鲁中肉羊SMAD1和ESR2基因多态性与产羔数关联分析

为探究SMAD1、ESR2基因多态性与鲁中肉羊产羔数之间的关系,采用Sequenom MassARRAY誖SNP技术检测鲁中肉羊SMAD1、ESR2基因单核苷酸多态性,并与产羔数进行关联分析。结果表明:SMAD1基因g.12485895A>G存在AA、AG和GG基因型,基因型频率分别为0.05、0.45和0.50;ESR2基因g.73324006C>T存在CC和CT基因型,基因型频率分别为0.98和0.02。g.12485895A>G位点在鲁中肉羊表现为中度多态(0.25T位点为低度多态(PIC<0.25);卡方适合性检验表明,g.12485895A>G位点在鲁中肉羊处于哈代温伯格不平衡状态(P<0.05),g. 73324006C>T位点处于哈代温伯格平衡状态(P>0.05)。g.12485895A>G位点多态性与鲁中肉羊产羔数没有显著关联(P>0.05), g.73324006C>T位点多态性与鲁中肉羊产羔数显著关联(P<0.05)。综上可知,SMAD1基因g.12485895A>G位点和鲁中肉羊产羔数性状没有显著关联(P>0.05),ESR2基因g.73324006C>T位点对鲁中肉羊产羔数性状选育具有一定的指导意义。     

Use of high‐density SNP data to identify patterns of diversity and signatures of selection in broiler chickens

The development of broiler chickens over the last 70 years has been accompanied by large phenotypic changes, so that the resulting genomic signatures of selection should be detectable by current statistical techniques with sufficiently dense genetic markers. Using two approaches, this study analysed high‐density SNP data from a broiler chicken line to detect low‐diversity genomic regions characteristic of past selection. Seven regions with zero diversity were identified across the genome. Most of these were very small and did not contain many genes. In addition, fifteen regions were identified with diversity increasing asymptotically from a low level. These regions were larger and thus generally included more genes. Several candidate genes for broiler traits were found within these ‘regression regions’, including IGF1, GPD2 and MTNR1AI. The results suggest that the identification of zero‐diversity regions is too restrictive for characterizing regions under selection, but that regions showing patterns of diversity along the chromosome that are consistent with selective sweeps contain a number of genes that are functional candidates for involvement in broiler development. Many regions identified in this study overlap or are close to regions identified in layer chicken populations, possibly due to their shared precommercialization history or to shared selection pressures between broilers and layers.     

采用四引物ARMS-PCR方法检测奶牛乳腺组织DGAT1基因单核苷酸多态性

试验旨在快速有效地检测泌乳奶牛的二酰甘油转酰基酶1（diacylgycerol acyltransferase 1,DGAT1）乳脂量性状的优势等位基因K232A单核苷酸多态性,确定DGAT1基因型进而确定泌乳性状,为中国荷斯坦奶牛分子标记辅助选择提供技术支持。选取6头泌乳初期（3头为高乳产量牛,3头为低乳产量牛）中国北方荷斯坦奶牛为研究对象,提取乳腺组织基因组DNA,分别设计1对外引物和1对内引物,建立一种四引物ARMS-PCR体系快速检测奶牛乳腺组织DGAT1基因单核苷酸多态性。结果发现,外引物扩增片段长度为512 bp,为PCR反应的阳性对照,基因型为232K扩增片段长度为369 bp,基因型为232A扩增片段长度为181 bp。PCR结果显示,6头牛的乳腺组织样本均由外部引物扩增出长度为512 bp的片段,泌乳期高乳产量奶牛和泌乳期低乳产量奶牛乳腺组织的特异性扩增片段长度均为181 bp。表明本研究选取的6头奶牛样本DGAT1基因K232A多态性均为232A型。提示该PCR鉴定方法能够快速有效地鉴定奶牛DGAT1基因型,可用于中国荷斯坦奶牛分子标记辅助选择。     

PCR-RFLP molecular confirmation of color dilution alopecia in dogs in Brazil

Color dilution alopecia (CDA) is a dermatopathy observed exclusively in animals having a diluted coat color. In dogs, color dilution occurs as a result of a single-nucleotide variation (SNV) c.-22G > A in the melanophilin gene. We standardized a PCR–restriction-fragment length polymorphism (PCR-RFLP) technique to identify this mutation and determine its frequency in dogs in Brazil. The standardized PCR-RFLP technique could efficiently identify the SNV c.-22G > A in the melanophilin gene, with mutated allele frequencies of 0.1, 0.1, and 0.0875 in Dachshund, Miniature Pinscher, and Yorkshire Terrier breeds, respectively, with no statistical difference among the breeds (p = 0.252). The mutation was identified in 2 homozygous Dachshund dogs with alopecia, confirming the clinical characteristic of CDA. The standardization of a simpler and more accessible molecular technique for recognition of the SNV c.-22G > A in the melanophilin gene allows identification of heterozygous (phenotypically normal) dogs that can be excluded from reproduction, to avoid the birth of dogs with diluted coat color and consequently CDA.     

