基于珙桐SNP位点的dCAPS标记开发

本研究收集了珙桐、喜树等19份植物材料,运用分子生物学和生物信息学手段比对分析了rpoB,rpoC,matK等8个基因碱基序列上的单核苷酸多态性(SNP)位点,在atpF-atpH基因上找到了合适的SNP位点并设计了dCAPS引物,经PCR扩增和酶切验证后,将SNP转化为衍生型酶切扩增多态性序列(dCAPS)标记,可促进SNP标记在珙桐的遗传图谱构建、基因定位、种质资源鉴定、遗传多样性研究等领域的应用。     

Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice (Oryza sativa L.)

The waxy gene, which encodes the granule bound starch synthase enzyme, is one of the key genes influencing starch synthesis in the rice endosperm. To investigate functional differences between GBSS alleles, we cloned and sequenced GBSS cDNA from a series of cultivars that differed substantially in apparent amylose content and starch viscosity characteristics. We found two single nucleotide polymorphisms in exons 6 and 10 that resulted in amino acid substitutions. These substitutions are associated with differences in apparent amylose content and viscosity characteristics. Subsequent sequencing of these regions from additional cultivars confirmed their association with particular rice quality characteristics. These point mutations could prove useful as molecular markers in the production of cultivars with superior eating, cooking and processing quality, and contribute to our understanding of the various structural and functional differences among granule bound starch synthase alleles.     

单核苷酸多态性在作物遗传及改良中的应用

单核苷酸多态性（single nucleotide polymorphism,SNP）是等位基因间序列差异最为普遍的类型,可作为一种高通量的遗传标记。已建立了PCR扩增目标序列及其产物测序和电子SNP（eSNP）等多种发现和检测SNP的方法。玉米和大豆等作物也已开展了SNP分析。一些栽培作物种质的多样性不断减少,其结果使连锁不平衡（linkage disequilibrium,LD）增加,这有利于目的基因座上SNP单元型（haplotype）与表型的相关性分析。SNP已在作物基因作图及其整合、分子标记辅助育种和功能基因组学等领域展示了广泛的应用价值。 Abstract:Single nucleotide polymorphism(SNP) is the most common type of sequence difference between alleles,which can be used as a kind of high-throughput genetic marker.Several different routes have been developed to discover and identify SNP.These include the direct sequencing of PCR amplicons,electronic SNP(eSNP) and so on.SNP assays have been made in many crop species such as maize and soybean.The elite germplasm of some crops have been narrowed in genetic diversity,increasing the amount of linkage disequilibrium(LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes.SNP analysis has been broadly used in the field of plant gene mapping,integration of genetic and physical maps,DNA marker-assisted breeding and functional genomics.     

Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers.

We searched for SNPs in 417 regions distributed throughout the genome of three Oryza sativa ssp. japonica cultivars, two indica cultivars, and a wild rice (O. rufipogon). We found 2800 SNPs in approximately 250,000 aligned bases for an average of one SNP every 89 bp, or one SNP every 232 bp between two randomly selected strains. Graphic representation of the frequency of SNPs along each chromosome showed uneven distribution of polymorphism-rich and -poor regions, but little obvious association with the centromere or telomere. The 94 SNPs that we found between the closely related cultivars 'Nipponbare' and 'Koshihikari' can be converted into molecular markers. Our establishment of 213 co-dominant SNP markers distributed throughout the genome illustrates the immense potential of SNPs as molecular markers not only for genome research, but also for molecular breeding of rice.     

单核苷酸多态性及其在鸡QTL定位上的应用

单核苷酸多态性是指DNA序列上的单个碱基变异,它具有分布广、多态信息含量大、易于检测和统计分析等优点,能较好用于基因图谱构建和数量性状QTL定位研究,被称为继RFLP和微卫星标记之后的第3代基因遗传标记。本文综述了单核苷酸多态性的性质及检测技术、利用候选基因SNP进行鸡QTL定位研究的现状,并对未来SNP的应用前景进行了展望。Abstract:Single nucleotide polymorphism (SNP) refers to the change of single nucleotide in DNA sequence.Because of its high density in genomes and easy in detection and analysis statistically,SNP can be used in genetic linkage map construction and QTL mapping.Here,the characters and detecting technology of SNP,as well as the status and foreground of the use of candidate gene SNP in chicken QTL mapping are introduced.     

