绵羊(ATAG)n四碱基微卫星位点的筛选及其在亲子鉴定中的应用

本试验旨在筛选绵羊高度多态性四碱基微卫星遗传标记,建立适用于绵羊亲子关系鉴定的实验体系。试验以小尾寒羊为主要研究对象,从已有的绵羊参考基因组序列出发,基于全基因组序列筛选法共筛选出53个（ATAG）_n四碱基重复微卫星位点,然后通过基因分型数据共筛选出30个扩增效果好、多态信息含量（PIC）丰富的四碱基重复微卫星位点;30个位点的基因分型结果表明,共扩增出253个等位基因,平均等位基因数为8.433,等位基因数均>5,多态信息含量在0.566~0.898,观测杂合度（Ho）范围在0.548~0.903,期望杂合度（He）范围在0.631~0.921,平均期望杂合度为0.776;哈代-温伯格平衡检验30个位点均处于遗传平衡状态。随后根据PCR的扩增效率从获得的30个多态性位点中筛选出22个微卫星位点用于亲权排除概率的计算,根据多态信息含量的大小由高到低依次增加位点数进行组合。结果表明,在两个亲本的基因型均未知的情况下,标记位点数为15个时,累积排除概率可达到99.99%,其中单个位点的第一非亲排除率（non-exclusion probability of the first parent,NE-1P）介于0.321~0.663之间。利用建立的亲子鉴定体系对16只具有系谱记录的小尾寒羊样本进行检测,结果共鉴定出4个具有高置信度的绵羊家系,鉴定结果与系谱记录完全一致。本试验为绵羊分子系谱的构建、亲子鉴定以及保障绵羊育种工作的正常开展奠定重要基础。     

绵羊SPP1和PLCB3基因多态性与产羔数的关联分析

试验旨在探究SPP1基因g.36651870T>C位点多态性、PLCB3基因g.41871219T>C位点多态性与绵羊产羔数之间的关系,以期寻找绵羊产羔数性状相关的分子标记.利用全基因组重测序结合Sequenom MassARRAY?SNP技术对多羔羊(小尾寒羊、湖羊、策勒黑羊)和单羔羊(滩羊、苏尼特羊、萨福克羊和草原...     

绵羊SMAD1基因组织表达及其多态性与产羔数关联分析

【目的】绵羊产羔数是一个极其复杂的性状,受诸多因素影响。绵羊排卵数是最重要的因素之一,而SMAD蛋白在哺乳动物卵泡发育和颗粒细胞生长分化过程中发挥着重要作用。因此本文基于前期绵羊基因组重测序数据,深入研究SMAD家族成员1 (SMAD family member 1, SMAD1)基因在不同繁殖力小尾寒羊组织表达模式,并探讨其多态性与绵羊产羔数的关系,以揭示其影响产羔数的机制,为绵羊分子育种提供科学依据。【方法】首先,采用反转录PCR技术检测SMAD1基因在不同繁殖力小尾寒羊群体大脑,小脑,下丘脑,垂体,子宫,卵巢,输卵管,心脏,肝脏,脾脏,肺脏,肾脏,肾上腺,甲状腺,大肠,小肠,胰腺,瘤胃,肾脂 19种组织的表达模式,然后利用实时荧光定量PCR技术检测SMAD1基因在不同繁殖力小尾寒羊下丘脑,垂体,子宫,卵巢,输卵管5种繁殖组织的相对表达量。随后采用Sequenom MassARRAY ^?SNP技术对SMAD1基因5个单核苷酸多态性(single nucleotide polymorphism, SNP)位点在小尾寒羊（n=380）、湖羊（n=101）、苏尼特羊（n=21）、草原型藏羊（n=161）、策勒黑羊（n=52）、滩羊（n=22）6个绵羊品种中进行基因型检测,计算其群体遗传学指标,利用SPSS19.0软件分析5个SNPs与小尾寒羊产羔数的相关性。【结果】PCR结果显示,SMAD1基因在不同繁殖力小尾寒羊全身性组织均有表达,在小尾寒羊单羔群体卵巢的表达量极显著高于多羔群体（P<0.01）,小尾寒羊单羔群体下丘脑、垂体的表达量显著高于多羔群体（P<0.05）;基因分型结果表明,g.12485895A>G,g.12487558G>A和g.12487190G>T位点在单、多羔绵羊品种中基因型和等位基因频率均差异显著（P<0.05）;而g.12508874T>C和g.12487467A>G位点在单、多羔绵羊品种中基因型和等位基因频率均差异不显著（P>0.05）;群体遗传学分析表明,g.12485895A>G、g.12487558G>A、g.12508874T>C、g.12487467A>G、g.12487190G>T位点在小尾寒羊、湖羊、策勒黑羊、苏尼特羊、草原型藏羊中均为中度多态（0.25<PIC<0.5）,g.12508874T>C位点在滩羊群体表现为低度多态（PIC<0.25）;卡方适合性检验表明,g.12485895A>G和g.12487467A>G位点在小尾寒羊和草原型藏羊中处在Hardy-Weinberg不平衡状态（P<0.05）,g.12487558G>A在小尾寒羊和湖羊群体处于Hardy-Weinberg不平衡状态（P<0.05）,g.12487190G>T位点在草原型藏羊处于Hardy-Weinberg不平衡状态（P<0.05）。其他位点在6个绵羊品种中均处在Hardy-Weinberg平衡状态（P>0.05）;关联分析表明,g.12485895A>G,g.12487558G>A,g.12508874T>C和g.12487467A>G位点与小尾寒羊产羔数无显著关联。g.12487190G>T位点TT基因型母羊前三胎的产羔数均显著高于GT和GG基因型母羊（P<0.05）。将该位点与小尾寒羊FecB（A746G）不同基因型进行组合后发现,AA-GG、AA-GT、AA-TT型母羊产羔数显著低于其他组合基因型母羊（P<0.05）。【结论】SMAD1与小尾寒羊繁殖密切相关,g.12487190G>T可作为小尾寒羊产羔性状选育的潜在分子标记。     

