P. persica （L.） Batsch were selected （UDP96- 003, ch04g09 and UDP97-402）. In addition, genetic distances between the three molecular markers and the gene controlling flesh color around the stone of P. persi- ca （L.） Batsch were calculated, which were 16.7, 10.1 and 17.0 cM, respectively. [Conclusion] This study laid the foundation for further selection of co-dominant molecular markers with closer genetic distance.

离体条件下桃（Prunuspersica）脱病毒技术的改进

为提高离体条件下挑脱病毒的效果，本研究比较了茎尖分生组织培养和微型嫁接两种脱病毒技术。采用倒“Ｔ”型和“孔穴”嫁接法，将“红港”嫁接在“ＣＴ２６９２”上，其成活率分别为１８．２％和４９．５％。试验还表明，“红港”的茎尖分生组织的分化率取决于分生组织块的大小。供试３个桃品种“红港”、“Ｍａｙｏｒｅｓｔ”和“Ｓｕｎｃｒｅｓｔ”的茎尖分生组织在本试验所筛选出的改进培养基上的分化率最高，该培养基含Ａｎｄｅｒｓｏｎ矿质成份（１９７８），Ｓｐａｔａ维生素（１９６９）。激素配比为０．２ｍｇ／１ＢＡ，０．０１ｍｇ／１ＩＢＡ和０．１ｍｇ／１ＧＡ。关键词：

两种抑制剂对肥城桃(Prunus persica)多酚氧化酶抑制类型的研究

本文用丙酮和硫酸铵沉淀的方法将肥城桃 (Feichengpeach)多酚氧化酶部分纯化 ,纯化倍数为 5 1 1倍。研究了二乙基二硫代氨基甲酸钠和硫脲对该酶活性的抑制类型。

紫叶桃(Prunus persica f. atropurpurea)的愈伤组织诱导和培养

紫叶桃是重要的园林彩叶树种,叶色美丽,观赏期长,具有很高的园林应用价值。以紫叶桃的叶片和嫩茎段为外植体,用不同浓度生长调节物质进行愈伤组织诱导试验,建立紫叶桃的再生体系。结果表明,诱导愈伤组织时,MS+2 mg/L BA+1 mg/L 2,4-D对叶片诱导效果最佳,其诱导率达80%;MS+1 mg/L BA+1 mg/L 2,4-D对茎段诱导效果最佳,其诱导率达100%。在MS+1 mg/L BA+1 mg/L NAA培养基上愈伤组织能直接分化出不定芽。

Molecular Identification and Cultivar Fingerprints of Prunus persica (L.) Batsch Germplasms

[Objective] The aim was to study the molecular identification and cultivar fingerprints of Prunus persica (L.) Batsch germplasms.[Method] Sixty peach genotypes,representing China common local cultivars and European samples were screened by microsatellites (simple sequence repeats,SSRs) and Inter-Simple Sequence Repeat (ISSR) markers.[Result] 26 reproducible bands were amplified by Nine SSR primers,and 24 of which were polymorphic; 236 bands were amplified by 30 ISSR primers,and 113 of which were polymorphic.31 genotypes were discriminated with 1-3 distinct polymorphic bands generated from the primers ISSR and SSR.Seven cultivar-specific ISSR fragments and two SSR unique alleles obtained from this study were available to be converted into Sequence Characterized Amplified Region (SCAR) markers.The genetic similarity coefficient (GS) estimated from these molecular data averaged were 0.939 (ranged from 0.856 to 0.983) for ISSR and 0.646 (ranged from 0.240 to 1.000) for SSR,respectively.The combined grouping association indicated that most local Chinese peach cultivars and exotic accessions were clustered together.This could be related to the mode of introduction and maintenance of the peach cultivars involving limited foundation germplasm,exchange of cultivars between plantations,and periodic development of new recombinant cultivars following sexual reproduction.[Conclusion] The results obtained in this work would help to improve the conservation,molecular identification and management of peach germplasm in breeding.

