基于EST数据库进行SNP分子标记开发的研究进展及在猕猴桃属植物中的应用研究

对基于EST数据库开发SNP标记的特点、开发策略等进行了综述,并介绍了在中华猕猴桃复合体(Actinidia chinesis Planch.)中开发EST-SNP的基本思路和初步结果,为后续分子实验验证及其在自然居群中的应用奠定基础,并为其它相关物种的EST-SNP分子标记开发提供借鉴.     

Cryopreservation of shoot tips,evaluations of vegetative growth,and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’ (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis ‘Jinmi’, respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.     

利用AFLP分析西南特色猕猴桃的遗传多样性

为分析西南地区特色猕猴桃(Actinidia Lindl.)的遗传多样性,建立并优化了猕猴桃DNA 多态性分析的AFLP 体系,AFLP 标记体系为300 ng 基因组DNA 用EcoR Ⅰ/Mse Ⅰ (15 U/5 U)于37℃下酶切2 h,加接头后的混合物稀释5 倍用于预扩增,预扩增产物再稀释10 倍后进行选择性扩增.结果表明,筛选的22 对引物进行扩增反应,共得到979 条条带,其中多态性条带为649 条.采用优化的AFLP 体系,对西南地区的10 种猕猴桃50 个样本进行了遗传多样性分析,包括普通品种中华猕猴桃、美味猕猴桃,及特色品种硬毛猕猴桃、毛被猕猴桃、革叶猕猴桃、四萼猕猴桃、狗枣猕猴桃、葛枣猕猴桃、京梨猕猴桃和紫果猕猴,结果表明种内和种间的聚类关系明显;斑果组(sect. Maculatae)和净果组(sect. Leiocarpae)的种间有聚类交叉,并呈现地理区域性分布.     

猕猴桃溃疡病流行的生态因子及药剂对病菌的抑菌作用

经5年系统研究得出,影响猕猴桃溃疡病发生流行的特点是,海拔750m以上的果园发病重,向阳坡发病重于背阳坡,不同品种发病差异显著,金丰品种易感病,金魁品种高度抗病;树龄越大,病株率越高;不同枝龄比较,一年生枝条病枝死亡率和枝条死亡率最高.6种药剂以加瑞农和硫酸链霉素抑菌效果最好.     

高海拔地区猕猴桃耐贮性研究初报

产于海拔700m地区的猕猴桃果实在相同的贮藏条件下比海拔100m地区的果实耐贮2～3个月,这与高海拔地区各种生态因子的综合作用有关.果皮的组织解剖表明,高海拔地区果实的果皮木栓组织一般5～7个细胞层,低海拔地区的一般4～5个细胞层.     

Adventitious root formation of kiwifruit in relation to sampling date, IBA and Agrobacterium rubi inoculation

During the winters of 2001 and 2002, the effects of three strain of Agrobacterium rubi (A1, A16 and A18), a range of IBA concentrations (0, 2000, 4000 and 6000 ppm) alone and in combination with three strains of Agrobacterium rubi and date of cutting collection on the rooting of hardwood stem cuttings of kiwifruit cv. Hayward were evaluated. Treatments of hardwood stem cuttings of kiwifruit cv. Hayward with the bacteria, IBA and IBA plus bacteria were found to promote rooting. Highest rooting percentage was obtained from cuttings treated with 4000 ppm IBA plus A18 in cv. Hayward in both years. Higher rooting percentages were observed when shoots were collected in February rather than in January.     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.     

Gender variation in Actinidia deliciosa,the kiwifruit

Summary Flower and fruit characters were measured in ten female, five male and five fruiting male selections of A. deliciosa var deliciosa (A. Chev) Liang and Ferguson. Flowers from female vines had functional pistils, which contained many ovules. Stamens appeared to be fully developed but produced only empty pollen grains. Flowers from male vines had functional stamens that produced high percentages of pollen grains with stainable cytoplasmic contents. Pistils did not contain ovules and were generally small with vestigial styles. Fruiting male vines had both staminate and bisexual flowers. Staminate flowers were similar to those found on strictly male vines. Bisexual flowers produced ovules and stainable pollen. Pistils were smaller than in pistillate flowers. Although the three flower sexes differed in style length, ovary dimensions and ovules per carpel, staminate and bisexual flowers were similar in number of flowers per inflorescence, stamen filament length, pollen stainability, inflorescence rachis length and carpel number, and differed from pistillate flowers in these characters. The three flower sexes had similar sepal and petal numbers. The fruit of fruiting males were considerably smaller than those of females. Low ovule number appears to be the major factor limiting fruit size in the fruiting males studied. Prospects for developing hermaphroditic kiwifruit cultivars through breeding are discussed.     

An ELISA method to identify the phytotoxic Pseudomonas syringae pv. actinidiae exopolysaccharides: A tool for rapid immunochemical detection of kiwifruit bacterial canker

The phytotoxic exopolysaccharides produced by Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit, were isolated and partially identified. Their phytotoxic activity was evaluated on host and non-host plants and their role in the complex mechanisms of host-pathogen interaction was also discussed. The phytotoxic exopolysaccharides, which are natural antigens, were used to arise specific antibodies by rat immunization. The antibodies were used to develop a rapid and specific method to unambiguously detect P.s. pv. actinidiae exopolysaccharides isolated from bacterial culture and infected plant samples. Indeed, the antibodies recognized the exopolysaccharides produced by other two strains of P. s. pv. actinidiae but did not cross reacted with those isolated from P. s. pv. syringae and Pseudomonas viridiflava culture filtrates. Finally, the same antibodies significantly recognized the exopolysaccharides extracted from infected kiwi leaves.     

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 μM CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules.