Molecular characterization of a polygalacturonase inhibitor from Pyrus communis L. cv Bartlett.

A polygalacturonase inhibitor glycoprotein with an apparent molecular mass of 43 kD was purified from pear (Pyrus communis L. cv Bartlett) fruit. Chemical deglycosylation of this protein decreased the molecular mass to 34 kD. Gas chromatographic analysis suggests that N-linked glycosylation accounts for the majority of sugar moieties. Partial amino acid sequence analysis of the purified polygalacturonase inhibitor protein provided information used to amplify a corresponding cDNA by polymerase chain reactions. Multiple cloned products of these reactions were sequenced and the same open reading frame was identified in all of the products. It encodes a 36.5-kD polypeptide containing the amino acid sequences determined by protein sequencing and predicts a putative signal sequence of 24 amino acids and seven potential N-glycosylation sites. The expression of polygalacturonase inhibitor is regulated in a tissue-specific manner. Activity and mRNA level were much higher in fruit than in flowers or leaves.     

Purification of d'Anjou Pear (Pyrus communis L.) Polyphenol Oxidase

Polyphenol oxidase (PPO) was extensively purified to homogeneity from d'Anjou pear (Pyrus communis L.) by extraction in the presence of the phenolic binder AG 2-X8 andTriton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Purification of pear PPO was achieved after chromatography on Phenyl Sepharose CL-4B, DEAE-cellulose, and hydroxylapatite columns. Only after the columns were run at room temperature rather than at 4°C were sharp peaks and good resolution obtained. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to successful purification. Three separate fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide slab gel electrophoresis and corresponded to relative mobilities of 0.41, 0.43, and 0.73. The effect of dimethylsulfoxide on enzyme activity was investigated and found to increase significantly the activity of purified pear PPO over the control.     

微卫星标记对高原蕨麻猪的遗传多样性研究

目的探讨蕨麻猪的亲缘关系,遗传多样性,是否受外来血缘的影响。方法用9个微卫星DNA标记对5群猪进行等位基因频率、遗传距离、系统发生树构建和主成分等分析。结果微卫星标记在125个个体中,共检测出148个等位基因,蕨麻猪最少(55个)。蕨麻猪与兰州猪的遗传距离DA和标准遗传距离DS最大,分别为0.6781和1.3312,DA、DS分别用UPGMA和NJ构建了四种系统发生树,都是蕨麻猪聚为一类,其余4种猪聚为一类。主成分1(PC1)为62.174%,看作是蕨麻猪的代表,与其他4群猪差异较大。结论蕨麻猪与兰州、武威、临洮和青海四群猪相比,遗传距离大,主成分差异大,亲缘关系较远,蕨麻猪比较纯,没有受到这4群猪血缘的影响。     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Inheritance and linkage of isozyme loci in pear (Pyrus communis L.)

 The polymorphism of 11 enzymes was analysed in 11 progenies from controlled crosses between pear varieties, using acrylamide and starch electrophoresis gels. Twenty-two loci were identified and segregation was scored for 20 of them. Three pairs of duplicated loci forming intergenic hybrid bands were detected, these correspond to equivalent duplicated genes in apple. A total of 49 active alleles and 1 null allele were identified. Joint segregation analysis revealed three linkage groups, which could all be related to existing groups on the apple map. The conservation of isozyme patterns, duplicated genes and linkage groups indicates a high degree of synteny between apple and pear. Received: 8 July 1996 / Accepted: 19 July 1996     

