基因组选择技术在农业动物育种中的应用

基因组选择(genomic selection, GS)是畜禽经济性状遗传改良的重要方法。随着高密度SNP芯片和二代测序价格的下降,GS技术越来越多被应用于奶牛、猪、鸡等农业动物育种中。然而,降低全基因组SNP分型成本、提高基因组育种值(genomic estimated breeding value,GEBV)估计准确性仍然是GS研究的主要难题。本文从全基因组SNP分型策略和GEBV估计模型两个方面进行了综述,并对目前GS技术在主要畜禽品种中的应用现状进行了介绍,以期为GS在农业动物育种中的深入开展提供借鉴和参考。     

一种基于高密度遗传标记的亲子鉴定方法及其应用

系谱是人类遗传及动植物育种研究与实践的重要信息来源之一。系谱记录错误是育种生产中普遍存在的一种记录错误,影响基因定位、遗传值及表型值预测等相关研究结果的可靠性。现有的方法软件可以利用遗传标记信息对疑似亲子进行亲子鉴定,但这些软件方法操作复杂,限制标记数量,如Cervus。针对当前高密度SNP标记在人类及动植物研究中广泛应用的现状,文章提出了一种基于全基因组高密度SNP数据的亲子鉴定新方法,命名为EasyPC。对EasyPC及Cervus的运行效率进行了对比,并用中国荷斯坦牛(n=2180)和杜洛克猪(n=191)的全基因组SNP芯片数据对EasyPC进行了验证。结果表明：EasyPC运行效率高于Cervus,牛和猪群体系谱错误率分别为20%和6%,与相关研究报道相符。通过使用全基因组SNP标记对群体孟德尔错误率的经验分布进行分析,该方法不仅可以简单、快速、准确地判别系谱的正确性,而且还可以对错误系谱进行校正。EasyPC为解决全基因组研究中基因型及系谱数据前处理过程中的系谱校正问题提供了一种新的途径。     

随机SNP在全基因组关联研究人群分层分析中的应用

在复杂疾病的全基因组关联研究中,人群分层现象会增加结果的假阳性率,因此考虑人群遗传结构、控制人群分层是很有必要的。而在人群分层研究中,使用随机选择的SNP的效果还有待进一步探讨。文章利用HapMap Phase2人群中无关个体的Affymetrix SNP 6.0芯片分型数据,在全基因组上随机均匀选择不同数量的SNP,同时利用f值和Fisher精确检验方法筛选祖先信息标记（Ancestry Informative Markers,AIMs）。然后利用HapMap Phase3中的无关个体的数据,以F-statistics和STRUCTURE分析两种方法评估所选出的不同SNP组合对人群的区分效果。研究发现,随机均匀分布于全基因组的SNP可用于识别人群内部存在的遗传结构。文章进一步提示,在全基因组关联研究中,当没有针对特定人群的AIMs时,可在全基因组上随机选择3000以上均匀分布的SNP来控制人群分层。     

小型猪葡萄糖转运子4外显子4a的基因多态性分析

目的分析我国特有的3个小型猪品系巴马小型猪、中国农大小型猪、五指山小型猪葡萄糖转运子4基因外显子4a的单核苷酸多态性（SNPs）分布特点,为我国小型猪在糖尿病和代谢性疾病研究中提供基础资料。方法以3个品系小型猪基因组DNA为模板,应用特异性引物进行PCR扩增,扩增产物纯化测序,进行BLAST比对分析。结果在小型猪GLUT-4基因外显子4a上有2个SNP位点：SNP1：GCT→GCC（Ala133 Ala）,3个品系均发生了变化,均为纯合突变,其突变率为（22/22,100%）。SNP2：GGC→GGT（Gly146 Gly）,巴马小型猪突变率为（6/6,100%）,均为纯合突变;五指山小型猪突变率为（6/6,100%）,均为纯合突变;中国农大小型猪突变率为（10/10,100%）,其中包括6例纯合突变（6/10,60%）和4例杂合突变（4/10,40%）。结论SNPs1,在所有测定的小型猪品系中检出,这可能是所测小型猪的共有特征。SNPs2可能是小型猪品系之间的差异。     