Polymorphism attribution of cSNPs in cancer-related genes located in loss regions with a high frequency of HCC between HBV and health groups

Cancer-related genes harbored in the loss regions containing a high frequency of hepatocellular carcinoma (HCC) were selected. Related information was gathered and the coding single nucleotide polymorphism (cSNP) sequences were obtained from the single nucleotide polymorphism (SNP) database. The appropriate primers and oligonucleotide probes were then designed in accordance with the SNP sites, and subsequently, the gene chips for detecting SNPs were constructed. Genomic DNA was extracted from blood samples of healthy controls and from patients with HBV infection. The sequences, including the SNPs, were amplified via polymerase chain reaction (PCR) and labeled using digoxigenin deoxyuridine tri-phosphate (Dig-dUTP). The labeled products were then hybridized with the SNP chips. Results confirmed that the differences in allele frequencies of three SNPs EGFL3 (rs947345), Caspase9 (rs2308950), and E2F2 (rs3218171) were distinct between HBV-infected patients and controls, suggesting that these SNPs ocuring in high frequency in HBV-infected individuals may be associated with susceptibility to HCC. Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2006, 39(3): 1–5 [译自: 南开大学学报(自然科学版)]     

Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.     

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 .     

不同性别类型黄瓜ACC合酶基因的单核苷酸多态性标记和酶切扩增长度多态性标记

黄瓜的性别分化与乙烯密切相关,1-氨基环丙烷-1-羧酸(ACC)合酶是乙烯生物合成过程中的关键酶.根据ACC合酶基因家族的保守序列设计PCR引物,从8个不同性别类型(雌雄同株、强雌性和全雌性)黄瓜品种中克隆了长度为1188bp的ACC合酶基因(CS-ACS2)片段(GenBank登记号为:DQ115884～DQ115886和DQ115875～DQ115879).经测序分析,3个雌雄同株性别类型品种的序列完全相同.与之相比,5个强雌性和全雌性品种中存在8个单核苷酸多态性(SNPs)标记,SNPs标记为4个A←→G和4个T←→C之间的转换.在8个SNPs中,有1个SNP位于内含子区域,其余7个SNPs都位于外显子区域.在7个位于外显子区域的SNPs中,有3个为非编码区的SNPs,4个为cSNPs.而在4个cSNPs中,有3个导致了编码的氨基酸序列改变.研究结果表明,与雌雄同株性别类型相比,雌性系中均存在单核苷酸的变异,这提示ACC合酶基因CS-ACS2的单核苷酸变异可能与黄瓜雌性系的发生形成有关.另一方面,根据SNP多态性还发展了一个酶切扩增长度多态性(CAPS)标记C-MT700.利用CAPS标记C-MT700能将强雌性优良品种MT-705与其他黄瓜品种相区别,该标记在黄瓜育种生产上具有一定的应用价值.此外,研究获得的SNPs标记和CAPS标记丰富了黄瓜的分子标记种类.     

脂肪因子visfatin的调节与功能多样性

内脏脂肪素(visfatin)是一种由内脏脂肪细胞分泌的分子质量为52 ku的蛋白质细胞因子．基因序列分析显示其cDNA编码序列与前B细胞集落增强因子(PBEF)同源且在进化中高度保守．Visfatin被发现具有多种迥然不同的生物活性：通过与胰岛素受体相互作用,在不同的情况下visfatin可表现出类胰岛素或抗胰岛素样作用;在细胞质中,visfatin具有烟酰胺磷酸核糖转移酶(Nampt)活性,能够催化烟酰胺腺嘌呤二核苷酸(NAD)的生物合成;作为分泌型的细胞因子,visfatin还可以诱导多种炎性因子的表达,如TNFα、IL-1β和IL-6．Visfatin与一些代谢疾病和急、慢性炎性疾病的关系日益受到重视,如糖尿病、肥胖、急性肺损伤、类风湿性关节炎、败血症、心肌梗塞和炎性肠病等．最近,对visfatin的启动子区及其单核苷酸多态性(SNP)的研究,进一步深化了人们对其在疾病发病机制中作用的认识．重点讨论visfatin的结构、功能多态性以及它与多种疾病的关系．     

Polymorphisms in cytokine genes and risk of Helicobacter pylori infection among Jamaican children