Penetration of naphthaleneacetic acid into pear (Pyrus communis L.) leaves

The penetration of naphthaleneacetic acid (NAA) into pear (Pyruscommunis L. cv. Bartlett) leaves as influenced by selected treatingsolution and environmental factors was studied under definedand controlled conditions. Penetration through the adaxial leafsurface was linear with time, whereas, movement through theabaxial surface was rapid initially and then the rate diminishedwith time. Penetration through both leaf surfaces was proportionalto external concentration and was temperature dependent. MoreNAA penetrated at pH values below than above the pK_a. Light(1000 ft-c) stimulated NAA penetration. The light enhanced penetrationof NAA was depressed by 3-tert-butyl-5-chloro-6-methyluracil,3-(p-chlorophenyl)-1,1-dimethylurea, phenylmercuric acetate,2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazone andcarbonylcyanide p-trifluoromethoxyphenylhydrazone. We concludedthat NAA penetration was controlled by both physical and metabolicfactors. ¹ Journal Article No. 5725 from the Michigan Agricultural ExperimentStation. This investigation was supported-in-part by NIH TrainingGrant No. 5 TOI GM 00246 from General Medical Sciences, PublicHealth Service Grant CC 00246 from the National CommunicableDisease Center, Atlanta, Georgia, and Food and Drug AdministrationGrant FD 00223. ² Present address: Department of Plant & Soil Sciences,University of Massachusetts, Amherst, Massachusetts, 01002. (Received November 9, 1971; )     

微卫星DNA标记分析野生鲤鱼群体的遗传多样性

利用30个微卫星分子标记对海南鲤(HN)、长江鲤(CY)、月亮湖鲤(YL)、黑龙江鲤(FY)、呼伦湖鲤(ZL)、贝尔湖鲤(BR)6个野生鲤鱼群体的观测杂合度(Ho)、期望杂合度(He)、多态信息含量(PIC)和有效等位基因数(Ae)等进行了遗传检测,根据基因频率计算遗传相似系数和Nei氏标准遗传距离,χ2检验估计Hardy-Weinberg平衡,用近交系数(FST)和基因流(Nm)分析群体的遗传分化及其来源。同时,使用PHYLIP3.63软件绘制基于Nei氏标准遗传距离的UPGMA进化树。6个群体共检测到8,136个扩增片段,长度在125bp～414bp,30个基因座扩增出等位基因数从3～13个不等,共计210个等位基因,平均每个基因座扩增得到7个等位基因。结果显示:(1)6个野鲤群体的多态性指标均适中,多态信息含量依次0.44、0.52、0.53、0.57、0.63和0.64,有效等位基因数1.04～4.72个不等,平均有效等位基因数依次为2.19、2.60、2.42、2.43、2.45和2.33,无偏期望杂合度平均值为0.50、0.59、0.56、0.56、0.57和0.54;(2)遗传相似系数BR与ZL最高(0.8511),BR与HN最低(0.6688),聚类结果与地理分布呈一定相关性。     

Proteomic analysis of core breakdown disorder in Conference pears (Pyrus communis L.)

2-DE was applied to study core breakdown disorder in controlled atmosphere stored 'Conference' pears. This physiological disorder is characterized by internal browning of the fruit tissue and the development of cavities. Suitable protein phenol extraction/ammonium acetate-methanol precipitation and 2-DE protocols for a wide pH range were established for pear tissue. The protein expression profiles of healthy, sound (intact tissue of pears with core breakdown) and brown tissue were analyzed with the univariate non-parametric Kolmogorov-Smirnov test and multivariate statistical techniques such as principal component analysis and partial least square discriminant analysis. Both statistical approaches revealed interesting differentially expressed proteins between healthy and disordered pears. LC-ESI-MS/MS identification of differentially expressed proteins between healthy and sound tissue revealed their participation in the energy metabolism, the antioxidant system and ethylene biosynthesis. Up-regulated characteristic proteins in brown tissue were mainly involved in energy metabolism and defense mechanisms. Proteomics coupled to univariate and multivariate statistical techniques seems to be an efficient approach to get a better insight into the different mechanisms and pathways leading to the core breakdown disorder.     

