单核苷酸多态性在茶树中应用的研究进展

茶树原产于中国西南地区,是一种重要的经济作物。茶叶作为中国的传统饮品,是世界上三大无酒精饮料之一。长期的自然演化及自交不亲和使得茶树的种质资源极其丰富。单核苷酸多态性(single nucleotide polymorphism,SNP)是基因组水平上单核苷酸突变引起的基因序列多样性,是继限制性酶切片段多态性(restriction fragment length polymorphism,RFLP)和简单序列重复(simple sequence repeat,SSR)的第3代分子标记,广泛存在于生物基因组中。SNP分子标记作为最重要的分子标记技术对遗传学研究有极其重要的作用。本文介绍了SNP分子标记的特征,综述了SNP在茶树中的开发检测方法,研究应用于分子标记、基因定位、关联分析等,旨在为理解和研究SNP分子标记在茶树中的研究应用提供参考。     

单核苷酸多态性在多倍体作物油菜中的研究进展

单核苷酸多态性（single nucleotide polymorphism,SNP）是新型分子标记,广泛存在于生物基因组中,在人类及动物的许多研究领域得到了应用,但是在油菜等一些重要的多倍体植物中应用较少。本文以油菜为例,对作物中常用的SNP开发及检测方法作了概述,其中包括第二代测序方法和SNP芯片等快速发展的新技术;综述了SNP在油菜基因定位、遗传多样性研究、全基因组关联分析等研究中的应用进展;讨论了作物SNP研究的发展趋势;以期为SNP在油菜等重要多倍体作物中广泛应用提供参考。     

The number of single nucleotide polymorphisms and on-farm data required for whole-herd parentage testing in dairy cattle herds

New platforms utilizing single nucleotide polymorphisms (SNP) offer operational advantages over the conventional microsatellite-based ones, making them a promising alternative for parentage exclusion. Through simulation and empirical data, a 40-SNP panel (where the minor allele frequency was 0.35 on average) was shown to be a comparable or better diagnostic tool than the current 14-microsatellite panel that is used to parentage test New Zealand dairy animals. The 40 SNP alone did not have sufficient power of exclusion to match more than 75% of the progeny to the correct sire and dam. Utilizing mating records and grouping progeny and dams by birth and calving dates, respectively, decreased the number of sire-dam combinations that each progeny was tested against and dramatically increased the utility of the SNP. These results highlight the importance of combining genotypes with on-farm data to maximize the ability to assign parentage in the New Zealand dairy herd.     

Efficient parentage assignment and pedigree reconstruction with dense single nucleotide polymorphism data

Large numbers of dairy cattle are now routinely genotyped for dense single nucleotide polymorphism (SNP) arrays for the purpose of predicting genomic estimated breeding values. Such SNP arrays contain very good information for parentage assignment and pedigree reconstruction. The main challenge in using this information for parentage assignment and pedigree reconstruction is development of computationally efficient strategies that enable a candidate animal to be assigned its sire and dam with the large volume of data. Here we describe an efficient algorithm for parentage assignment with SNP data and demonstrate very accurate assignment with 50,000-SNP and 3,000-SNP panels. The computer code implementing the algorithm is given in the Appendix.     

Random Forests approach for identifying additive and epistatic single nucleotide polymorphisms associated with residual feed intake in dairy cattle