猪CACNA1S基因部分序列的克隆、测序及SNP检测

CACNA1S是钙离子通道主效亚基α1亚单位的编码基因,该基因突变会导致人（Homo sapiens）低钾性周期性麻痹和恶性高温综合征．目前CACNAIS基因的研究主要集中在人和模式动物上,而在家畜中的研究少见报道．本研究以金华猪（115头）、皮特兰猪（30头）、金华猪与皮特兰猪杂交产生的金皮F2代杂种猪（126头）、大约克猪（23头）为研究对象,根据人CACNA1S基因序列设计引物,对猪基因组DNA进行PCR扩增、克隆测序,并与人相应序列作同源性比较,然后采用PCR-SSCP技术检测序列中可能存在的单核苷酸多态（single nucleotide polymorphism,SNP）位点,并对有合适内切酶存在的突变点应用PCR-RFLP技术进行验证．结果表明：（i）用6对引物对猪基因组DNA进行扩增,共克隆得到猪CACNAIS基因约5211bp的DNA序列,其中外显子区域,猪与人同源性为82．6％,序列已递交GeneBank收录（登录号DQ767693）;（ii）在克隆得到的DNA序列中,共检测到57个单核苷酸突变点,其中外显子区域存在24个;（iii）突变位点的PCR-RFLP验证与PCR-SSCP检测结果一致,经与GenBank公布的猪CACNAIS基因EST小片段（Bx914582,Bx666997）比较,相应长度核酸序列内本实验检测到的11个SNP位点中,有8个位点的碱基突变与2个EST片段间存在的碱基差异相同．     

高原肺水肿易感基因多态性的全基因组关联分析

目的：高原肺水肿严重影响高原人群的健康。筛选高原肺水肿易感基因以用于高原肺水肿易感者的评估及防护。方法：利用Affymetrix SNP Array6．0芯片对23例高原肺水肿患者和17个健康对照进行全基因组SNP分型,利用PLINK软件进行了全基因组关联分析,利用Go和Pathway软件进行分析及作图。结果：全基因组关联分析获得39个相对显著的SNPs位点（P〈10^-4）。通过对这些SNP位点附近27个基因的c0和Pathway富集分析,发现这些基因主要参与细胞增殖调控过程、氮代谢过程和G蛋白耦联受体蛋白信号转导通路等。结论：本文发现的多态性位点及相关基因可能与高原肺水肿易感性相关。     

人类基因组致病SNP与良性SNP分布模式探究

基因组变异与生物学功能,乃至人体健康紧密相关。不同种类的基因组变异会对人类健康产生不同的影响,可能出现致病、良性或未知的临床症状。深入研究临床上表现为致病性或良性的基因组变异,能够有效加深我们对基因组变异与疾病发生发展的认识,对评估基因组变异所带来的影响具有一定的意义。本研究选择Clinvar数据库中与疾病相关的SNP,探究临床表现为致病与良性的SNP位点分别在基因组上的分布模式以及对蛋白序列的影响。本研究发现,致病SNP与良性SNP在外显子与内含子上的分布以及蛋白结构域内外的分布上具有非常显著差异,致病SNP在外显子和蛋白结构域内的分布密度更高。从蛋白编码区的SNP突变类型来看,致病SNP位点中的同义突变与非同义突变所占比例分别是0.64%和99.36%,而良性SNP位点中两类突变的比例相差较小,检验结果显示致病SNP位点中非同义突变更多。此外,本研究通过分析致病SNP与良性SNP中的同义突变与非同义突变在蛋白质结构域内外的分布情况,发现两类SNP变异中同义突变的分布没有显著差异,但非同义突变的分布具有显著差异,其中致病SNP变异分布在蛋白质结构域内外的比例是53.22%和46.78%,良性SNP变异分布比例为31.96%和68.04%。随后,我们对非同义突变所在基因进行了功能注释和富集分析,发现两类变异所在基因的功能范围几乎一致。     