A genetic linkage map in southern‐by‐spring oat identifies multiple quantitative trait loci for adaptation and rust resistance

We developed 178 recombinant inbred lines from a southern‐by‐spring oat population designated as “TxH.” These lines were genotyped to generate a high‐quality linkage map that resolved 6,902 markers into 21 linkage groups that matched closely with the latest hexaploid oat consensus map. Three major quantitative trait loci (QTLs) affecting heading date were found in locations that are consistent with known QTLs and candidate genes, and two other QTLs affecting heading date were found in novel locations. Five QTLs affecting plant height were found. Both sets of QTLs are responsible for transgressive segregation observed for these two traits. Four QTLs affecting resistance to crown rust, caused by the pathogen Puccinia coronata f. sp. avenae, were identified. Two of these QTLs are consistent with known clusters of rust resistance genes, while two may represent new locations of novel rust resistance genes. A complete set of SNP sequences suitable for generating markers for molecular selection is provided.     

Molecular typing of Streptococcus agalactiae isolates from fish

The genetic variability among Streptococcus agalactiae isolates recovered from fish was characterized using single-stranded conformation polymorphism (SSCP) analysis of the intergenic spacer region (ISR), and amplified fragment length polymorphism (AFLP) fingerprinting. A total of 46 S. agalactiae cultures isolated from different fish species and geographic origins as well as related reference strains were included in the study. ISR-SSCP divided the S. agalactiae isolates analysed into five distinct genotypes. Genotype 1 grouped all Kuwait isolates while genotype 4 clustered the majority of non-Kuwait isolates (USA, Brazil and Honduras). AFLP analysis offered a higher resolution level by dividing the isolates into 13 different genotypes. Two different AFLP profiles were identified within the Kuwait isolates. When data from both ISR-SSCP and AFLP were combined through a multidimensional analysis (MDS), a good correlation between geographical origin and genotypes was observed. Both AFLP and ISR-SSCP revealed genetic differences between S. agalactiae isolates from fish. While AFLP offered a higher resolution, ISR-SSCP also provided valid information being a simpler and faster method.     

Status of black grass (Alopecurus myosuroides) resistance to acetyl-coenzyme A carboxylase inhibitors in France

We assessed the contributions of target site‐ and non‐target site‐based resistance to herbicides inhibiting acetyl‐coenzyme A carboxylase (ACC) in Alopecurus myosuroides (black grass). A total of 243 A. myosuroides populations collected across France were analysed using herbicide sensitivity bioassay (24 300 seedlings analysed) and ACC genotyping (13 188 seedlings analysed). Seedlings resistant to at least one ACC‐inhibiting herbicide were detected in 99.2% of the populations. Mutant, resistant ACC allele(s) were detected in 56.8% of the populations. Among the five resistant ACC alleles known in A. myosuroides, alleles containing an isoleucine‐to‐leucine substitution at codon 1781 were predominant (59.5% of the plants containing resistant ACC alleles). Comparison of the results from herbicide sensitivity bioassays with genotyping indicated that more than 75% of the plants resistant to ACC‐inhibiting herbicides in France would be resistant via increased herbicide metabolism. Analysis of herbicide application records suggested that in 15.9% of the populations studied, metabolism‐based resistance to ACC‐inhibiting herbicides was mostly selected for by herbicides with other modes of action. Our study revealed the importance of non‐target site‐based resistance in A. myosuroides. Using herbicides with alternative modes of action to control populations resistant to ACC‐inhibiting herbicides, the recommended management approach, may thus be jeopardised by the widespread occurrence of metabolism‐based resistance mechanisms conferring broad‐spectrum cross‐resistance.     

全基因组SNP分型技术在畜禽遗传育种研究中的应用

随着畜禽资源分子鉴定、物种进化、全基因组育种等热点领域的逐渐兴起,准确的全基因组SNP分型成为了畜禽基因组研究的关键。基因芯片、重测序、简化基因组测序及靶向捕获测序等全基因组SNP分型技术已广泛应用于畜禽基因组研究中。本文概述了全基因组SNP分型技术的原理及其在全基因组关联分析、选择信号分析和畜禽遗传资源背景分析等方面的应用,以期为畜禽基因组研究和育种应用提供借鉴和参考。     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

弓形虫基因分型研究进展

弓形虫可感染多种动物引起严重的弓形虫病,为了对不同宿主感染的弓形虫株基因型差异及不同基因型弓形虫虫株的致病力差异进行进一步研究,弓形虫基因型的分析多采用限制性片段长度多态性PCR（PCR-RFLP）、随机扩增多态性DNA、多位点PCR-RFLP分析、DNA序列分析和微卫星DNA序列分析等技术,扩增位点多选择SAG1、SAG2、SAG3、BTUB和GRA6等遗传标记。传统的弓形虫基因型主要有3个,随着基因型研究的不断深入,越来越多的基因型被发现。本文就弓形虫基因分型研究进展进行讨论分析。