Feed efficiency is an economically important trait in the beef and dairy cattle industries. Residual feed intake (RFI) is a measure of partial efficiency that is independent of production level per unit of body weight. The objective of this study was to identify significant associations between single nucleotide polymorphism (SNP) markers and RFI in dairy cattle using the Random Forests (RF) algorithm. Genomic data included 42,275 SNP genotypes for 395 Holstein cows, whereas phenotypic measurements were daily RFI from 50 to 150 d postpartum. Residual feed intake was defined as the difference between an animal’s feed intake and the average intake of its cohort, after adjustment for year and season of calving, year and season of measurement, age at calving nested within parity, days in milk, milk yield, body weight, and body weight change. Random Forests is a widely used machine-learning algorithm that has been applied to classification and regression problems. By analyzing the tree structures produced within RF, the 25 most frequent pairwise SNP interactions were reported as possible epistatic interactions. The importance scores that are generated by RF take into account both main effects of variables and interactions between variables, and the most negative value of all importance scores can be used as the cutoff level for declaring SNP effects as significant. Ranking by importance scores, 188 SNP surpassed the threshold, among which 38 SNP were mapped to RFI quantitative trait loci (QTL) regions reported in a previous study in beef cattle, and 2 SNP were also detected by a genome-wide association study in beef cattle. The ratio of number of SNP located in RFI QTL to the total number of SNP in the top 188 SNP chosen by RF was significantly higher than in all 42,275 whole-genome markers. Pathway analysis indicated that many of the top 188 SNP are in genomic regions that contain annotated genes with biological functions that may influence RFI. Frequently occurring ancestor-descendant SNP pairs can be explored as possible epistatic effects for further study. The importance scores generated by RF can be used effectively to identify large additive or epistatic SNP and informative QTL. The consistency in results of our study and previous studies in beef cattle indicates that the genetic architecture of RFI in dairy cattle might be similar to that of beef cattle.     

Prediction of unobserved single nucleotide polymorphism genotypes of Jersey cattle using reference panels and population-based imputation algorithms

The availability of dense single nucleotide polymorphism (SNP) genotypes for dairy cattle has created exciting research opportunities and revolutionized practical breeding programs. Broader application of this technology will lead to situations in which genotypes from different low-, medium-, or high-density platforms must be combined. In this case, missing SNP genotypes can be imputed using family- or population-based algorithms. Our objective was to evaluate the accuracy of imputation in Jersey cattle, using reference panels comprising 2,542 animals with 43,385 SNP genotypes and study samples of 604 animals for which genotypes were available for 1, 2, 5, 10, 20, 40, or 80% of loci. Two population-based algorithms, fastPHASE 1.2 (P. Scheet and M. Stevens; University of Washington TechTransfer Digital Ventures Program, Seattle, WA) and IMPUTE 2.0 (B. Howie and J. Marchini; Department of Statistics, University of Oxford, UK), were used to impute genotypes on Bos taurus autosomes 1, 15, and 28. The mean proportion of genotypes imputed correctly ranged from 0.659 to 0.801 when 1 to 2% of genotypes were available in the study samples, from 0.733 to 0.964 when 5 to 20% of genotypes were available, and from 0.896 to 0.995 when 40 to 80% of genotypes were available. In the absence of pedigrees or genotypes of close relatives, the accuracy of imputation may be modest (generally <0.80) when low-density platforms with fewer than 1,000 SNP are used, but population-based algorithms can provide reasonably good accuracy (0.80 to 0.95) when medium-density platforms of 2,000 to 4,000 SNP are used in conjunction with high-density genotypes (e.g., >40,000 SNP) from a reference population. Accurate imputation of high-density genotypes from inexpensive low- or medium-density platforms could greatly enhance the efficiency of whole-genome selection programs in dairy cattle.     

A genome-wide association study of calf birth weight in Holstein cattle using single nucleotide polymorphisms and phenotypes predicted from auxiliary traits

Previous research has found that a quantitative trait locus exists affecting calving and conformation traits on Bos taurus autosome 18 that may be related to increased calf birth weights, which are not routinely recorded in the United States. Birth weight data from large, intensively managed dairies in eastern Germany with management systems similar to those commonly found in the United States were used to develop a selection index predictor for predicted transmitting ability (PTA) of birth weight. The predictor included body depth, rump width, sire calving ease, sire gestation length, sire stillbirth, stature, and strength. Genetic and phenotypic correlations and heritabilities from the United States were substituted for the German values, and birth weight PTA predicted for 31,984 bulls with US genetic evaluations. A genome-wide association study was conducted on the predicted birth weight PTA with the 2-step genomic BLUP procedure used for routine evaluations in the United States. Allele substitution effects were predicted for 43,188 single nucleotide polymorphisms (SNP). Genotypes were available for 53,644 predictor animals. Gene set enrichment analysis was performed on the 100 SNP that had the largest effects expressed in additive genetic standard deviations. Several SNP related to growth and development were found among the 25 SNP with the largest effects, including markers located within or near (≤100 kbp) ABCA12, FLRT2, LHX4, MAP3K5, NRAC, NTNG1, PIGN, and ZNF75A. The gene set enrichment analysis identified the Kyoto Encyclopedia of Genes and Genomes “Regulation of actin cytoskeleton” pathway (bta04810) as being enriched. That pathway includes the ROCK gene, which is involved in placental function in the human, as well as other developmental genes (e.g., FAK and PAK). Prediction equations derived from one population are useful for identifying genes and gene networks associated with phenotypes that are not directly measured in a second population. This approach will identify only genes associated with the traits used to construct the birth weight predictor, and not loci that affect only birth weight.     