Genotyping Shorthorn cattle for generalised glycogenosis

OBJECTIVE: To develop a procedure for routine genotyping of Shorthorn cattle for the generalised glycogenosis allele in exon 18 of the acidic alpha-glucosidase gene. PROCEDURE: Allele-specific amplification and double mismatch amplification procedures for the discrimination of the exon 18 alleles were evaluated using leucocytes and hair roots as sources of target DNA. RESULTS: Allele-specific amplification was effective for genotyping Shorthorn cattle at the 2454 site when purified DNA was used as target for the polymerase chain reaction. However, when the target DNA was derived from hair roots, differences in the relative yield of wild-type and mutant amplicons were observed. The double mismatch amplification procedure was effective in genotyping all subjects, independent of the source of DNA. The unique cleavage sites for Drd I and PshA I within exon 18 are present and absent respectively in the wildtype amplicon, and are lost and acquired, respectively, in the mutant amplicon. In addition, the Drd I and PshA I mismatching cleavage sites incorporated into the primers serve as internal controls for Drd I and PshA I cleavage. CONCLUSION: The double Drd I/PshA I mismatch amplification procedure using hair root samples as the source of DNA is a robust method for genotyping Shorthorns for generalised glycogenosis.     

宽皮柑橘单核苷酸多态性的高分辨率熔解曲线分型

 高分辨率熔解曲线分析（High resolution melting analysis,HRM）可以检测单碱基改变引起的DNA双链熔解温度（T_m）值变化,从而可以对样本在单核苷酸多态性分子标记（Single nucleotide polymor- phism,SNP）上进行基因分型。通过分析NCBI数据库中宽皮柑橘的表达序列标签（Expressed sequence tag,EST）数据鉴别SNP位点,并用小片段扩增法高分辨率熔解曲线分型技术（High resolution melting analysis of small amplicons）分析11个宽皮柑橘（Citrus reticulata）品种以及柳橙（Citrus sinensis Osbeck var.‘Liucheng’）的5个SNP位点的基因型。结果显示,小片段扩增法高分辨率熔解曲线分型可以快速、清楚地分辨纯合与杂合基因型,在校正温度差异后也可以很好地分辨同一个SNP位点不同的纯合型。统计分析表明样本在所有SNP位点上均存在多态性,5个SNP位点的平均多态性信息含量（PIC）为0.3190,显示样本在这组SNP位点上具有较高的杂合率。     

后基因组时代下作物的SNP分型方法

单核苷酸多态性(single nucleotide polymorphisms,SNPs)是生物体最普遍的一种多态差异,在植物功能基因组研究和作物遗传改良方面有着广泛的应用。利用全基因组水平SNP标记谱进行遗传变异的研究、群体结构分析、关联性分析、作物分子设计育种,以及对大规模SNP数据进行验证、评估等,都迫切需要发展和利用各种不同的SNP分型手段实现。本文综述了目前常用的一些SNP分型方法,简要介绍了检测原理及操作流程,并对后基因组时代下作物的高通量SNP数据的分析进行了讨论。     

High‐throughput detection of highly benzimidazole‐resistant allele E198A with mismatch primers in allele‐specific real‐time polymerase chain reaction

BACKGROUND: A point mutation often confers resistance of organisms against medical drugs and agricultural pesticides. Allele‐specific nucleotide polymerase chain reaction (ASPCR) and allele‐specific quantitative real‐time PCR using SYBR Green (ASQPCR) are widely and effectively applied to detect and monitor this type of resistance. However, the former is unsuitable for high‐throughput detection, and the latter often reduces the accuracy of detection. RESULTS: In order to decrease background amplification, a rapid and high‐throughput genotyping method with mismatch primers was developed (ASQPCR‐MP) and applied specifically to survey the frequency of the highly benzimidazole‐resistant MBC^HR mutation (E198A) in the β‐tubulin gene of Sclerotinia sclerotiorum (Lib.) de Bary populations. Genomic DNA from 223 sclerotia was analysed. Similar genotype results were also obtained using ASPCR with mismatch primers and a mycelial growth inhibition assay. It was found that ASQPCR‐MP clearly differentiated MBC^HR and benzimidazole‐sensitive MBC^S phenotypes. Moreover, ASQPCR‐MP took less than 6 h to complete. CONCLUSION: ASQPCR‐MP appears suitable for large epidemiological studies involving resistant genotypes and requiring high‐throughout formats. Copyright © 2009 Society of Chemical Industry     

One tube nested-PCR在SNP基因型分型中的应用

nested–PCR是一种在普通PCR基础上发展起来的专门用于检测单核苷酸多态性(SNP)的技术。结合nested–PCR技术在水稻中检测SNP的研究,以1个水稻香味基因Fgr和3个稻瘟病基因Pi–ta、Pi9、Pigm为例,把nested–PCR的4条引物同时加在一管PCR反应中进行扩增,在其序列内针对SNP位点设计功能标记,用来扩增含有突变位点的DNA片段。通过优化引物浓度梯度和改良反应程序,达到了一步快速检测SNP基因型的目的。     

鲁中肉羊GTF2A1基因多态性及其与产羔数的关联分析

该研究旨在分析绵羊GTF2A1基因g.89505005G>A位点多态性与产羔数之间的关系,以期寻找与绵羊产羔数有关的分子标记。针对前期利用基因组选择信号分析获得的候选基因GTF2A1及其g.89505005G>A位点,采用Sequenom MassARRAY SNP技术对产羔数存在差异的鲁中肉羊群体进行该位点的多态性检测,并与产羔数进行关联分析。结果表明:鲁中肉羊GTF2A1基因g.89505005G>A位点存在AA、AG和GG三种基因型,且以GG基因型为主;g.89505005G>A位点多态性与鲁中肉羊第1胎、第2胎以及第3胎产羔数均存在显著关联(P<0.05),AA型各胎产羔数均高于GG型(P<0.05)。综上,g.89505005G>A位点A等位基因可能是提高绵羊产羔数的一个潜在有效的DNA标记。