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。

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.

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.

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.

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.

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)转录组SSR信息分析及其分子标记开发

利用MISA软件筛选桃(Prunus persica[L.]Batsch)转录组数据,获得的109 248条Unigene,检测出24 102个SSR位点,分布于22 689条Unigene中,出现频率为20.76%。SSRs位点中主导类型是以AG/CT(占总SSRs的18.70%)为主的二核苷酸重复,占总SSRs的33%;其次是三核苷酸重复,出现频率为27%;四核苷酸重复,占总SSRs的25%。利用Primer 5共设计出9 657对SSR引物。随机选择50对引物进行PCR扩增,其中20对扩增出清晰可重复的条带,并且在同属材料(苹果试材)中也能获得理想结果,引物多态性检测发现,在15份桃试材中,10对引物具有多态性,利用UPGMA作图,可将15个桃材料很好区分。结果表明,桃转录组测序产生的Unigene是开发SSR标记的有效信息,为SSR标记在桃的遗传分析上提供可靠的标记选择。

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.

HPLC法测定不同产地桃仁中苦杏仁苷的含量

目的:建立不同产地桃仁中苦杏仁苷含量的高效液相测定方法,测定不同产地桃仁药材中苦杏仁苷的含量。方法:色谱柱HypersilBDS C18(250mm×4.6mm,5μm),流动相为乙腈-水(17∶83)。结果:苦杏仁苷在4.94～80μg/mL范围内线性关系良好,r=0.9996,平均回收率为99.87%,RSD=1.01%。含量测定结果表明,不同产地桃仁药材中苦杏仁苷含量存在差异。结论:该方法简便快捷,准确灵敏,重复性好,可用于桃仁药材的质量控制。

不同方法测定桃树抗寒性关系研究

以生产中抗寒性表现较强、中等、较弱的3个桃品种为试材,应用电解质渗透法(EL)和电阻抗图谱法(EIS)研究了不同时期枝条抗寒性和未经冷冻处理枝条EIS参数的变化,并对未经冷冻处理枝条EIS参数、冷冻处理枝条EIS参数测定的半致死温度与EL测定的半致死温度的关系进行了统计分析。结果表明:未经冷冻处理3个桃品种的胞外电阻率(r_e)和胞内电阻率(r_i)均随着外界温度的降低而升高,随着外界温度的升高而降低;弛豫时间(τ)与r_(e)、r_(i)变化趋势相反;通过对4个电阻抗参数与EL测定的半致死温度统计分析得出,EIS(r_(i))是最佳参数,其相关系数为-0.828,通径系数为-7.385,线性回归方程为y=-4.063 9x+4.333 8,R^(2)=0.686。EL和EIS参数法测定3个桃品种的抗寒性与生产上抗寒性强弱表现一致;通过对2种方法统计分析得出,EL与EIS 4个参数法测定抗寒性均呈极显著正相关,其中弛豫时间法相关系数、通径系数和决定系数均最高,因此,弛豫时间是预测冷冻处理桃树枝条抗寒性的适宜参数。

Relationships Between the Distribution of Relative Canopy Light Intensity and the Peach Yield and Quality

The aim of the present experiment was to study the relationship between the distribution of relative light intensity in canopy and yield and quality of Wanmi peach. The optimum relative canopy light intensity was judged to be 36.3% for high quality peaches, when canopy volumes of Wanmi peach trees with a relative light intensity 〈 30% accounted for 7.7 and 47.9% of the total canopy volume in June and September, respectively. The canopy volume with a relative light intensity 〉 80% was 27.7 and 3.1% of the total canopy volume in June and September, respectively. Peach canopies were divided into 0.5 m × 0.5 m × 0.5 m cubes, with the relative light intensity being measured at different positions of the canopy during the growing season. Yield and fruit quality were also measured at these positions at harvest. The results showed that the relative light intensity decreased gradually from outside to inside and from top to bottom of the tree canopy. Fruit were mainly distributed in the upper and middle portions of the canopy, 1.5-3.0 m above ground. Regression results showed that single fruit weight and soluble solid content were positively related to relative light intensity.