限制性内切酶组合对阪崎克罗诺杆菌的脉冲场凝胶电泳基因分型效果的影响

目的利用多种限制性内切酶提高脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)技术对阪崎克罗诺杆菌(Cronobacter Sakazakii)的分型效果,找到更多的以PFGE为基础的阪崎克罗诺杆菌分子分型信息。方法本研究选用XbaⅠ、SpeⅠ、NheⅠ和BlnⅠ分别酶切34株克罗诺杆菌分离株的DNA,并经PFGE得到相应的基因图谱,通过束平均数、束菌株百分比、结与菌株比值与Simpson多样性指数对各限制内切酶以及结合酶的相对分辨率进行评价。结果 NheⅠ是PFGE单酶切分型中效果最好的一种限制性内切酶;4种限制性内切酶相结合的分型方法可提高PFGE阪崎克罗诺杆菌基因分型效果。结论运用PFGE技术对菌株进行亚分型时,选用的酶种类越多,则结果越可靠,使用XbaⅠ、SpeⅠ、NheⅠ、BlnⅠ4种内切酶结合分析,可以得到理想的菌株亚分型结果。     

单核苷酸多态性技术在羊绒、羊毛纤维鉴别中的应用

随着羊毛纤维剥鳞、拉伸等先进技术的发展,原先一直使用的根据鳞片结构不同,鉴别羊绒羊毛纤维的光学显微镜法逐渐显现出局限性.文章介绍了一种利用多态性技术(SNP),通过聚合链式反应(PCR)的基因扩增和限制性片段长度多态性(RFLP)技术的酶切,根据各种纤维的线粒体DNA经扩增酶切后基因序列的不同,来鉴定山羊绒与绵羊毛的新技术.     

贝母属药用植物叶绿体基因组单核苷酸多态性位点生物信息学分析

目的分析贝母属药用植物叶绿体基因组单核苷酸多态性(single nucleotide polymorphism,SNP)位点的分布特点、鉴别能力和应用价值,为中药材贝母精准鉴定方法的建立提供技术支撑。方法应用多重序列比对、SNP筛选、酶切位点分析等生物信息学分析手段,对贝母属6种药用植物的15条叶绿体基因组序列进行分析。结果贝母属叶绿体基因组DNA同源性达98.38%,通过分析共发现SNP位点4058个,其中川贝母类鉴别候选位点71个,瓦布贝母鉴别候选位点个25个,太白贝母鉴别候选位点个120个,浙贝母鉴别候选位点个79个,湖北贝母鉴别候选位点个61个,平贝母鉴别候选位点794个。结论叶绿体基因组SNP分子标记因其密度高、鉴别力强和便于分析的特点,具有较大的应用价值。     

