A powerful tool for genome analysis in maize: development and evaluation of the high density 600 k SNP genotyping array

Background

High density genotyping data are indispensable for genomic analyses of complex traits in animal and crop species. Maize is one of the most important crop plants worldwide, however a high density SNP genotyping array for analysis of its large and highly dynamic genome was not available so far.

Results

We developed a high density maize SNP array composed of 616,201 variants (SNPs and small indels). Initially, 57 M variants were discovered by sequencing 30 representative temperate maize lines and then stringently filtered for sequence quality scores and predicted conversion performance on the array resulting in the selection of 1.2 M polymorphic variants assayed on two screening arrays. To identify high-confidence variants, 285 DNA samples from a broad genetic diversity panel of worldwide maize lines including the samples used for sequencing, important founder lines for European maize breeding, hybrids, and proprietary samples with European, US, semi-tropical, and tropical origin were used for experimental validation. We selected 616 k variants according to their performance during validation, support of genotype calls through sequencing data, and physical distribution for further analysis and for the design of the commercially available Affymetrix® Axiom® Maize Genotyping Array. This array is composed of 609,442 SNPs and 6,759 indels. Among these are 116,224 variants in coding regions and 45,655 SNPs of the Illumina® MaizeSNP50 BeadChip for study comparison. In a subset of 45,974 variants, apart from the target SNP additional off-target variants are detected, which show only a minor bias towards intermediate allele frequencies. We performed principal coordinate and admixture analyses to determine the ability of the array to detect and resolve population structure and investigated the extent of LD within a worldwide validation panel.

Conclusions

The high density Affymetrix® Axiom® Maize Genotyping Array is optimized for European and American temperate maize and was developed based on a diverse sample panel by applying stringent quality filter criteria to ensure its suitability for a broad range of applications. With 600 k variants it is the largest currently publically available genotyping array in crop species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-823) contains supplementary material, which is available to authorized users.     

高分辨率熔解——SNP及突变研究的最新工具

随着分子生物学技术的不断发展,人们得以不断深入的研究单核苷酸多态性（SNP）以及突变与表型和疾病的相关性;相应的,人们对SNP及突变研究的关注也推动了相关研究手段的不断发展,使其在方法学上得以不断推陈出新。高分辨率熔解（high resolution melt,HRM）是近年来发展出的最新的SNP及突变研究工具,HRM因其高通量、低成本、不受检测位点的局限,且真正实现SNP的闭管检测而广受国外科研工作者的关注。该文对HRM技术与其他相关研究手段进行了比较,并对HRM技术的特点、应用进行了阐述。     

单核苷酸多态性检测方法的研究进展

单核苷酸多态性（single nucleotide polymorphism, SNP）的研究已成为人类后基因组时代的主要内容之一。因此建立高度自动化和高通量的SNP检测分析技术十分重要。文章系统地介绍了最新发展的几种SNP检测技术的原理和检测平台,详细阐述了等位基因特异性杂交、内切酶酶切技术、引物延伸法、寡核苷酸连接反应等SNP检测原理,以及平板读数仪、基因芯片、微球阵列技术和质谱仪等检测平台,并对SNP高通量检测技术的发展进行了展望。     

Automated SNP genotype clustering algorithm to improve data completeness in high-throughput SNP genotyping datasets from custom arrays

High-throughput SNP genotyping platforms use automated genotype calling algorithms to assign genotypes. While these algorithms work efficiently for individual platforms, they are not compatible with other platforms, and have individual biases that result in missed genotype calls. Here we present data on the use of a second complementary SNP genotype clustering algorithm. The algorithm was originally designed for individual fluorescent SNP genotyping assays, and has been optimized to permit the clustering of large datasets generated from custom-designed Affymetrix SNP panels. In an analysis of data from a 3K array genotyped on 1,560 samples, the additional analysis increased the overall number of genotypes by over 45,000, significantly improving the completeness of the experimental data. This analysis suggests that the use of multiple genotype calling algorithms may be advisable in high-throughput SNP genotyping experiments. The software is written in Perl and is available from the corresponding author.     

应用创造酶切位点法检测单碱基突变

应用引物错配技术结合单碱基突变位点而配合成一个酶切位点,使之成为可用PCR-RFLP方法分析的突变位点,是对单碱基突变位点进行基因型鉴定的有效而简捷的手段。本文以鸡胞外脂肪酸结合蛋白(Extracelluar fatty acid binding protein,EX-FABP)基因单碱基突变的基因型检测为例,探讨了应用创造酶切位点PCR（Created Restriction Site PCR,CRS-PCR）检测单碱基突变基因型的思路、方法和策略。Abstract:Created Restriction Site PCR (CRS-PCR) is a simple and efficient method to identify SNP genotypes.One or more mismatch bases are used in a primer to create a restriction site by combining SNP site after PCR.The CRS-PCR products can be genotyped with a way the same as PCR-RFLP.In the study,Extracelluar fatty acid binding protein (EX-FABP)　gene was served as an example for establishing the CRS-PCR method.Strategy of CRS-PCR was also discussed.     