Volatiles from <i>Prunus persica</i>Flowers and Their Correlation with Flower-Visiting Insect Community in Wanbailin Ecological Garden, China

Object: To investigate the VOCs from living Prunus persica flowers of different branches and their correlation with floral insects. Special, dominant and 20 VOCs were analysed from living Prunus persica flowers captured by closely and circularly headspace way in adsorbent tubes and ATD-GC/MS on 29 April in Wanbailin Ecological Garden, China. VOCs from high altitude tree are more than low altitude in sunny site, and floral volatile constituents from upslope branches are less than downslope of the same tree on high altitude. Special floral VOCs were alpha-dimethyl-benzenemethanol, isopropyl palmitate, ethylbenzene, p-xylene, acetophenone, 3-ethyl-2-methyl-Heptane on sunny slope, and propylene glycol, decanal, hexadecane on shady slope. Dominant VOCs founded during 8 temporal quanta were toluene, hexane, 2-ethyl-1-hexanol, dodecane, pentadecane. Floral VOCs’ number from sunny slope was significantly negative correlated with flower-visiting insect community richness, abundance, diversity on sunny or shady slope, which from shady slope was significantly positive correlated with flower-visiting insect community parameters on both slopes tried dividing insects visiting floral branches from Prunus persica in sunny site with n/2 + 1 or n/2 - 1, and shady site with n/2 before sampled volatiles day. On sampled volatiles day, 2-ethyl-1-hexanol from sunny slope was significantly positive correlated with flower-visiting insect community parameters on shady slope (P Prunus persica on sunny slope, and even floral branches kept on shady slope.

The Potential Efficacy of Glycyrrhizic Acid and Its Nanostructure Against Brown Rot of Peach fruits

Production of peaches(Prunus persica(L.)Batsch)for both local market and export is increasing each year in Egypt.Brown rot disease,caused by Monilinia laxa and Monilinia fructigena,is considered one of the most important postharvest rots affecting peaches in Egypt and economic losses are increasing.Antifungal activity of glycyrrhizic acid nanoparticles(GA-NPs)and glycyrrhizic acid(GA)at 0.2 and 0.4 mmol/L was investigated as a control for both these brown rot pathogens on peach fruits in both in vitro and in vivo studies.In the in vitro studies,GA-NPs were the most effective as shown by the ability to decrease linear growth of both brown rot pathogens in potato dextrose agar(PDA)amended with 0.4 mmol/L GA-NPs.Micrographs of M.fructigena exposed to 0.4 mmol/LGA showed mycelial deformations,nodule formation,detachment of the cell wall,shrinkage and inhomogeneous cytoplasmic materials with large vacuoles.Mycelium of M.laxa exposed to 0.4 mmol/LGA-NPs resulted in thinner and distorted hyphae,nodule formation,cell wall thinning,and swellings.The GANPs and GA treatments improved fruit quality by maintaining firmness and total soluble solids(TSS).GA-NPs were more effective in decreasing decay incidence than their bulk material.The 0.4 mmol/L GA-NPs completely inhibited the disease on naturally infected peach fruits for both seasons of 2018 and 2019.Furthermore,0.4 mmol/L GA-NPs reduced the disease incidence in inoculated fruits by 95(M.laxa)and 88%(M.fructigena)in 2018 season and 96(M.laxa)and 85%(M.fructigena)in 2019 season.In conclusion,GA-NPs could enhance the resistance of peaches against brown rot caused by M.laxa and M.fructigena.

Identification and expression analysis of chlorogenic acid biosynthesis key gene PpHCT in peach

Shikimic acid/quinic acid hydroxy cinnamyl transferase(HCT)is one of the key enzymes in the phenylpropanoid pathway.However,the role of the HCT gene in chlorogenic acid(CGA)biosynthesis in peach fruit remains unclear.For this,we identified the accumulation pattern of CGA in four peach cultivars,cloned and characterized 11 PpHCT gene members,and further analyzed the expression patterns of these PpHCT genes during fruit development.The contents of CGAs in the four peach cultivars all exhibited a trend of increasing and then decreasing during the fruit growth and development.Moreover,the contents of CGAs in the peel and flesh were tissue-specific.Gene structure analysis indicated that the PpHCT genes were highly conserved,containing two exons and one intron.The protein structure analysis demonstrated that the PpHCT proteins contained two conserved motifs(HXXXD,DFGWG)and a transferase domain(PF02458),which belonged to the BAHD acyltransferase family.The cis-acting element analysis suggested that the promoters of PpHCT genes contained many light-related,hormone-related,stress-related,tissue-specific,and circadian-related elements,and they could participate in a variety of biological processes.Phylogenetic analysis showed that the HCT proteins of peach were closely related to the HCT proteins of plum and had a close evolutionary relationship.The qRT-PCR analysis indicated that the expression levels of PpHCT1 and PpHCT2 showed an opposite trend to the accumulation of CGA,whereas the expression levels of PpHCT4,PpHCT5,PpHCT7,PpHCT8,and PpHCT11 demonstrated the same trend as CGA accumulation.It was worth noting that only PpHCT4 and PpHCT5 were highly expressed in the two high-CGA cultivars but showed low levels of expression in the two low-CGA cultivars.Therefore,it was hypothesized that these two genes might be key genes to the synthesis of CGA in peach fruit.Those findings provide a theoretical basis for further study on the biological functions of the HCT gene and help to reveal the molecular mechanism of CGA.