苏州市食源性金黄色葡萄球菌耐药分析及基因分型

目的 对苏州市2014~2017年分离的食源性金黄色葡萄球菌进行耐药谱和分子分型分析, 初步建立PFGE(pulsed-field gel electrophoresis)分型数据库。方法 采用微量肉汤稀释法检测金黄色葡萄球菌(Staphylococcus aureus, SAU)对19种常用的抗生素的耐药性, 采用Sma I进行酶切, 脉冲场凝胶电泳进行分子分型。菌株指纹图谱用BioNumerics v6.6软件进行分析。结果 81株SAU中检出10株耐甲氧西林金黄色葡萄球菌(methicillin-resistance Staphylococcus aureus, MRSA), 检出率12.3%。有72株菌存在不同程度的耐药, 耐药率达到88.9%, 其中对青霉素的耐药率最高(82.7%), 其次是氨苄西林(66.7%), 其后是克林霉素和红霉素(均为50.6%)。耐受3种和3种以上药物的多重耐药菌株共38株, 多重耐药率达46.9%。MRSA的多重耐药率高于甲氧西林敏感型金黄色葡萄球菌(methicillin-sensitive Staphylococcus aureus, MSSA), 差异具有统计学意义(P<0.05)。除8株菌不可被PFGE分型外, 其余73株菌可以被分为37个不同型别, 其中有4个优势型别共27株菌, 在可分型菌株中占比达37.0%。这些同源菌株分离自不同时间和地点, 有些还存在一定程度的变异。结论 食源性金黄色葡萄球菌耐药现象普遍存在, 多重耐药率较高, PFGE型别较为多样但有优势型别存在, 耐药谱和PFGE型别关联性较差。     

广西香港海鸥菌分离菌株脉冲场凝胶电泳分子分型研究

目的 探讨香港海鸥菌分子分型方法,了解广西水产品监测所分离的香港海鸥菌的相关性.方法 以NotⅠ限制性内切酶对2005年分离的香港海鸥菌酶切后进行脉冲电泳,用BioNumerics 5.1聚类分析获得电泳图谱.结果 7株香港海鸥菌分为6个分子型,其中从南宁分离的与从河池分离的2株香港海鸥菌高度同源,相似度达100％.结论 PFGE可应用于香港海鸥菌分子分型,有助于发现香港海鸥菌流行规律和传播途径,水鸟可能是香港海鸥菌传播环节的一种重要媒介.     

进出境食品中单核增生李斯特菌的血清分型与脉冲场凝胶电泳分型分析

目的研究进出境食品中的单核增生李斯特菌(LMO)的血清分型与脉冲场凝胶电泳(PFGE)分型的特性及其关系。方法采用标准方法对39株分离的LMO进行PFGE分型,分别用ApaI和Asc I酶切,电泳图谱进行聚类分析,以上菌株同时进行血清分型检测。结果 39株LMO分成6个血清型;经ApaI酶切后,分成29种带型,相似度在57.4%～100%;Asc I酶切后,分成34种带型,相似度在48.6%～100%。讨论 AscI酶切的PFGE分离效果优于ApaI酶切,与血清分型的一致性低于ApaI酶切;PFGE分型效果优于血清分型。     

Short communication: Genotyping and single nucleotide polymorphism analysis of bovine leukemia virus in Chinese dairy cattle

Bovine leukemia virus (BLV) causes enzootic leucosis in cattle and is classified into 10 genotypes with a worldwide distribution, except for several European countries, Australia, and New Zealand. Although BLV is widespread in Chinese cows with the positive rate of 49.1% at the individual level, very little is known about the BLV genotype in dairy cattle in China. To determine BLV genetic variability in cows in China, 112 BLV-positive samples from 5 cities in China were used for BLV molecular characterization in this study. Phylogenetic analysis using the neighbor-joining method on partial env sequence encoding gp51 obtained from 5 Chinese cities and those available in GenBank (n = 53, representing BLV genotype 1–10) revealed the Chinese strains belonged to genotype 6. Seven unique SNP were identified among Yancheng, Shanghai, and Bengbu strains out of the total 12 SNP identified in Chinese strains. The genotyping coupled with SNP analysis of BLV can serve as a useful molecular epidemiological tool for tracing the source of pathogens. This study highlights the importance of genetic analysis of geographically diverse BLV strains to understand BLV global genetic diversity.     