Effects of Temperature and Several Chemicals on Metabolic Changes During Dormancy Release in NJ72 Nectarine

Poor, delayed and ununiform budbreak is a major problem for peaches in greenhouse. To clarify the mechanism of breaking bud dormancy in nectarines, the effect of temperature and three dormancy-breaking agents on metabolic changes during dormancy release in two-year old NJ72 nectarine (Prunus persica L. Batch) was investigated. The result showed temperature and chemicals affected the budbreak and the metabolism of NJ72 nectarine during dormancy. Endogeneous peroxide content in buds increased soon after low temperature treatment. Meanwhile, catalase activity was also shown to increase significantly at low temperature treatment, coincided with increase of the activity of peroxidase and superoxide dismutase. The rate of respiration in flower buds increased at low temperature during dormancy. The rate of the pentose phosphate pathway increased, while the rate of the Embden-Meyerhof pathway decreased and the rate of tricarboxlic acid cycle changed little. Glucose 6-phosphate dehydrogenase activity increased at low temperature during dormancy. At the same time we found an accumulation of peroxide after treatment with dormancy-breaking chemicals. In flower buds treated with dormancy-breaking agents, thiourea, KNO3 and NH4NO3, catalase activity was inhibited soon after treatment, whereas peroxidase activity increased, and the changes of superoxide dismutase remained little. In this study, it was found that the rates of respiration in flower buds increased by chemicals sprays during dormancy. The activity of glucose 6-phosphate dehydrogenase, the key enzyme in the pentose phosphate pathway (PPP), increased by spraying with dormancy-breaking agents, concomitantly with the activation of the pentose phosphate pathway.

Effects of Different Storage Methods on the Fruit Quality and Physiological Changes in Yihong Honey Peach

Taking Yihong,the main cultivar of mid-ripening honey peaches(Prunus persica)in Hunan,as material,this study explored the effects of different storage methods on the fruit quality and physiological changes in Yihong honey peach by the combination of polyethylene film packaging with low-temperature storage.The results showed that the weight loss rate of packaged storage at normal temperature(T1)was significantly lower than that of unpackaged storage at normal temperature(CK),but there was no significant difference in other indicators.In the middle of storage,the respiration rate and ethylene release of packaged storage at 0℃(T3)were extremely significantly lower or significantly lower than those of unpackaged storage at 0℃(T2),packaged storage at 0℃after cold adapting at 8℃for 5 d(T4),and packaged storage in a fluctuating temperature range of 0~3.5℃(T5).After storage,the structure of the middle lamella in the cell wall of T3 was compact and uniform,and the mitochondrial structure was complete,while the cell walls and mitochondria of T4 and T5 were severely deformed and disintegrated.The activities of antioxidant enzymes peroxidase(POD)and superoxide dismutase(SOD)of T3 was significantly higher than those of T4 and T5,and the content of malondialdehyde(MDA)and polyphenol oxidase(PPO)of T3 was also the lowest.Therefore,0°C packaged storage can effectively delay the senescence of Yihong honey peach and prolong its freshness period.

Effect and Functional Mechanism of the Action of Exogenous Gibberellin on Flowering of Peach

This study was conducted to assess the effect of gibberellin and its possible mechanism of action on peach flower formation. At flower induction, 100 mg L^-1 of gibberellic acid 3 （GA3） was sprayed on the leaves of peach [Prunus persica （L.） Batsch.] cv. Bayuecui. Using anatomy, immunohistochemistry, and semi-quantitation, the in situ distribution of GAs and the expression of the key genes involved in peach flower formation in the apical meristem were studied during flowering differentiation. The results showed that induction of flowering in the Bayuecui peach occurred prior to 10 July in Beijing, China. Flower induction and further differentiation of the peach flower organs were significantly inhibited by leaf-spraying of GA3 at a concentration of 100 mg L^-1 during the induction stage. The flowering rate was only 11.67% after treatment. The distribution of GA1 in the apical meristem varied during the process of flower bud differentiation. From 13 June to 25 July, the GA1 signal from control plants was detected mainly in the vascular bundles at the base of the flower buds. No GA1 signal was detected in the apical meristem. After treatment with GA3, the distribution was similar to that of the control from 13 June to 3 July. On 13 July, a GA1 signal was detected in the apical meristem accompanied by an increase in the GA1 signal in the vascular bundles at the base of the flower buds. The GA1 signal weakened significantly in both the vascular bundles and the apical meristem on 25 July. The expression of the genes PpLEAFY and MADS6 in flower buds could be detected only on 10 October in the GA3-treated plants. The critical period for flower induction of Bayuecui peach in Beijing was in early July, during which time, leaf-spraying with 100 mg L-1 GA3 could effectively inhibit flower induction and further differentiation of the flower buds. GA1 in the gibberellin family was the suppressor for flower induction in peach. Its action was affected by the stage of flower bud differentiation. Expression of the key genes PpLEAFY and MADS6 involved in flower formation was inhibited by GA3 treatment.