Genetic Diversity and Structure of Two Prominent Zebu Cattle Breeds Adapted to the Arid Region of India Inferred from Microsatellite Polymorphism

This study aims to assess the genetic diversity and population structure of two major zebu dairy breeds (Tharparkar and Rathi) adapted to the arid region of Rajasthan state of India. Various variability estimates indicate the existence of sufficient within-breed genetic diversity. Mean estimates of F-statistics are significantly different from zero: F _IS = 0.112 ± 0.029, F _IT = 0.169 ± 0.033, F _ST = 0.065 ± 0.017. The overall positive value of F _IS (0.112) and an F _IT value (0.169) that is more than the F _ST (0.065) indicate departure from random mating. The drift-based estimates reflect a moderate yet significant level of breed differentiation between the Tharparkar and Rathi breeds. The evaluation of an exact test, showing that allele frequencies across all the loci differed significantly, supports the population differentiation. This is paralleled by the outcome of neighbor-joining clustering based on allele-sharing distance measures. The allocation of a high percentage of individuals (95.7%) to their population of origin and correspondence analysis further substantiates the existence of a cohesive genetic structure in both the breeds.     

Foliar fertilization to control iron chlorosis in pear (Pyrus communis L.) trees

The effectiveness of foliar fertilization to re-green chlorotic leaves in iron-deficient pear trees has been studied. Trials were made to assess the influence of (i) the level of Fe deficiency, (ii) the leaf surface treated (adaxial or abaxial), and (iii) two different surfactants, L-77 and Mistol. Treatments were ferrous sulphate alone, ascorbic, citric and sulphuric acids, applied either alone or in combination with ferrous sulphate, Fe-DTPA and water as a control. Solutions were applied with a brush and leaves were treated twice each year. None of the treatments caused a full recovery from Fe deficiency chlorosis. Treatments containing Fe caused the largest re-greening effects, and FeSO₄ had a similar re-greening effect to Fe(III)-DTPA. Increases in leaf Chl were more pronounced with abaxial leaf surface applications and in severely deficient leaves. Using Fe(III)-DTPA in foliar sprays does not seem to be justified, since their effects are not better than those of FeSO₄. The joint use of Fe(III)-DTPA and L-77 and that of FeSO₄ and citric acid do not seem to be suitable. With a single foliar application, FeSO₄ combined with acids gave slightly better results than FeSO₄ alone. Acidic solution applications without Fe may be effective in alleviating chlorosis in some cases, especially in the case of citric acid. In the current state of knowledge, foliar fertilization cannot offer yet a good alternative for full control of Fe chlorosis, although its low environmental impact and cost make this technique a good complementary measure to soil Fe-chelate applications and other chlorosis alleviation management techniques. Abbreviations: Chl – chlorophyll; EDDCHA – ethylenediamine di(5-carboxy-2-hydroxyphenylacetic) acid; EDDHA – ethylenediamine di(o-hydroxyphenylacetic) acid; EDDHMA – ethylenediamine di(o-hydroxy-p-methylphenylacetic) acid; EDDHSA – ethylenediamine di(2-hydroxy-5-sulfophenylacetic) acid     

The flavonol glycosides in the fruit of Pyrus communis L. cultivar Bon Chrétien

1. Two new flavonol glycosides were isolated from the fruit of Pyrus communis L. cultivar Bon Chrétien. These were identified as isorhamnetin 3-rhamnogalactoside and a derivative of isorhamnetin 3-glucoside which was associated (possibly acylated) with an unknown aliphatic organic acid. 2. The melting point of isorhamnetin 3-glucoside isolated from Bon Chrétien pears is different from that of isorhamnetin 3-glucoside previously isolated from Argemone mexicana and Calendula officinalis. 3. Isorhamnetin 3-rhamnoglucoside was isolated from the fruit of Pyrus communis L. cultivar Bon Chrétien. This glycoside appears to be identical with narcissin, previously isolated from Narcissus tazetta and Lilium auratum. 4. Isoquercitrin, previously reported to be present in pear leaves, was isolated from the fruit of Bon Chrétien pears. 5. The isolated glycosides were present in the peels and flesh of the fruit, but were absent from the cores.     

