Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

Background

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.

Results

A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.

Conclusions

Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.     

miRNA相关单核苷酸多态性

miRNA相关单核苷酸多态性(miRNA-related single nucleotide polymorphisms或mirSNP)是可以导致miRNA基因调控功能缺失或紊乱的一类功能型SNP的总称。不论是miRNA靶基因结合位点,还是miRNA基因或miRNA加工基因上的mirSNP,都有可能影响miRNA对靶基因的调控。miRNA基因及miRNA加工基因上的mirSNP主要通过阻碍miRNA的生物合成而发挥功能,而靶基因结合位点上的mirSNP主要通过导致自由能的改变或功能构象的消失,影响miRNA与靶序列结合而丧失其原有的调控功能。mirSNP大多位于人类基因组基因间区和内含子区,与包括肿瘤在内的众多复杂性疾病密切关联。mirSNP不论对于复杂性疾病发病机制研究还是诊疗预后分子标志的确定都具有极其重要的研究价值。     

Detection of single nucleotide polymorphisms

Single nucleotide polymorphism (SNP) detection technologies are used to scan for new polymorphisms and to determine the allele(s) of a known polymorphism in target sequences. SNP detection technologies have evolved from labor intensive, time consuming, and expensive processes to some of the most highly automated, efficient, and relatively inexpensive methods. Driven by the Human Genome Project, these technologies are now maturing and robust strategies are found in both SNP discovery and genotyping areas. The nearly completed human genome sequence provides the reference against which all other sequencing data can be compared. Global SNP discovery is therefore only limited by the amount of funding available for the activity. Local, target, SNP discovery relies mostly on direct DNA sequencing or on denaturing high performance liquid chromatography (dHPLC). The number of SNP genotyping methods has exploded in recent years and many robust methods are currently available. The demand for SNP genotyping is great, however, and no one method is able to meet the needs of all studies using SNPs. Despite the considerable gains over the last decade, new approaches must be developed to lower the cost and increase the speed of SNP detection.     

单核苷酸多态概述

单核苷酸多态ＳＮＰ是遍布于基因组中的一种ＤＮＡ序列变化类型,人类基因组中平均约每一千碱基中有一个。单核苷酸多态是一种双等位型多态,群体中出现的频率大于１％或２％者视为多态,低于１％或２％的则视为突变。由于其具有高信息量、高密度又便于自动化操作的特点,单核苷酸多态在遗传性疾病基因的克隆和药物的设计与开发方面具有广阔的应用前景。本文对单核苷酸的概念、特点、应用前景,及其研究应用的一些问题作一综述。     

Cytokine single nucleotide polymorphisms in Iranian populations

Cytokines are important immunomodulatory molecules involved in immune responses against microorganisms; they also have an important role in the setting of immune system disorders. Cytokine single nucleotide polymorphisms have been extensively studied in different, normal populations as well as in association with disease. Cytokine gene polymorphisms are potentially important as genetic predictors of disease susceptibility, clinical outcome, and as a tool for anthropological studies. In this study, samples have been collected from 455 healthy individuals located in different regions of Iran (Tehran, Yazd, Sistan and Balochistan). Allele and genotype frequencies of cytokine SNP, including: IL-1alpha, IL-1beta, IL-1R, IL-1RA, IL-2, IL-4, IL-4RA, IL-6, IL-10, IL-12, TNF-alpha, TGF-beta and IFN-gamma were investigated, using the PCR-SSP method. Allele frequencies in Tehran and Yazd populations were similar, except for TGF-beta. Allele frequencies in Sistani & Baloch populations were similar at all positions, except for IL-1beta at position of -511 and IFN-gamma genes at position UTR5644; there were some differences in allele frequencies comparing these populations with the Yazd population, including: IL-4, IL-6, IL-10, TGF-beta and TNF-alpha. Although some significant differences were observed for some cytokines, it seems that the cytokine gene polymorphism profile of the Iranian population is similar to that of Caucasians, particularly the Italian population.     

Linkage analysis using single nucleotide polymorphisms

We performed multipoint linkage analysis using 83 markers from the SNP Consortium (TSC) SNP linkage map in 3 regions covering 190 cM previously scanned with microsatellite markers and found to be linked to type 2 diabetes. Since the average linkage disequilibrium present in the TSC SNP marker clusters is relatively low, we assumed the intracluster genetic distances were a reasonable small nonzero distance (0.03 cM) and performed linkage analysis using GENEHUNTER PLUS and ASM linkage analysis software. We found that for the pedigree structures and missing data patterns in our samples the average information content in all three regions and the LOD score curves in two regions obtained from the TSC SNP markers were similar to results obtained from microsatellite marker maps with 10 cM average spacing. We also give an algorithm which extends the Lander-Green algorithm to permit multipoint linkage analysis of clusters of tightly linked markers with arbitrarily high levels of intracluster linkage disequilibrium.     