Background: Infection by Helicobacter pylori is often acquired during childhood. Recent studies suggest that inflammatory cytokines may play a role in susceptibility to, and disease phenotype caused by, H. pylori infection, but the association of host genetic variability with risk of H. pylori infection has not been studied in children. Methods: We investigated the relationship between the risk of H. pylori antibody positivity and cytokine gene polymorphisms among 199 two‐year‐old Jamaicans. H. pylori seropositivity was determined by a validated research enzyme‐linked immunosorbent assay. Real‐time Taqman® polymerase chain reaction was used to determine variants at 17 loci in 11 cytokine genes (IL1A, IL1B, IL2, TNF, TLR4, IL4, IL6, IL10, IL10RA, IL12A and IL13). We estimated the odds ratio and the 95% confidence interval for the association of genetic polymorphisms with H. pylori seropositivity, using logistic regression. Results: Forty (20.1%) of 199 children were seropositive. Children's H. pylori seropositivity correlated highly with maternal H. pylori seropositivity (OR = 7.98, 95% CI = 1.05–60.60, p = .02). Children carrying IL1A?889T had a lower risk of H. pylori positivity, compared to those carrying ?889C, with each T allele associated with 43% risk reduction (OR = 0.57, 95% CI = 0.33–0.99, p‐trend = .05). No other loci we examined were associated with the risk of H. pylori seropositivity. Conclusions: The IL1A?889 T allele, known to express a higher level of cytokine IL‐1α, is associated with a lower risk of H. pylori infection among Jamaican children. Our finding supports the hypothesis that an upregulation of pro‐inflammatory cytokines may protect against persistent H. pylori colonization.     

Three single nucleotide polymorphisms leading to non-synonymous amino acid substitution in the human ribonuclease 2 and angiogenin genes exhibit markedly less genetic heterogeneity in six populations

Angiogenin and ribonuclease 2 (RNase 2) are members of the human RNase superfamily. Although three potential single nucleotide polymorphisms (SNPs) in these genes, which could give rise to an amino acid substitution in the protein, have been identified, relevant population data are not available, and accordingly they have not been applied to clinical-genetic analysis. For this purpose, a novel genotyping method for each SNP using the mismatched PCR-restriction fragment length polymorphism technique has been developed. Using this method, the genotype distribution of each SNP was investigated in six populations: Japanese (n = 167), Korean (n = 90), Mongolian (n = 92), Ovambos (n = 86), Turkish (n = 87), and German (n = 70). In all the populations, only one genotype was found in each SNP. Irrespective of differences in ethnic groups, the angiogenin and RNase 2 genes appear to exhibit markedly less genetic heterogeneity with regard to these SNPs.     

不同品种猪肌肉生长抑制素基因单核苷酸多态性分析

用PCR-RFLPs和PCR-SSCP分析方法,对"双肌臀”大白猪、大白猪、长白猪、杜洛克、汉普夏、皮特兰、二花脸、东北民猪、湖北白猪和部分杂交猪等不同品种猪肌肉生长抑制素基因3＇编码区、5＇调控区及内含子1区3个单核苷酸多态性位点(SNPs)进行了分析.结果表明,3＇编码区的SNP发生的频率较低,在274头猪中未检出突变纯合体.对5＇调控区的SNP,引进猪种(大白猪、长白猪、杜洛克、汉普夏和皮特兰)及其杂交猪以等位基因T为主,二花脸和湖北白猪则以等位基因A为主,均偏离Hardy-Weinberg平衡状态(P＜0.01).东北民猪的3种基因型近乎相等,处于Hardy-Weinberg衡状态.对内含子1区的SNP,大白猪及其与长白猪的杂交猪等位基因G占优势,二花脸和湖北白猪则以等位基因A为主,均偏离Hardy-Weinberg平衡状态(P＜0.01);东北民猪和大二猪的等位基因G和A近乎相等,处于Hardy-Weinberg平衡状态."双肌臀”大白猪在5＇调控区和内含子1区这两个位点的A等位基因稍高于普通大白猪.5＇调控区和内含子1区SNPs所产生的等位基因表现出连锁遗传现象.     