High throughput SNP genotyping with two mini-sequencing assays

Single nucleotide polymorphisms (SNPs) are veryimportant markers that can be used in many areas such asevolutionary genetics [1], disease-susceptibility genes[2,3], personalized medicine and forensics. Only about20% of human polymorphisms are length polymorphisms,whereas about 80% of human polymorphisms areSNPs. Kruglyak et al. [4] reported that there were about11,000,000 SNPs in the world population. There are many kinds of SNP genotyping technology[5,6]: some are only suitable to low …     

Susceptibility genes & modifiers for cardiac arrhythmias

Cardiac arrhythmias in the absence of structural heart diseases can be subdivided in those cases which are acquired and those which are linked to genetic defects on cardiac ion channels and regulatory subunits. Although acquired arrhythmias do not contain any obviously genetic component the observation of frequently occurring single nucleotide polymorphism (SNPs) identified in cardiac ion channel genes lead to the question if these natural variants can influence the development of acquired forms of cardiac arrhythmias and thus serve as genetic susceptibility markers. This review summarizes the results of genetic association and linkage studies in drug induced long-QT syndrome and atrial and ventricular fibrillation and discusses advantages and future directions of this topic in cardiac research.     

高通量飞行时间质谱基因分型方法的研究

为了考察飞行时间质谱基因分型方法 (MALDI-TOF) 的位点分型成功率和分型结果质量的关系,分析了 96 个 SNPs 位点的近 10 000 个基因分型数据 (用 MALDI-TOF “4 重”实验方法检测 ). 结果显示,位点分型成功率和分型结果的质量显著正相关 . 分型成功率低于 82% 的 SNP 位点,其高质量结果占的比例开始逐渐降低 . 提示 82% 的分型成功率可以作为衡量分型结果质量的数据点 . 为了进一步提高通量并降低成本,在 MALDI-TOF “ 4 重”实验方法的基础上,发展了两种“准 8 重”实验方法 . 用新的实验方法检测了 95 个样本的 32 个 SNPs 位点 . 结果显示“混合准 8 重”实验方法与“ 4 重”实验方法相比无显著差异,而“复点准 8 重”的结果差于“ 4 重”分型方法 .     

基于连接酶—ELISA反应的单核苷酸多态性分型新方法

随着大量与人类疾病和药物治疗相关的单核苷酸多态性（Single-nucleotide polymorphism,SNP）的发现,出现了多种SNP分型检测的方法和技术。然而,大多数方法由于受限于检测灵敏度低或对检测设备和实验条件要求较高,不适宜于在一般实验条件下进行常规临床检测。通过建立一种基于连接酶-ELISA的SNP快速分型新方法,以非小细胞肺癌个体化治疗中,酪氨酸激酶抑制剂药物的生物标记基因—表皮生长因子受体基因（EGFR）为检测对象,对EGFR,c.2573T〉G（L858R）,EGFR,c.2582T〉A（L861Q）和EGFR,c.2155 G〉T（G719C）3个SNP位点进行了突变检测。经过18~28个循环的PCR扩增,能够通过琼脂糖凝胶电泳和ELISA反应,根据电泳条带的有无和ELISA显色值清晰判断检测位点的基因型,并且能够从混合等位基因样本中检测出5%的突变型等位基因。结果表明,方法具有较高的特异性和灵敏度,适合于在常规实验条件下从不均一的样本中进行突变等位基因的检测。     

基于国产微滴数字PCR的SNP快速检测技术研究

目的 在法医学领域,现有的SNP检测主要依赖进口,检测工作量大、耗时长且成本较高。微滴数字PCR (droplet digital PCR,ddPCR)作为新一代的PCR技术,可以快速检测低浓度样本DNA,且有较强的抗干扰能力。本研究旨在国产ddPCR平台建立SNP分型检测体系并对其性能进行评估,以探讨ddPCR技术在法医学检验领域的应用价值。方法 在ddPCR平台建立高原适应性EPAS1单倍型(rs115321619、rs73926263、rs73926264、rs73926265和rs55981512)检测体系,测试各位点引物探针特异性,对体系的准确性、稳定性、灵敏度、检材适应性进行评估,并比较了ddPCR和SNaPshot微测序检测体系的抗抑制性,最后对样本地区来源进行测试。结果 ddPCR在2.5 h内即可快速获取检测结果,体系准确性和稳定性好,检测灵敏度为0.312 5 ng,且抗抑制性能力突出。70份测试样本检测结果与背景信息一致。结论 基于ddPCR的SNP检测体系具有准确可靠、简便快速、抗抑制能力强等优势,在法医学快速检验领域有较强的应用潜力,适合法医现场检验需求。     

High throughput genotyping technologies.

A comprehensive genetic map containing several hundred microsatellite markers resulted from a large microsatellite mapping project. This was the first real study that introduced high throughput methods to the genetic community. This map and the concurrent technological advances, which will briefly be reviewed, led to further numerous mapping investigations of simple and complex diseases. The annotated draft sequence of approximately three billion base pairs (bp) of the human genome has been completed much sooner than many imagined, due to considerable technological advancements and the international enterprise that resulted. This was a major development for the genetics community, but is only the precursor to the next phase of studying and understanding the variation within the human genome. The awareness of the differences may help us understand the effects on the genetics of the variation between individuals and disease. It is these variations at the nucleotide level that determine the physiological differences, or phenotypes of each individual, including all biological functions at the cellular and body level. Single nucleotide polymorphisms (SNPs) will provide the next high density map, and be the genetic tool to study these genetic variations. There are many sources of SNPs and exhaustive numbers of methods of SNP detection to be considered. The focus in this paper will be on the merits of selected, varied SNP typing methodologies that are emerging to genotype many individuals with the required huge number of SNPs to make the study of complex diseases and pharmacogenomics a practical and economically viable option.     

A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis

To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure.     

High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.)

The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina''s GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.