Hot topic: performance of bovine high-density genotyping platforms in Holsteins and Jerseys

Two high-density single nucleotide polymorphism (SNP) genotyping arrays have recently become available for bovine genomic analyses, the Illumina High-Density Bovine BeadChip Array (777,962 SNP) and the Affymetrix Axiom Genome-Wide BOS 1 Array (648,874 SNP). These products each have unique design and chemistry attributes, and the extent of marker overlap and their potential utility for quantitative trait loci fine mapping, detection of copy number variation, and multibreed genomic selection are of significant interest to the cattle community. This is the first study to compare the performance of these 2 arrays. Deoxyribonucleic acid samples from 16 dairy cattle (10 Holstein, 6 Jersey) were used for the comparison. An independent set of DNA samples taken from 46 Jersey cattle and 18 Holstein cattle were used to ascertain the amount of SNP variation accounted by the 16 experimental samples. Data were analyzed with SVS7 software (Golden Helix Inc., Bozeman, MT) to remove SNP having a call rate less than 90%, and linkage disequilibrium pruning was used to remove linked SNP (r² ≥ 0.9). Maximum, average, and median gaps were calculated for each analysis based on genomic position of SNP on the bovine UMD3.1 genome assembly. All samples were successfully genotyped (≥98% SNP genotyped) with both platforms. The average number of genotyped SNP in the Illumina platform was 775,681 and 637,249 for the Affymetrix platform. Based on genomic position, a total of 107,896 SNP were shared between the 2 platforms; however, based on genotype concordance, only 96,031 SNP had complete concordance at these loci. Both Affymetrix BOS 1 and Illumina BovineHD genotyping platforms are well designed and provide high-quality genotypes and similar coverage of informative SNP. Despite fewer total SNP on BOS 1, 19% more SNP remained after linkage disequilibrium pruning, resulting in a smaller gap size (5.2 vs. 6.9 kb) in Holstein and Jersey samples relative to BovineHD. However, only 224,115 Illumina and 241,038 Affymetrix SNP remained following removal of SNP with a minor allele frequency of zero in Holstein and Jersey samples, resulting in an average gap size of 11,887 bp and 11,018 bp, respectively. Combining the 354,348 informative (r² ≥ 0.9), polymorphic (minor allele frequency ≥ 0), unique SNP data from both platforms decreased the average gap size to 7,560 bp. Genome-wide copy number variant analyses were performed using intensity files from both platforms. The BovineHD platform provided an advantage to the copy number variant data compared with the BOS 1 because of the larger number of SNP, higher intensity signals, and lower background effects. The combined use of both platforms significantly improved coverage over either platform alone and decreased the gap size between SNP, providing a valuable tool for fine mapping quantitative trait loci and multibreed animal evaluation.     

云南省食源性金黄色葡萄球菌脉冲场凝胶电泳分型分析

目的采用脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)方法分析云南省2013～2015年食源性金黄色葡萄球菌分子分型带型,初步建立金黄色葡萄球菌PFGE分型的数据库。方法用限制性内切酶Smal I酶切113株金黄色葡萄球菌染色体DNA以进行PFGE分析,并利用BioNumerics软件对分离株的指纹图谱进行聚类分析。结果 113株食源性金黄色葡萄球菌的PFGE图谱的相似性系数在56.67%～100%之间,经聚类分析得到89个PFGE型别。结论建立云南省食源性金黄色葡萄球菌PFGE分型数据库,PFGE带型呈现多样性,对今后云南省金黄色葡萄球菌引起食物中毒的诊断和溯源工作有重要意义。     

Predictive ability of direct genomic values for lifetime net merit of Holstein sires using selected subsets of single nucleotide polymorphism markers