植物全基因组选择技术的研究进展及其在玉米育种上的应用

全基因组选择技术通过全基因组中大量的单核苷酸多态性标记（SNP）和参照群体的表型数据建立 BLUP 模型估计出每一标记的育种值,称为估计育种值（GEBV）,然后仅利用同样的分子标记估计出后代个体育种值并进行选择。该文就近年来国内外有关影响基因组选择效率的主要因素——参考群体的类型与大小、模型的建立方法、标记的类型及其数目、性状遗传力,以及对基因组选择效率的影响等方面的研究进展进行综述,并介绍了全基因组选择技术在玉米育种上应用概况以及对未来的展望。     

全基因组测序在重要家畜上的研究进展

全基因组测序研究主要包括通过不同测序技术和组装比对方法,获得某物种的全基因组序列图谱,及在此基础上构建物种全基因组遗传变异图谱进行个体或群体遗传多样性、选择信号或起源进化等方面的研究。利用单核苷酸多态性(SNP)、插入和缺失(Indel)和拷贝数变异(CNV)等遗传变异作为分子标记,全基因组测序研究已经在家畜起源进化、驯化、适应性机制、重要经济性状候选基因、群体历史动态等方面取得了许多重要的研究成果。本文主要对近几年全基因组测序在常见家畜(猪、马、牛、羊等及其近缘物种)的取得的重要研究成果进行了综述,并讨论了全基因组测序的优势、缺点及在生产中意义。此外,对基因组测序研究的未来发展进行了归纳及展望,以期为今后家畜重要经济性状的功能基因定位和物种起源、驯化研究提供参考。     

倍性复杂植物的2种倍性鉴定方法的建立及应用(专题约稿）

研究以猕猴桃属内不同植物的幼嫩叶片为材料,利用流式细胞术和全基因组SNP(single nucleotide polymorphism)位点杂合子等位基因深度比率(heterozygous allele depth ratio)分布2种方法进行猕猴桃倍性鉴定。对取样叶片的生长状态、防止细胞核黏连的PVP(聚乙烯吡咯烷酮)浓度、滤网目数及过滤次数、不同倍性样本全基因组SNP分型的参数调整等因素进行探索。结果表明,流式细胞术检测中取未展开的幼嫩叶片获得完整细胞核的数目最多;5%PVP对减少细胞核之间的黏连最适宜;500目滤网过滤3次效果最好。SNP的分型主要与模拟基因组的组装质量和过滤识别SNP的参数设置有关。流式细胞术鉴定倍性的关键技术是使用未展开的幼嫩叶片以保证足够数量的完整细胞核及减少细胞核之间的黏连。同一植物材料的染色体倍性在60Co-γ辐照处理前后未发生改变。全基因组SNP位点杂合子频率分布图判断的倍性与流式细胞术鉴定结果一致。2种鉴定结果可以相互验证,使倍性的判断变得更加准确,为加快猕猴桃育种提供了基础。     

Linkage mapping of gene-associated SNPs to pig chromosome 11

Single nucleotide polymorphisms (SNPs) were discovered in porcine expressed sequence tags (ESTs) orthologous to genes from human chromosome 13 (HSA13) and predicted to be located on pig chromosome 11 (SSC11). The SNPs were identified as sequence variants in clusters of EST sequences from pig cDNA libraries constructed in the Sino-Danish pig genome project. In total, 312 human gene sequences from HSA13 were used for similarity searches in our pig EST database. Pig ESTs showing significant similarity with HSA13 genes were clustered and candidate SNPs were identified. Allele frequencies for 26 SNPs were estimated in a group of 80 unrelated pigs from Danish commercial pig breeds: Duroc, Hampshire, Landrace and Large White. Eighteen of the 26 SNPs genotyped in the PiGMaP Reference Families were mapped by linkage analysis to SSC11. The EST-based SNPs published here are new genetic markers useful for linkage and association studies in commercial and experimental pig populations. This study represents the first gene-associated SNP linkage map of pig chromosome 11 and adds new comparative mapping information between SSC11 and HSA13. Furthermore, our data facilitate future studies aimed at the identification of interesting regions on pig chromosome 11, positional cloning and fine mapping of quantitative trait loci in pig.     

