梨果实酸/低酸性状的SSR分析

以八月红(Pyrus communis L.‘Bayuehong')×砀山酥梨(P.× bretshneider‘Dangshansuli')F1代杂交分离群体(共118株)为试材,测定了成熟果实的酸含量,从表型上分析了梨果实含酸量的遗传规律.利用225对源自梨和苹果基因组的SSR引物,结合分离群体分离分析法(Bulk...     

主要组织相容性复合体在鸟类保护生物学中的应用

在保护生物学研究中,主要组织相容性复合体（major histocompatibility complex,MHC）的遗传多样性越来越受到人们的重视。在研究比较广泛和深入的哺乳动物和鱼类中,MHC被认为与抗病能力、配偶选择和亲缘识别有关。然而,MHC基因对哺乳动物行为产生影响的机制是否适用于鸟类,特别是鸟类野生种群,尚不清楚。文章对MHC多态性机制、MHC基因分型方法及其与鸟类疾病抗性、配偶选择的关系等进行了综述,为研究人员对鸟类保护生物学的深入研究提供参考。     

Comparison of genotyping by sequencing and microsatellite markers for unravelling population structure in the clonal fungus Verticillium dahliae

Microsatellite genotyping of a large sample of isolates of Verticillium dahliae from diverse locations recently identified seven distinct genotypic clusters. However, these clusters were not put in the context of phenotypes known to be correlated with clonal lineages in V. dahliae. The objective of this study was to compare clusters defined by microsatellite markers with clonal lineages defined by single‐nucleotide polymorphisms (SNPs) and vegetative compatibility groups (VCGs). Genotyping isolates known to belong to specific clonal lineages (based on SNPs) with microsatellite markers determined the correspondence of clusters and lineages. All but one cluster corresponded to a known clonal lineage, allowing analysis of correlations of phenotypes with microsatellite genotypes from other studies. As shown previously, most race 1 isolates are in lineage 2A, and most isolates with the defoliating pathotype are in lineage 1A. Phylogenetic incompatibility was used to test for recombination or homoplasy caused by hypervariable microsatellite loci; incompatibility was highly correlated with the number of alleles per locus, suggesting that homoplasy caused by parallel evolution of microsatellite alleles is the cause of incompatibility. Microsatellite genotyping of lineage 1A isolates from cotton and olive in Spain over a 29‐year period revealed remarkably little variation; these markers did not mutate enough to provide insight on the spatial and temporal expansion of this clone. Overall, this study showed that microsatellite genotyping can be used to identify clonal lineages in V. dahliae, which has predictive power for inferring phenotypes of phytopathological relevance such as race and pathotype.     

Identification and differentiation of Phyllosticta species causing freckle disease of banana using high resolution melting (HRM) analysis

Freckle disease of banana is caused by three closely related species of Phyllosticta, namely P. musarum, P. maculata and P. cavendishii. In this study, a high resolution melting (HRM) analysis assay was developed and its potential to identify these three fungal species is reported. The assay, which targets the ITS of the nuclear rDNA of the fungal species, generates three distinct melt profiles for the three Phyllosticta species. It is also able to distinguish a combination of up to three co‐infecting species by generating a deviant melt curve. Thirty‐five fungal cultures and infected herbarium leaf specimens, previously characterized using nucleotide sequencing as belonging to one of the three Phyllosticta species, were used for validation of the HRM analysis assay. The normalized curves generated differentiated all samples, with samples from each species correctly identified. The assay was further evaluated against 18 uncharacterized infected leaf specimens from various geographic locations and the results were verified by subsequent nucleotide sequencing. This HRM analysis assay allows rapid identification and differentiation of the three Phyllosticta species using a single primer pair in a one‐step closed‐tube system without labelled fluorescence probes. This novel assay format has potential for simultaneously identifying and differentiating other closely related species of plant pathogens, as well as the classification of infected historic specimens.     