Decreased ascorbic acid levels and brown core development in pears (Pyrus communis L. cv. Conference)

Changes in ascorbic acid content are measured in the cortex tissue of Conference pears stored at various compositions of carbon dioxide and oxygen. Enhanced carbon dioxide levels cause ascorbic acid concentrations to decline. Soon after ascorbic acid has declined below a certain value, browning of the core tissue can be observed. Reducing carbon dioxide levels before this value is reached causes ascorbic acid levels to increase again and prevents browning to a great extent. In preliminary experiments with a photoacoustic laser-based detection system, it was shown that pears that show browning produce ethane, which is most likely a result of membrane peroxidation. Storage conditions, ascorbic acid levels and browning in pears are discussed in relation to diffusion characteristics, energy metabolism and energy maintenance levels of the fruit.     

The initiation and organization of microtubules in germinating pear (Pyrus communis L.) pollen

To study microtubule organization in germinating pear (Pyrus communis L., cv., Bartlett) pollen, we removed the pollen wall by freeze-fracturing before treating the resultant pollen protoplasts by conventional immunofluorescence procedures. Results reveal that axial bundles of microtubules are present in the generative cell of both inactivated and activated pollen grains. Microtubules are not present in the vegetative cells of inactivated pollen, but they are present in the vegetative cells of activated pollen grains. Microtubule nucleation occurs in the vegetative cell cortex. Subsequently, the microtubules grow as branching arrays through most of the vegetative cell cortex except at the apertures where they form localized converging or criss-cross patterns. Eventually, in a germinated pollen grain, the microtubules form network-like arrays through most of the pollen grain and a collar of short arrays at the base of the pollen tube. It is suggested that the role of vegetative cell microtubules in pollen germination is indirect through their mediation of the conformational changes in actin organization that are essential for pollen germination.     

Iron deficiency-induced changes in the photosynthetic pigment composition of field-grown pear (Pyrus communis L) leaves

In this work we characterize the changes induced by iron deficiency in the pigment composition of pear (Pyrus communis L.) leaves grown under high light intensities in field conditions in Spain. Iron deficiency induced decreases in neoxanthin and β-carotene concomitantly with decreases in chlorophyll a, whereas lutein and carotenoids within the xanthophyll cycle were less affected. Iron deficiency caused major increases in the lutein/chlorophyll a and xanthophyll cycle pigments/chlorophyll a molar ratios. The chlorophyll a/chlorophyll b ratio increased in response to iron deficiency. The carotenoids within the xanthophyll cycle in iron-deficient and in iron-sufficient (control) leaves underwent epoxidations and de-epoxidations in response to ambient light conditions. In control leaves dark-adapted for several hours, most of the xanthophyll cycle pigment pool was in the epoxidated form vio-laxanthin, whereas iron-deficient leaves had significant amounts of zeaxanthin. Iron-deficient leaves also exhibited an increased non-photochemical quenching, supporting the possibility of a role for pigments within the xanthophyll cycle in photoprotection.     

Iron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) trees.

Experiments have been carried out with field-grown pear trees to investigate the effect of iron chlorosis on the composition of the leaf apoplast. Iron deficiency was associated with an increase in the leaf apoplastic pH from the control values of 5.5-5.9 to 6.5-6.6, as judged from direct pH measurements in apoplastic fluid obtained by centrifugation and fluorescence of leaves incubated with 5-CF. The major organic acids found in leaf apoplastic fluid of iron-deficient and iron-sufficient pear leaves were malate, citrate and ascorbate. The total concentration of organic acids was 2.9 mM in the controls and increased to 5.5 mM in Fe-deficient leaves. The total apoplastic concentration of inorganic cations (Ca, K and Mg) increased with Fe deficiency from 15 to 20 mM. The total apoplastic concentration of inorganic anions (Cl-, NO3-, SO4(2-) and HPO4(2-)) did not change with Fe deficiency. Iron concentrations decreased from 4 to 1.6 microM with Fe deficiency. The major Fe species predicted to exist in the apoplast was [FeCitOH](-1) in both Fe-sufficient and deficient leaves. Organic acids in whole leaf homogenates increased from 20 to 40 nmol x m(-2) with Fe deficiency. The accumulation of organic anions in the Fe-deficient leaves does not appear to be associated to an increased C fixation in leaves, but rather it seems to be a consequence of C transport via xylem.     

