Use of Degenerate Primers in a RT-PCR Assay for the Identification and Analysis of Some Filamentous Viruses, with Special Reference to Clostero- and Vitiviruses of the Grapevine

RT-PCR with degenerate primers was used for the screening of the genome of some members of the Closterovirus, Vitivirus and Trichovirus genera. Two sets of primers, targeted to conserved sequences of the heat shock protein 70 homologue of closteroviruses or to the RNA dependent RNA polymerase genes of tricho- and vitiviruses, amplified the expected fragments from total RNA extracts or double-stranded RNAs of infected plants. Amplified cDNAs were cloned, sequenced and phylogenetically analyzed. Results support the allocation of grapevine viruses A, B, D and heracleum latent virus (HLV) in the genus Vitivirus, whereas, the detection of a HSP70 homologue in grapevine leafroll-associated viruses agrees with their assignment in the genus Closterovirus. The use of degenerate primers for the identification of grapevine viruses belonging to Vitivirus and Closterovirus genera is envisaged.     

绵羊FecB突变检测方法的建立及高繁滩羊核心群的构建

旨在建立快速、高效、精准检测FecB突变的荧光qPCR技术,为滩羊多羔性能研究、群体改良及肉用多羔滩羊新品系培育提供技术支撑。基于荧光qPCR技术基本原理,设计特异性引物对,开发基于荧光qPCR检测绵羊FecB突变的方法;对85只已知FecB基因型的健康、适繁雌性滩羊血液基因组DNA样品分别采用PCR-Sanger测序法、TaqMan探针法和荧光qPCR法进行FecB基因分型,统计3种方法的准确率;采用开发的荧光qPCR方法对939只健康、适繁滩羊进行FecB基因分型,统计不同FecB基因型经产母羊的产羔率。结果表明,使用TaqMan探针法、PCR-Sanger测序法和开发的荧光qPCR法对已知FecB基因型样品检测的比较中,荧光qPCR法对各基因型鉴定的准确度均达100%,高于前两者。939只滩羊FecB基因分型结果显示,6只滩羊为FecB突变纯合型（BB）、57只为杂合型（B+）、876只为野生型（++）,BB型、B+型和++型滩羊的基因型频率分别为0.64%、6.07%和93.29%,其中B等位基因频率为0.04,+等位基因频率为0.96;监测母羊群体的产羔数发现,FecB突变纯合型滩羊产羔率为166.67%,突变杂合型滩羊产羔率为153.12%,野生型滩羊产羔率为105.78%,纯合突变型和杂合突变型母羊群体的产羔率极显著高于野生型群体（P<0.01）。综上所述,与TaqMan探针法及PCR-Sanger测序法相比,本研究建立的绵羊FecB突变荧光qPCR分型方法具有简便易行、廉价高效的优点,在绵羊的分子选育中具有较高应用价值;同时本研究证实了滩羊FecB突变可显著提高母羊产羔率,为多羔滩羊的分子选育提供了坚实的技术支撑。     

普陀樟SRAP-PCR反应体系的建立

以舟山群岛的普陀樟（Cinnamomum japonicum var.chenii）新鲜叶片为实验材料,采用L16（45）正交试验设计,对影响普陀樟SRAP-PCR反应的Mg2+浓度、dNTPS、Taq DNA聚合酶、引物和模板DNA用量5因素组合进行了筛选。结果表明：适于普陀樟的SRAP-PCR反应体系（20μL）为2.5 mmol/L Mg2+、0.2 mmol/L dNTPs、1.5 U Taq DNA聚合酶、0.4μmol/L引物、10 ng模板DNA、2μL 10×PCR buffer,该体系位点清晰,扩增稳定;利用该反应体系从100对SRAP引物组合中筛选出多态性好的引物24对。     

基于白木香ESTs的沉香属SSR分子标记引物筛选

通过对白木香样品的454GS FLX Titanium高通量测序,获取了大量白木香EST序列,从中搜索出956个SSR,根据引物设计标准,设计了44对EST-SSR引物。在合适的PCR反应体系下,以海南白木香样品的DNA为模板,对设计的EST-SSR引物进行筛选,发现有22对EST-SSR引物能较好地扩增出产物。在沉香属3个种9份样品中进一步对这些可扩增的引物进行多态性初步验证,所筛选引物均可在所有沉香属样品中使用,为后续白木香居群及沉香属植物遗传多样性分析奠定基础。     

旱稻孢囊线虫的快速分子检测

根据旱稻孢囊线虫(Heterodera elachista)和常见孢囊线虫的ITS序列比对分析,设计1对旱稻孢囊线虫特异性引物He–F/He–R,特异性片段长度为281 bp。运用该特异性引物及建立的DNA提取方法和PCR体系,可特异性检测旱稻孢囊线虫单条2龄幼虫,可以从混合有1条旱稻孢囊线虫的0.1 g水稻根组织中特异性检测出目的DNA片段。特异性引物He–F/He–R与通用引物D2A/D3B结合,运用一步双重PCR检测方法可快速鉴定单孢囊,也可从初始分离的田间土壤总线虫样品中直接检测出旱稻孢囊线虫。     