基于PFGE技术的酸乳中乳酸菌的基因分型

采用选择性培养基分离酸乳中的保加利亚乳杆菌和嗜热链球菌,并利用生理生化和糖发酵实验结合16S rDNA同源性分析对分离所得菌株进行鉴定,获得保加利亚乳杆菌和嗜热链球菌分离菌株。利用脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）分别对保加利亚乳杆菌和嗜热链球菌分离株进行基因分型。结果表明：所检测的酸乳样品中均含有保加利亚乳杆菌和嗜热链球菌;仅有2 种酸乳的嗜热链球菌为同一菌株,其余酸乳中的保加利亚乳杆菌及嗜热链球菌均为不同菌株。PFGE可以对保加利亚乳杆菌和嗜热     

团头鲂MHCⅡ α基因的SNP位点开发、鉴定及与抗病性状关联分析

用嗜水气单胞菌(Aeromonas hydrophila)感染团头鲂(Megalobrama amblycephala)区分抗病和易感组,通过PCR扩增及测序在MHCⅡα基因上筛选单核苷酸多态位点(single nucleotide polymorphisms,SNP),利用高分辨率熔解曲线法和限制性内切酶酶切法对SNP进行分型,分析其多态性及与抗病性状之间的关系。在MHCⅡa基因上共筛选出35个候选SNP位点,占MHCⅡa基因总碱基的1.45%,包含30个转换位点、5个颠换位点。其中外显子部分有16个,内含子部分7个,5′非编码区有1个,3′非编码区有11个。有13个SNP位点位于编码区,占总氨基酸位点的5.56%,位于839bp的T/A颠换和1 663bp的A/G转换是无义突变,其余11个SNP位点为有义突变。α1结构域的SNP位点分别占总碱基和总氨基酸位点的4.88%和10.98%,明显高于α2结构域的1.08%和3.22%。MHCⅡα基因的21个抗原结合位点(peptide binding region,PBR)中,有4个位点变异,变异率为19.05%;而non-PBR的变异率仅为8.20%(5/61)。用SPSS软件对分型成功的5个SNP位点在易感和抗病组各100尾中的基因型频率和等位基因频率进行了统计。通过卡方检验分析发现位于1 395bp(T/A)位点的基因型频率和等位基因频率在易感和抗病组中差异极显著,位于221bp(G/T)和1 859bp(G/T)位点的基因型频率和等位基因频率在易感组和抗病组中差异显著。本研究结果表明团头鲂MHCⅡα基因的多态性和抗细菌性败血症性状显著关联。     

牛A群轮状病毒G10型XJ-2022株分离鉴定及基因型分析

【目的】 试验旨在对新疆某规模牛场患腹泻疾病的犊牛进行病原学鉴定及基因型分析。【方法】 采用抗原诊断试剂盒方法对在新疆某牛场随机采集的15份腹泻犊牛粪便样品进行检测,对抗原检测结果为阳性的样品进行反复冻融和过滤处理,然后将样品接种于Marc-145细胞进行病毒的分离和传代。对分离毒株进行间接免疫荧光试验（IFA）和负染电镜观察进一步确定病原。对分离株的VP6和VP7基因进行PCR扩增测序,并对其进行基因相似性比对和进化树分析。【结果】 抗原诊断试剂盒结果显示,3份粪便样品呈牛轮状病毒（Bovine rotavirus,BRV）抗原阳性。将阳性粪便样品分别接种于Marc-145细胞,仅有1份样品连续盲传至第9代出现明显的细胞病变效应（CPE）。IFA结果显示,接种该分离株的Marc-145细胞有亮绿色荧光,对照组未见荧光。电镜观察可见约65 nm的圆形病毒粒子,并命名为XJ-2022株。经PCR扩增获得VP6和VP7基因的目的条带,长度分别为1 356和342 bp。基因相似性比对和遗传进化分析表明,VP6基因与人源A群轮状病毒参考株DB2015-066（LC367318.1）相似性最高且遗传进化亲缘关系最近;VP7基因与牛源G10轮状病毒参考株XJX2（MN937506.1）相似性最高,遗传进化亲缘关系最近,确定该分离株为A群G10型轮状病毒。【结论】 试验成功分离到基因型为A群G10型BRV XJ-2022株,该毒株为新疆地区首次发现的多宿主来源的基因重配病毒。     

SNP检测方法在动物研究中的应用

单核苷酸多态性(SNP)作为第三代分子标记技术,以其数量丰富、遗传稳定性高、易于快速自动化检测等优点备受关注。该文综述了基于不同原理的SNP分型检测方法,包括几种常见的测序技术、基于酶学及杂交原理的分析检测技术和基于色谱及质谱的相关技术。阐释了方法的原理、分析了方法的优缺点及适用范围。总结了目前在动物遗传育种、亲缘鉴定、动物品种及产品溯源等方面的应用研究现状。为下一步开发更灵敏准确、简便易行、高通量及低成本的SNP检测方法及拓展SNP的应用领域提供参考。