氨基酸水溶肥促进桃苗生长

氨基酸水溶肥是具有营养效果好、无污染等优点的新型绿色肥料。以桃(Prunus persica L.)苗为材料,通过盆栽试验探究施用不同稀释倍数(600、900、1200、1500倍)的氨基酸水溶肥对桃苗生长的影响,以期筛选出效果最佳的喷施浓度。结果表明,施用氨基酸水溶肥增加了桃苗各部位的生物量,其根、茎、叶及地上部分生物量在稀释倍数为1500倍时最高,较对照分别增加了54.83%、51.28%、35.73%和42.12%;施用氨基酸水溶肥提高了桃苗光合色素含量,当稀释倍数为1500倍时,桃苗的光合色素含量最大;施用氨基酸水溶肥提高了桃苗的抗氧化酶活性,但降低了桃苗的可溶性蛋白质含量。因此,稀释1500倍的氨基酸水溶肥为促进桃苗生长的最佳喷施浓度。

丛枝菌根真菌对桃幼苗生长及硒富集的影响

采用盆栽试验,研究了摩西球囊霉(Glomus mosseae)、脆无梗囊霉(Acaulospora delicata)、隐类球囊霉(Paraglomus occultum)和幼套球囊霉(Glomus etunicatum)4种丛枝菌根真菌对桃[Prunus persica(L.)Batsch]幼苗生长及硒富集特性的影响。结果表明,施用脆无梗囊霉增加了桃幼苗的生物量,促进了桃幼苗的生长,使桃幼苗的根系和地上部分生物量分别比未施用增加了11.28%和9.18%,也在一定程度上提高了光合色素含量、过氧化酶活性及过氧化氢酶活性。施用摩西球囊霉、隐类球囊霉和幼套球囊霉3种丛枝菌根真菌降低了桃幼苗的生物量、光合色素含量及抗氧化酶活性或对其没有显著影响。施用脆无梗囊霉也在一定程度上增加了桃幼苗的总硒含量及有机硒含量,其中地上部分的总硒含量和有机硒含量分别较未施用增加了8.01%和9.34%。施用摩西球嚢霉、隐类球囊霉和幼套球囊霉降低了桃幼苗的总硒含量及有机硒含量或对其没有显著影响。

电解质渗透法和电阻抗参数法测定桃树抗寒性关系

以抗寒性不同的6个桃树品种为试材,研究了电阻抗图谱(EIS)与电解质渗透法(EL)2种方法测定冷冻枝条半致死温度的相关关系,以期找到评价桃树抗寒性的简便方法,为新品种的早期鉴定以及适地栽培提供参考依据。结果表明:2种方法测定6个桃品种的抗寒性与生产上抗寒性强弱表现一致;电阻抗参数法可以在抗寒锻炼后期和脱锻炼初期进行抗寒性的测定;EL法与EIS 4个参数法测定抗寒性均呈极显著正相关,其中τ法相关系数最高,为0.904,τ法通径系数为-1.389,线性方程为y=1.0126x-2.0761,R^(2)为0.8178。

中国桃主要品种资源及其野生近缘种的分子身份证构建

【目的】以国家果树种质郑州葡萄、桃圃保存的237份中国桃地方品种、育成品种及其野生近缘种为试材,进行种质分子身份证构建工作,并对构建方法进行探讨。【方法】采用筛选后的SSR标记对品种进行区分,然后根据引物对不同品种扩增条带分子量的大小进行编码、组合。【结果】从80对引物中筛选出来自桃8条染色体上的16对SSR引物,在供试种质间共检测出等位基因203个,每对引物平均检测到等位基因数为12.7个。根据等位基因既位于地方品种、育成品种,又位于近缘野生种的选择方法,筛选出123对等位基因并赋值后可用于构建种质的分子身份证。【结论】在237份种质中有202份具有的可辨分子身份证编码,继续对不同引物组合对种质的筛选效率进行分析,筛选出8对核心引物使176份种质具有可辨的分子身份证,平均每对引物区分种质达22.1份,即在保证每对引物区分效率较高的基础上,兼顾了总的区分数量。最后,为使区分品种简便化,依据引物的多样性指数先后选择引物组合,达到用最少的引物组合区分不同品种的目的。

桃炭疽病病原鉴定及木醋液防治研究

炭疽病是湖北省桃[Prunus persica(L.)Batsch]产区一种重要的桃树病害。为有效控制该病的危害,试验采用分子生物学技术对湖北省桃园炭疽病的病原进行了分子鉴定,并在实验室内测定了不同浓度的木醋液对炭疽病病原菌的抑菌效果。结果表明,木醋液稀释300倍后对炭疽病病原菌的抑菌效果仍在70%以上;同时田间试验结果表明,木醋液能较好地控制炭疽病危害,在桃园绿色防控上具有广阔的应用前景。

湖北地方红肉桃种质资源收集、评价及利用

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