日本结缕草（Zoysia japonica）磷脂酶D基因部分 保守序列的克隆及其对机械损伤的响应

以日本结缕草Zenith为材料,通过同源克隆的方法克隆出其磷脂酶D(PLD)基因部分保守序列,序列长399 bp,并通过实时荧光定量PCR检测PLD基因在机械损伤胁迫下随时间变化的表达模式.结果表明,磷脂酶D在胁迫2h内大量表达,随后逐渐降低,在9h已接近于对照水平,说明PLD基因在损伤初期大量参与了应对机械损伤胁迫的防御与修复过程,在损伤后期其作用减弱或被代替;电导率的测定结果表明,细胞膜结构在胁迫初期遭到极大程度的破坏,胁迫9h后膜损伤程度开始减轻.结合PLD基因和电导率的表达模式,推测PLD基因在6h内会激活一系列感受信号及下游信号分子,9h后其相应防御机制发挥作用来减轻胁迫造成的损害.     

冬瓜SSR-PCR体系优化及引物筛选

本研究利用L16(54)正交试验设计研究了影响冬瓜SSR-PCR的五个因子(10×Buffer(含Mg2+),d NTP,DNA,引物,r Taq),研究结果表明Buffer(含Mg2+)和r Taq对PCR影响最明显,最终筛选出最佳反应体系:10×Buffer 2.0μL(1×Buffer),d NTP 2.0μL(200μmol/μL),DNA 0.6μL(6 ng/μL),引物0.4μL(0.2μmol/L)。体系体积为20μL,循环数为35。引物筛选程序为:94℃预变性3 min,94℃变性45 s,58℃~68℃复性30 s,5个循环,每个循环退火温度降2℃,72℃延伸1 min,94℃变性30 s,50℃~58℃复性1 min,8个循环,每个循环退火温度降低1℃,72℃延伸1 min,94℃变性30 s,50℃复性30 s,72℃延伸1 min,共30个循环,72℃延伸7 min。本研究建立了适合冬瓜的SSR反应体系和引物多态性筛选程序。     

Distribution, habitat and biomass of Pittosporum undulatum, the most important woody plant invader in the Azores Archipelago

Pittosporum undulatum Ventenat (Pittosporaceae) is a tree or shrub native to Australia introduced in the Azores Islands in the 19th century, presently naturalized in the nine islands. According to a random survey of vascular plants in the Azores, the invader is present throughout the archipelago, in 62% of 547 1-km² samples. It was found in pure or mixed stands, forming groups and also as isolated trees. P. undulatum was frequently found in native scrubland (62%), mixed woodland (39%) and hedgerows (25%). The altitudinal range extended from sea level up to about 800 m a.s.l., with the highest frequency between 100 and 400 m. The woody species more strongly associated with P. undulatum included characteristic native and endemic species as well as non-indigenous and invasive taxa. Based on a forest inventory, 49% of the forested area in the Azores, about 24,000 ha, is occupied by P. undulatum. Considerable areas inside Island Natural Parks are covered by this species. The estimated annual P. undulatum biomass production in the Azores might range from only about 150 Mg in the small island of Corvo up to more than 60,000 Mg in Pico Island. The heating value of its wood and its chemical composition make it a good candidate for use in combustion or gasification processes. Since there are no resources available to control this large-scale invasion, using P. undulatum biomass for energy production might be an important factor to stimulate the progressive and sustainable cutting of its stands and its replacement by Macaronesian species.     

Use of random amplified DNA markers to analyse genetic variability and relationships of Coffea species

Summary The use of random amplified DNA fragments as genetic markers in Coffea was investigated. Arbitrary oligonucleotides were used as primers to amplify genomic DNA of different coffee accessions representing major Coffea species by polymerase chain reaction. Intraspecific variation was easily detected in C. canephora and C. liberica whereas the primers assayed failed to reveal polymorphism between C. arabica accessions. Extensive interspecific variation was observed. Genetic relationships between Coffea species are deduced from the degrees of similarity in amplified product profiles. Random amplified DNA markers appeared to be of high value for characterization, analysis and utilization of coffee genetic resources.     

Assessment of genetic relationships between <Emphasis Type="Italic">Rhus</Emphasis> L. species using RAPD markers

Genetic diversity of seven Rhus L. species was assessed using random amplified polymorphic DNA (RAPDs) markers. Initially, 90 primers were screened, of which 25 produced reproducible amplification products. These primers generated a total of 296 bands, with an average of 11.8 bands per primer. Out of 296 bands scored, 236 (80%) were polymorphic and 62 (20%) were monomorphic. Primers OPC-05 and OPD-05 generated 100% polymorphic bands. The resolving power of primers ranged from 9.4 to 26.8. Similarity matrix values ranged from 0.45 to 0.63. The dendrogram generated using Unweighted Pair Group Method using Arithmetic Averages (UPGMA) grouped all the species of Rhus in one major group with two sister groups, whilst R. pyroides Burch. and R. dentata Thunb. were outliers. R. gerrardii (Harv. ex Engl.) Diels, R. glauca Thunb. and R. pentheri Zahlbr. constituted one sister group, while R. natalensis Bernh. ex C. Krauss and R. gueinzii Sond. were included in the other. The degree of genetic diversity observed between seven species of Rhus with RAPD markers suggest that this approach could be used for studying the phylogeny of the genus.