Sequence polymorphisms in porcine homologs of murine coat colour‐related genes

Herein, we report the variability among 57 porcine homologs of murine coat colour‐related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F₂ family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F₂ family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig.     

猪Toll样受体4基因SNPs功能分析

Toll样受体4(Toll-like receptor 4,TLR4)在机体的免疫反应中发挥重要作用,该基因突变会影响受体的信号转导能力和机体的疾病抗性/易感性。文章在前期工作的基础上,进一步分析c.611 T>A(p.Leu204His)、c.1027C>A(p.Gln343Lys)和c.1605 G>T(p.Leu535Phe)3个错义突变对猪TLR4功能的影响。利用RT-PCR方法克隆猪TLR4基因全长编码区并引入定点突变;利用真核表达、双荧光素酶报告系统和Western blotting方法在瞬时转染的PK-15细胞内研究3个单核苷酸多态(Single nucleotide polymorphisms,SNPs)对猪TLR4配体识别和信号转导能力的影响;同时,利用创造酶切位点PCR-RFLP方法分析对TLR4活性有显著影响的点突变在民猪、大白、长白和中国东北野猪4个群体中的分布。结果,成功获得了民猪TLR4基因的全长编码区和3个单碱基变异体,构建了不同等位基因的真核表达载体,在PK-15细胞内确定了c.1605 G>T变异导致TLR4向下游传递信号的能力显著降低(P<0.01),该SNP只存在于民猪和野猪中且频率较高。猪TLR4基因c.1605 G>T变异影响Toll样受体的信号传递,可能和机体的疾病抗性/易感性有关。     

Development of an in silico coding gene SNP map in pigs

A total of 5450 sequences obtained from the NCBI pig SNP database were consolidated into 465 unique sequences (189 singleton sequences and 276 contigs). These 465 sequences contained 1787 putative SNPs and had strong sequence homology to 433 human protein-coding genes based on blast analyses. These genes were assigned to the pig QTL maps ( http://www.animalgenome.org/QTLdb/pig.html ) via the human and pig comparative maps established by a pig radiation hybrid (RH) map. The SNP information characterized from this study provides a useful functional gene variation resource to facilitate QTL data mining in the pig genome.     

The pig genome project has plenty to squeal about

Significant progress on pig genetics and genomics research has been witnessed in recent years due to the integration of advanced molecular biology techniques, bioinformatics and computational biology, and the collaborative efforts of researchers in the swine genomics community. Progress on expanding the linkage map has slowed down, but the efforts have created a higher-resolution physical map integrating the clone map and BAC end sequence. The number of QTL mapped is still growing and most of the updated QTL mapping results are available through PigQTLdb. Additionally, expression studies using high-throughput microarrays and other gene expression techniques have made significant advancements. The number of identified non-coding RNAs is rapidly increasing and their exact regulatory functions are being explored. A publishable draft (build 10) of the swine genome sequence was available for the pig genomics community by the end of December 2010. Build 9 of the porcine genome is currently available with Ensembl annotation; manual annotation is ongoing. These drafts provide useful tools for such endeavors as comparative genomics and SNP scans for fine QTL mapping. A recent community-wide effort to create a 60K porcine SNP chip has greatly facilitated whole-genome association analyses, haplotype block construction and linkage disequilibrium mapping, which can contribute to whole-genome selection. The future 'systems biology' that integrates and optimizes the information from all research levels can enhance the pig community's understanding of the full complexity of the porcine genome. These recent technological advances and where they may lead are reviewed.     