The objective of the present study was to assess the predictive ability of subsets of single nucleotide polymorphism (SNP) markers for development of low-cost, low-density genotyping assays in dairy cattle. Dense SNP genotypes of 4,703 Holstein bulls were provided by the USDA Agricultural Research Service. A subset of 3,305 bulls born from 1952 to 1998 was used to fit various models (training set), and a subset of 1,398 bulls born from 1999 to 2002 was used to evaluate their predictive ability (testing set). After editing, data included genotypes for 32,518 SNP and August 2003 and April 2008 predicted transmitting abilities (PTA) for lifetime net merit (LNM$), the latter resulting from progeny testing. The Bayesian least absolute shrinkage and selection operator method was used to regress August 2003 PTA on marker covariates in the training set to arrive at estimates of marker effects and direct genomic PTA. The coefficient of determination (R²) from regressing the April 2008 progeny test PTA of bulls in the testing set on their August 2003 direct genomic PTA was 0.375. Subsets of 300, 500, 750, 1,000, 1,250, 1,500, and 2,000 SNP were created by choosing equally spaced and highly ranked SNP, with the latter based on the absolute value of their estimated effects obtained from the training set. The SNP effects were re-estimated from the training set for each subset of SNP, and the 2008 progeny test PTA of bulls in the testing set were regressed on corresponding direct genomic PTA. The R² values for subsets of 300, 500, 750, 1,000, 1,250, 1,500, and 2,000 SNP with largest effects (evenly spaced SNP) were 0.184 (0.064), 0.236 (0.111), 0.269 (0.190), 0.289 (0.179), 0.307 (0.228), 0.313 (0.268), and 0.322 (0.291), respectively. These results indicate that a low-density assay comprising selected SNP could be a cost-effective alternative for selection decisions and that significant gains in predictive ability may be achieved by increasing the number of SNP allocated to such an assay from 300 or fewer to 1,000 or more.     

苏州市食品中单增李斯特菌污染状况及分子特征分析

目的 研究2016—2020年苏州市各类食品中单增李斯特菌的污染状况及分子特征。方法 将采样食品按WS/T464—2015《食物成分数据表达规范》进行分类,计算各类食品污染率,通过聚合酶链式反应(polymerase chain reaction, PCR)和凝胶电泳对毒力基因(inlA、inlB)和耐药相关基因(virR、dltA、dltB、dltC、dltD、mprF)进行检测,通过脉冲场凝胶电泳进行分子分型。结果 683件食品中共检出44件阳性样品,总体检出率6.4%。各类食品中以肉类食品的检出率最高(17.2%),肉类食品中以预制肉制品检出率最高(37.2%)。分离的44株单增李斯特菌毒力基因inlA携带率为100.0%,inlB携带率为97.7%,耐药相关基因virR、dltA、dltB、dltC、dltD、mprF的携带率分别为90.9%、100.0%、90.9%、100.0%、95.5%、97.7%。分离株的PFGE型别可分为11个基因型簇,相似度为53.3%～100.0%,有11组100.0%相似度的带型,其中有3组为同一时间,同一地点采集样品所分离菌株。结论 苏州市肉...     

A genome scan to detect quantitative trait loci for economically important traits in Holstein cattle using two methods and a dense single nucleotide polymorphism map

Genome scans for detection of bovine quantitative trait loci (QTL) were performed via variance component linkage analysis and linkage disequilibrium single-locus regression (LDRM). Four hundred eighty-four Holstein sires, of which 427 were from 10 grandsire families, were genotyped for 9,919 single nucleotide polymorphisms (SNP) using the Affymetrix MegAllele GeneChip Bovine Mapping 10K SNP array. A hybrid of the granddaughter and selective genotyping designs was applied. Four thousand eight hundred fifty-six of the 9,919 SNP were located to chromosomes in base-pairs and formed the basis for the analyses. The mean polymorphism information content of the SNP was 0.25. The SNP centimorgan position was interpolated from their base-pair position using a microsatellite framework map. Estimated breeding values were used as observations, and the following traits were analyzed: 305-d lactation milk, fat, and protein yield; somatic cell score; herd life; interval of calving to first service; and age at first service. The variance component linkage analysis detected 102 potential QTL, whereas LDRM analysis found 144 significant SNP associations after accounting for a 5% false discovery rate. Twenty potential QTL and 49 significant SNP associations were in close proximity to QTL cited in the literature. Both methods found significant regions on Bos taurus autosome (BTA) 3, 5, and 16 for milk yield; BTA 14 and 19 for fat yield; BTA 1, 3, 16, and 28 for protein yield; BTA 2 and 13 for calving to first service; and BTA 14 for age at first service. Both approaches were effective in detecting potential QTL with a dense SNP map. The LDRM was well suited for a first genome scan due to its approximately 8 times lower computational demands. Further fine mapping should be applied on the chromosomal regions of interest found in this study.