Characterisation of single nucleotide polymorphisms in sugarcane ESTs

Commercial sugarcane cultivars (Saccharum spp. hybrids) are both polyploid and aneuploid with chromosome numbers in excess of 100; these chromosomes can be assigned to 8 homology groups. To determine the utility of single nucleotide polymorphisms (SNPs) as a means of improving our understanding of the complex sugarcane genome, we developed markers to a suite of SNPs identified in a list of sugarcane ESTs. Analysis of 69 EST contigs showed a median of 9 SNPs per EST and an average of 1 SNP per 50 bp of coding sequence. The quantitative presence of each base at 58 SNP loci within 19 contiguous sequence sets was accurately and reliably determined for 9 sugarcane genotypes, including both commercial cultivars and ancestral species, through the use of quantitative light emission technology in pyrophosphate sequencing. Across the 9 genotypes tested, 47 SNP loci were polymorphic and 11 monomorphic. Base frequency at individual SNP loci was found to vary approximately twofold between Australian sugarcane cultivars and more widely between cultivars and wild species. Base quantity was shown to segregate as expected in the IJ76-514 × Q165 sugarcane mapping population, indicating that SNPs that occur on one or two sugarcane chromosomes have the potential to be mapped. The use of SNP base frequencies from five of the developed markers was able to clearly distinguish all genotypes in the population. The use of SNP base frequencies from a further six markers within an EST contig was able to help establish the likely copy number of the locus in two genotypes tested. This is the first instance of a technology that has been able to provide an insight into the copy number of a specific gene locus in hybrid sugarcane. The identification of specific and numerous haplotypes/alleles present in a genotype by pyrophosphate sequencing or alternative techniques ultimately will provide the basis for identifying associations between specific alleles and phenotype and between allele dosage and phenotype in sugarcane.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Empirical Bayes analysis of single nucleotide polymorphisms

Background  

An important goal of whole-genome studies concerned with single nucleotide polymorphisms (SNPs) is the identification of SNPs associated with a covariate of interest such as the case-control status or the type of cancer. Since these studies often comprise the genotypes of hundreds of thousands of SNPs, methods are required that can cope with the corresponding multiple testing problem. For the analysis of gene expression data, approaches such as the empirical Bayes analysis of microarrays have been developed particularly for the detection of genes associated with the response. However, the empirical Bayes analysis of microarrays has only been suggested for binary responses when considering expression values, i.e. continuous predictors.     

IFNGR1 single nucleotide polymorphisms in rheumatoid arthritis

On the basis of their biological function, potential genetic candidates for susceptibility to rheumatoid arthritis can be postulated. IFNGR1, encoding the ligand-binding chain of the receptor for interferon gamma, IFNγR1, is one such gene because interferon gamma is involved in the pathogenesis of the disease. In the coding sequence of IFNGR1, two nucleotide positions have been described to be polymorphic in the Japanese population. We therefore investigated the association of those two IFNGR1 single nucleotide polymorphisms with rheumatoid arthritis in a case-control study in a central European population. Surprisingly, however, neither position was polymorphic in the 364 individuals examined, indicating that IFNGR1 does not contribute to susceptibility to rheumatoid arthritis, at least in Caucasians.     

New strategy to detect single nucleotide polymorphisms

A great effort has been made to identify and map a large set of single nucleotide polymorphisms. The goal is to determine human DNA variants that contribute most significantly to population variation in each trait. Different algorithms and software packages, such as PolyBayes and PolyPhred, have been developed to address this problem. We present strategies to detect single nucleotide polymorphisms, using chromatogram analysis and consensi of multiple aligned sequences. The algorithms were tested using HIV datasets, and the results were compared with those produced by PolyBayes and PolyPhred using the same dataset. Our algorithms produced significantly better results than these two software packages.     

Characterization of 12 single nucleotide polymorphisms in weathervane scallop

We describe 27 single nucleotide polymorphisms (SNPs) in a commercially important bivalve, the weathervane scallop (Patinopecten caurinus), identified using a targeted‐gene approach. We further characterize 12 of these using 5′‐nuclease and allele‐specific PCR assays. Polymorphisms were identified in both mitochondrial and nuclear genes. These are the first SNPs developed for delineating population structure in the weathervane scallop and will provide a useful complement to currently available genetic markers.     