Cloning and mapping multiple S-locus F-box genes in European pear (Pyrus communis L.)

European pear, as well as its close relatives Japanese pear and apple, exhibits S-RNase-based gametophytic self-incompatibility. The male determinant of this self-incompatibility mechanism is a pollen-expressed protein containing an F-box domain; in the genera Petunia (Solanaceae), Antirrhinum (Plantaginaceae), and Prunus (Rosaceae), a single F-box gene determines the pollen S. In apple and Japanese pear, however, multiple S-locus F-box genes were recently identified as candidates for the pollen S, and they were named S-locus F-Box Brothers. These genes were considered good candidates for the pollen S determinant since they exhibit S-haplotype-specific polymorphisms, pollen-specific expression, and linkage to the S-RNase. In the present study, S-locus F-Box Brothers homologs have been cloned from two of the most agronomically important European pear varieties, “Abbé Fétel” (S_104-2/S₁₀₅) and “Max Red Bartlett” (S₁₀₁/S₁₀₂), and they have been mapped on a genetic linkage map developed on their progeny. Our results suggest that the number of F-box genes linked to the S-locus of the European pear is higher than expected according with previous reports for apple and Japanese pear, since up to five genes were found to be linked to a single S-haplotype. Moreover, two of these genes exhibited an incomplete linkage to the S-RNase, allowing the identification of low-frequency recombinant haplotypes, generated by a crossing-over event between the two genes. These F-box genes are most likely placed in close proximity of the S-locus but do not belong to it, and they can thus be excluded from being responsible for the determination of pollen S function.     

利用微卫星标记分析山东地方鸡品种的遗传多样性

微卫星是近几年来应用较多的一种分子标记,可有效地进行基因鉴定与系谱分析,并可估算群体间的遗传距离。通过选用5个微卫星标记,检测了山东省5个地方鸡种：日照麻鸡、寿光鸡、莱芜黑鸡、济宁百日鸡、鲁西斗鸡以及一个外来鸡种——安卡黄鸡和一个外省地方鸡种——广西黄鸡共7个鸡种的遗传多样性。根据测试结果计算了每个等位基因的频率,以基因频率为基础分析了品种内的遗传变异和品种间的DA遗传距离,并讨论了微卫星多态性在应用于群体遗传变异及亲缘关系等方面的意义。结果表明：共检测到40个等位基因,其中等位基因数最多的位点为。ADL0136(10个);等位基因数最低的位点为ADL0146(5个);而且每个位点的等位基因分布并不均匀,都有一种或几种优势基因存在。在7个品种中,杂合度最低的为寿光鸡,杂合度值为0．3327,因为此鸡种多年来一直由寿光市慈伦种鸡场进行纯繁保种,未与其他鸡种杂交,因此杂合度最小;其他鸡种杂合度也都低于0．4,据分析可能是由于日照麻鸡、济宁百日鸡群体较小;莱芜黑鸡是正在选育的一个品种,个体间遗传关系也不远;安卡黄鸡和广西黄鸡自从引人嘉明公司后,群体近交现象普遍,因此各鸡种杂合度都偏低。由此可见,通过对杂合度的计算,微卫星可以较好地反应群体内的变异。各品种PIC值的变动范围从0．6196(寿光鸡)到0．7027(莱芜黑鸡),PIC值的大小与杂合度的高低体现了较高的一致性。对DA遗传距离的计算表明：日照麻鸡与济宁百日鸡的距离最近,而鲁西斗鸡与其他鸡种距离均较远。用UPGMA法进行聚类分析,结果7个鸡种被聚为3类：山东的4个地方鸡种寿光鸡、日照麻鸡、莱芜黑鸡与济宁百日鸡聚为一类;安卡黄鸡和广西黄鸡聚在一起;鲁西斗鸡独自为一类。这与几个鸡种的分化与选育历史是一致的,因此聚类图能够比较正确地反映7个品种之间的亲缘关系。