不同施肥处理对桃容器苗生长及SPAD的影响

[目的]研究不同肥料种类及施肥量对桃容器苗生长及叶绿素相对含量(SPAD)的影响,为快速培育出优质桃苗木而筛选出最适合的施肥方法。[方法]采用13种不同施肥处理,研究不同时期不同处理下桃容器苗株高、地径、冠幅、分枝数量、一年生枝条长度等生长指标及SPAD的变化。[结果]在植物生长季施用平衡型复合肥料比磷钾型腐殖酸肥料更适合植株生长;平衡型复合肥与低浓度EM菌剂组合施用比复合肥单独施用,更有利于桃容器苗的生长。[结论]生长季施用浓度1∶1000~1∶500的氮、磷、钾平衡型(20∶20∶20)复合肥+浓度1∶200的EM菌剂最有利于桃容器苗的生长及快速成苗。

基于多元素和稳定同位素技术的桃产地溯源

目的研究利用桃中多元素和稳定同位素地域特征进行产地溯源的可行性。方法应用电感耦合等离子体质谱和稳定同位素比质谱技术,测试桃样品中54种元素和碳(^(13)C/^(12)C)、氮(^(15)N/^(14)N)、氢(^(2)H/^(1)H)、氧(^(18)O/^(16)O)4种稳定同位素比值,分析不同产地桃的特征指标,采用逐步判别分析,建立产地判别模型,开展平谷、顺平和丹东桃产地溯源。结果不同产地桃中碳和氧同位素比值差异显著(P<0.05),Na、Mg、Al、K、Mn、Cu、Zn、Rb、Sr、Cd和Ba 11种元素含量差异显著(P<0.05)。筛选C、H、O、Mg、Al、K、Fe、Cu、Zn、Cd10项指标建立产地判别模型,进行原始回代检验和留一交叉检验,判别准确率均为100%。结论研究表明,同时利用桃中多元素含量和稳定同位素比值特征指标建立产地判别模型,能够区分不同产地的桃,为桃产地溯源的可行性提供方法依据。

PpWRKY4通过影响PpCCD4表达调控桃果实类胡萝卜素积累

【目的】克隆桃PpWRKY4基因,探究其在桃果实类胡萝卜素代谢过程中的调控机制。【方法】以白肉桃中油桃14号果实为材料,分析了其果实中类胡萝卜素含量变化趋势,克隆桃中PpWRKY4基因,对其进行生物信息学和表达分析。通过亚细胞定位、酵母单杂交技术和双荧光素酶试验确定PpWRKY4对PpCCD4的调控机制。【结果】随着中油桃14号果实成熟,PpCCD4基因表达量逐渐升高,类胡萝卜素含量(w,后同)由1.97μg·g^(-1)降至0.68μg·g^(-1)。PpWRKY4基因CDS区共1764 bp,编码587个氨基酸,蛋白分子质量为64.06 ku,等电点为6.00;通过氨基酸序列比对,其包含两个WRKY保守基序,为Ⅰ类WRKY蛋白。PpWRKY4基因在桃果实发育前期高表达,后期表达量较低,与PpCCD4表达趋势相反。亚细胞定位结果表明PpWRKY4定位在细胞核。酵母单杂交技术和LUC试验表明PpWRKY4通过结合PpCCD4启动子抑制其表达。【结论】PpWRKY4通过负调控PpCCD4表达进而调控桃果实类胡萝卜素降解,研究结果为进一步解析桃果实类胡萝卜素积累分子机制提供理论基础。