Gelatinization temperature of rice explained by polymorphisms in starch synthase

The cooking quality of rice is associated with the starch gelatinization temperature (GT). Rice genotypes with low GT have probably been selected for their cooking quality by humans during domestication. We now report polymorphisms in starch synthase IIa (SSIIa) that explain the variation in rice starch GT. Sequence analysis of the eight exons of SSIIa identified significant polymorphism in only exon 8. These single nucleotide polymorphisms (SNPs) were determined in 70 diverse genotypes of rice. Two SNPs could classify all 70 genotypes into either high GT or low GT types which differed in GT by 8 °C. 'A' rather than 'G' at base 2412 determined whether a methionine or valine was present at the corresponding amino acid residue in SSIIa, whilst two adjacent SNPs at bases 2543 and 2544 coded for either leucine (GC) or phenylalanine (TT). Rice varieties with high GT starch had a combination of valine and leucine at these residues. In contrast, rice varieties with low GT starch had a combination of either methionine and leucine or valine and phenylalanine at these same residues. At least two distinct polymorphisms have apparently been selected for their desirable cooking qualities in the domestication of rice.     

Characterization of eight single nucleotide polymorphism markers in Aedes aegypti

Sequencing of part of seven genes from Aedes aegypti collected in 16 Brazilian cities revealed the existence of 53 single nucleotide polymorphisms (SNPs), representing one SNP every 52 base pairs. From these 53 SNPs, we selected eight that are independent and highly polymorphic. We describe the use of these markers for differentiation of Brazilian populations of A. aegypti. These are the first SNPs developed for delineating population structure in A. aegypti, and will be a useful complement to epidemiological studies.     

Haplotypes of single cancer driver genes and their local ancestry in a highly admixed long-lived population of Northeast Brazil

Admixed populations have not been examined in detail in cancer genetic studies. Here, we inferred the local ancestry of cancer-associated single nucleotide polymorphisms (SNPs) and haplotypes of a highly admixed Brazilian population. SNP array was used to genotype 73 unrelated individuals aged 80-102 years. Local ancestry inference was performed by merging genotyped regions with phase three data from the 1000 Genomes Project Consortium using RFmix. The average ancestry tract length was 9.12-81.71 megabases. Strong linkage disequilibrium was detected in 48 haplotypes containing 35 SNPs in 10 cancer driver genes. All together, 19 risk and eight protective alleles were identified in 23 out of 48 haplotypes. Homozygous individuals were mainly of European ancestry, whereas heterozygotes had at least one Native American and one African ancestry tract. Native-American ancestry for homozygous individuals with risk alleles for HNF1B, CDH1, and BRCA1 was inferred for the first time. Results indicated that analysis of SNP polymorphism in the present admixed population has a high potential to identify new ancestry-associated alleles and haplotypes that modify cancer susceptibility differentially in distinct human populations. Future case-control studies with populations with a complex history of admixture could help elucidate ancestry-associated biological differences in cancer incidence and therapeutic outcomes.     

Large-scale identification and characterization of genetic variants in asthma candidate genes

Asthma is a chronic inflammatory disorder of the airways, and a number of genetic loci are associated with the disease. Candidate gene association studies have been regarded as effective tools to study complex traits. Knowledge of the sequence variation and structure of the candidate genes is required for association studies. Thus, we investigated the genetic variants of 32 asthma candidate genes selected by colocalization of positional and functional candidate genes. We screened all exons and promoter regions of those genes using 12 healthy individuals and 12 asthma patients and identified a total of 418 single nucleotide polymorphisms (SNPs), including 270 known SNPs and 148 novel SNPs. Levels of nucleotide diversity varied from gene to gene (0.72×10⁻⁴–14.53×10⁻⁴), but the average nucleotide diversity between coding SNPs (cSNPs) and noncoding SNPs was roughly equivalent (4.63×10⁻⁴ vs 4.69×10⁻⁴). However, nucleotide diversity of cSNPs was strongly correlated to codon degeneracy. Nucleotide diversity was much higher at fourfold degenerate sites than at nondegenerate sites (9.42×10⁻⁴ vs 3.14×10⁻⁴). Gene-based haplotype analysis of asthma-associated genes in this study revealed that common haplotypes (frequency >5%) represented 90.5% of chromosomes, and they could be uniquely identified with five or fewer haplotype-tagging SNPs per gene. Therefore, our results may have important implications for the selection of asthma candidate genes and SNP markers for comprehensive association studies using large sample populations.     

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。