单核苷酸多态性在林木中的研究进展

摘要: 单核苷酸多态性(Single nucleotide polymorphisms, SNPs)是许多生物体最丰富的遗传变异形式。林木是重要的植物类群和陆地植物生态系统的重要组成部分, SNP作为新的分子标记已应用于松、杨、黄杉、桉和云杉等属的多个树种的遗传育种学研究, 获得了包括核苷酸多样性、连锁不平衡及群体结构等相关的遗传信息, 这些研究主要建立在对候选基因序列进行测序分析的基础上。基于SNP的关联遗传学分析或连锁不平衡(Linkage disequilibrium, LD)作图, 已成为研究林木复杂数量性状的理想工具, 对桉树和火炬松的关联遗传学研究发现, 多个基因内的SNP位点与不同的木材性状相关联。利用SNP标记对林木遗传参数的估算从不同程度上揭示了林木群体进化规律及其生态学意义。SNP标记在林木中应用的不断深入, 必将极大地推动林木遗传育种学研究的发展。     

dbSNP: a database of single nucleotide polymorphisms

In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Cancer for Biotechnology Information (NCBI) has established the dbSNP database. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. Submitted SNPs can also be downloaded via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/     

Association genetics in Corymbia citriodora subsp. variegata identifies single nucleotide polymorphisms affecting wood growth and cellulosic pulp yield

Wood is an important biological resource which contributes to nutrient and hydrology cycles through ecosystems, and provides structural support at the plant level. Thousands of genes are involved in wood development, yet their effects on phenotype are not well understood. We have exploited the low genomic linkage disequilibrium (LD) and abundant phenotypic variation of forest trees to explore allelic diversity underlying wood traits in an association study. Candidate gene allelic diversity was modelled against quantitative variation to identify SNPs influencing wood properties, growth and disease resistance across three populations of Corymbia citriodora subsp. variegata, a forest tree of eastern Australia. Nine single nucleotide polymorphism (SNP) associations from six genes were identified in a discovery population (833 individuals). Associations were subsequently tested in two smaller populations (130-160 individuals), 'validating' our findings in three cases for actin 7 (ACT7) and COP1 interacting protein 7 (CIP7). The results imply a functional role for these genes in mediating wood chemical composition and growth, respectively. A flip in the effect of ACT7 on pulp yield between populations suggests gene by environment interactions are at play. Existing evidence of gene function lends strength to the observed associations, and in the case of CIP7 supports a role in cortical photosynthesis.     

Rolling circle amplification for scoring single nucleotide polymorphisms

The analysis of the genetic basis of phenotypic traits is moving towards the complex diseases prevalent in wealthy populations. There is an increasing requirement for the detection of different types of sequence variation, particularly single-nucleotide polymorphisms (SNPs). SNPs occur about once every 100 to 300 bases. High-density SNP maps will help to identify the multiple genes associated with complex diseases such as cancer, diabetes, vascular disease, and some forms of mental illness.     

An efficient procedure for genotyping single nucleotide polymorphisms

Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.     

Haplotype tagging single nucleotide polymorphisms and association studies

OBJECTIVES: Discrete blocks of low haplotype diversity exist within the human genome. The non-redundant subset of 'haplotype tagging' single nucleotide polymorphisms (htSNPs) in such blocks can distinguish a majority of the haplotypes. Several approaches have been proposed to determine htSNPs, ranging from visual inspection to formal analytic procedures. Optimal htSNPs can be estimated using a small subgroup of an association study population that have been genotyped for a dense SNP map, and it is just these htSNPs that are genotyped in the remainder of the samples. We investigated by simulation how the size of the subsample affects the power of association studies, and what type of subjects it should include. METHODS: We used the program tagSNPs [Stram et al., Hum Hered 2003;55:27-36], which selects htSNPs to minimize the uncertainty in predicting common haplotypes for individuals with unphased genotype data. RESULTS: On average, 27% of the SNPs were designated as htSNPs. Genotyping as few as 25 unphased individuals to select the htSNPs did not appear to reduce the power of an association study, as compared with using all SNPs. For the disease models considered, selecting htSNPs based on cases, controls, or a mixture of both gave similar results. CONCLUSIONS: These results suggest that the genotyping effort in an association study can be substantially reduced with little loss of power by identifying htSNPs in a small subsample of individuals.