SNP mining porcine ESTs with MAVIANT, a novel tool for SNP evaluation and annotation

MOTIVATION: Single nucleotide polymorphisms (SNPs) analysis is an important means to study genetic variation. A fast and cost-efficient approach to identify large numbers of novel candidates is the SNP mining of large scale sequencing projects. The increasing availability of sequence trace data in public repositories makes it feasible to evaluate SNP predictions on the DNA chromatogram level. MAVIANT, a platform-independent Multipurpose Alignment VIewing and Annotation Tool, provides DNA chromatogram and alignment views and facilitates evaluation of predictions. In addition, it supports direct manual annotation, which is immediately accessible and can be easily shared with external collaborators. RESULTS: Large-scale SNP mining of polymorphisms bases on porcine EST sequences yielded more than 7900 candidate SNPs in coding regions (cSNPs), which were annotated relative to the human genome. Non-synonymous SNPs were analyzed for their potential effect on the protein structure/function using the PolyPhen and SIFT prediction programs. Predicted SNPs and annotations are stored in a web-based database. Using MAVIANT SNPs can visually be verified based on the DNA sequencing traces. A subset of candidate SNPs was selected for experimental validation by resequencing and genotyping. This study provides a web-based DNA chromatogram and contig browser that facilitates the evaluation and selection of candidate SNPs, which can be applied as genetic markers for genome wide genetic studies. AVAILABILITY: The stand-alone version of MAVIANT program for local use is freely available under GPL license terms at http://snp.agrsci.dk/maviant. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

水稻单核苷酸多态性及其应用现状

单核苷酸多态性（single nucleotide polymorphisms, SNPs）在水稻中数量多,分布密度高,遗传稳定性高。水稻SNPs的发现方法主要有对样本DNA的PCR产物直接测序、从SSR区段检测SNPs和从基因组序列直接搜索等。目前已有多种基因分型技术运用到了水稻SNPs检测,SNPs检测的高度自动化使水稻SNPs基因分型非常方便。单核苷酸多态性在水稻遗传图谱的构建、基因克隆和功能基因组学研究、标记辅助选择育种、遗传资源分类及物种进化等方面的应用具有巨大潜力。     

SNPMeta: SNP annotation and SNP metadata collection without a reference genome

The increase in availability of resequencing data is greatly accelerating SNP discovery and has facilitated the development of SNP genotyping assays. This, in turn, is increasing interest in annotation of individual SNPs. Currently, these data are only available through curation, or comparison to a reference genome. Many species lack a reference genome, but are still important genetic models or are significant species in agricultural production or natural ecosystems. For these species, it is possible to annotate SNPs through comparison with cDNA, or data from well‐annotated genes in public repositories. We present SNPMeta, a tool which gathers information about SNPs by comparison with sequences present in GenBank databases. SNPMeta is able to annotate SNPs from contextual sequence in SNP assay designs, and SNPs discovered through genotyping by sequencing (GBS) approaches. However, SNPs discovered through GBS occur throughout the genome, rather than only in gene space, and therefore do not annotate at high rates. SNPMeta can therefore be used to annotate SNPs in nonmodel species or species that lack a reference genome. Annotations generated by SNPMeta are highly concordant with annotations that would be obtained from a reference genome.     

The development of a genome wide SNP set for the Barnacle goose Branta leucopsis

Migratory birds are of particular interest for population genetics because of the high connectivity between habitats and populations. A high degree of connectivity requires using many genetic markers to achieve the required statistical power, and a genome wide SNP set can fit this purpose. Here we present the development of a genome wide SNP set for the Barnacle Goose Branta leucopsis, a model species for the study of bird migration. We used the genome of a different waterfowl species, Mallard Anas platyrhynchos, as a reference to align Barnacle Goose second generation sequence reads from an RRL library and detected 2188 SNPs genome wide. Furthermore, we used chimeric flanking sequences, merged from both Mallard and Barnacle Goose DNA sequence information, to create primers for validation by genotyping. Validation with a 384 SNP genotyping set resulted in 374 (97%) successfully typed SNPs in the assay, of which 358 (96%) were polymorphic. Additionally, we validated our SNPs on relatively old (30 years) museum samples, which resulted in a success rate of at least 80%. This shows that museum samples could be used in standard SNP genotyping assays. Our study also shows that the genome of a related species can be used as reference to detect genome wide SNPs in birds, because genomes of birds are highly conserved. This is illustrated by the use of chimeric flanking sequences, which showed that the incorporation of flanking nucleotides from Mallard into Barnacle Goose sequences lead to equal genotyping performance when compared to flanking sequences solely composed of Barnacle Goose sequence.