基于RAD重测序技术开发烟草品种SNP位点

利用限制性内切酶位点标签（RAD）技术,通过对10份供试烟草材料的基因组简化重测序,发掘了烟草高通量SNP位点,为烟草基因组学提供标记信息。结果表明,本研究共获得了44.33 Gb的Clean data数据,平均覆盖度1.01 X,共鉴定到291 770个SNP位点,SNP位点间的平均间距为10.066±29.801 kb。发掘到的SNP位点能够覆盖整个基因组,但在不同染色体部位上的分布密度存在一定差异,在17号染色上半臂的存在一段大范围的SNP密集区域。SNP变异类型以转换为主,通过功能注释在基因区域发现45 049处SNP位点。利用SNP分型信息,计算了供试品种间的遗传距离,平均为0.29,台烟8号的遗传背景与其他品种相对最远。该结果将为烟草QTL定位、候选基因发掘、亲本组配等研究提供科研依据。     

A procedure for olive oil traceability and authenticity: DNA extraction,multiplex PCR and LDR–universal array analysis

Traceability of olive oils is relevant not only in assessing their origin, but also in protecting against frauds. Here, we present an improvement of the assay previously developed for the genotyping of forty-nine frequently grown Mediterranean olive cultivars by ligation detection reaction (LDR)/universal array (UA), refining the entire procedure in order to address DNA extracted from monovarietal olive oils. Firstly, a simple and robust protocol to extract amplifiable DNA from olive oil was developed. Then, the SNP-containing DNA sequences were simultaneous amplified by multiplex PCR and used on a LDR-UA platform, which gave precise and accurate genotype results. Thirteen out of the seventeen investigated SNPs were amplifiable in multiplex PCR, and were sufficient to unequivocally discriminate the forty-nine cultivars. The availability of this semi-automated SNP genotyping assay should help food testing laboratories to verify the origin and authenticity of monovarietal extra-virgin olive oils. C. Consolandi and L. Palmieri equally contributed to this research paper.     

Genetic control of conventional labeling through the bovine meat production chain by single nucleotide polymorphisms using real-time PCR

Since January 2002, the European Union has adopted precise guidelines aimed at protecting the safety of meat and controlling the production chain. To this purpose, the conventional traceability of livestock and meat represents the main tool, but verification of traceability requires genetic support. At present, single nucleotide polymorphisms (SNPs) represent the most innovative molecular markers in genotyping studies. The aim of this study was to verify correct labeling in a bovine meat production chain by a real-time PCR protocol based on SNP analysis. Reference hair samples from 5,000 animals were randomly collected from 22 farms. Twelve hundred meat samples were collected at different steps of the bovine meat production chain. In particular, 1,000 meat samples were collected at the slaughterhouse and 200 samples from the same animals directly at the butcher's shop. The protocol was optimized and validated by testing a set of 16 SNP markers on 95 DNA samples from bovine sires of different breeds. Thereafter, the genotyping of 2,200 samples was conducted with a set of 12 selected SNPs to verify traceability of the meat production chain at three different stages: farm, slaughterhouse, and butcher's shop. Irregularities in conventional traceability were evidenced directly in 1.87% of the samples at the slaughterhouse. This percentage increased to 3.25% when sampling was conducted at the butcher's shop. This study demonstrates that despite the precautions adopted over the meat production chain, some critical points still exist that cause the loss of a correct association between registration numbers and samples.     

Development of an automation system for single nucleotide polymorphisms genotyping using bio-strand, a new three-dimensional microarray

Previously, we developed a novel three-dimensional microarray system called Bio-Strand, which may be used in various applications including single nucleotide polymorphisms genotyping. In Bio-Strand, samples for detection are immobilized on a one-dimensional thread, which is wound around a cylinder-shaped core to form a three-dimensional thread-and-core structure. The thread-and-core structure is then inserted into a plastic pipette tip, where hybridization and detection are performed. In this study, we have developed an automation system, NIAGALA Bio-Station SDx, which enables automated hybridization and detection during the genotyping procedure using Bio-Strand. Using this system, we have performed the single nucleotide polymorphism (SNP) genotyping of CYP2C, one of the important human cytochrome P450 genes and the results were completely consistent with the genotyping results determined by the TaqMan method.     

Short communication: predominance of beta-casein (CSN2) C allele in goat breeds reared in Italy

A protocol for the rapid and simultaneous genotyping of A, C, and 0 'CSN2 alleles in goat was developed by single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) technique. Screening the CSN2 variability in 7 goat breeds reared in Italy validated the genotyping test. The SSCP-PCR technique was also suitable for monitoring CSN2 polymorphism. In particular, the discrimination between CSN2*A and CSN2*C is important because the 2 corresponding protein variants cannot be separated by standard typing techniques. The monitoring of CSN2 variability in the goat breeds indicates the predominance of the C allele. In most breeds, CSN2*C occurred with the highest frequency, except in Saanen where CSN2*A and CSN2*C showed similar frequencies. Variant CSN2*C occurred with a frequency of 0.68 (Camosciata), 0.70 (Jonica), 0.71 (Garganica), 0.82 (Maltese), 0.87 (Cilentana), and 0.97 (Orobica). The alignment among the mature CSN2 sequences of different species suggests that CSN2*A is the ancestral allele compared with CSN2*C. Interestingly, the CSN2*A goat variant showed higher frequencies in selected breeds (Saanen and Camosciata).     

Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of 'classic' (sea to see) and 'new' (seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin (se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea-see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.     

Genetic evaluation of dairy cattle using a simple heritable genetic ground

The evaluation of an animal is based on production records, adjusted for environmental effects, which gives a reliable estimation of its breeding value. Highly reliable daughter yield deviations are used as inputs for genetic marker evaluation. Genetic variability is explained by particular loci and background polygenes, both of which are described by the genomic breeding value selection index. Automated genotyping enables the determination of many single‐nucleotide polymorphisms (SNPs) and can increase the reliability of evaluation of young animals (from 0.30 if only the pedigree value is used to 0.60 when the genomic breeding value is applied). However, the introduction of SNPs requires a mixed model with a large number of regressors, in turn requiring new algorithms for the best linear unbiased prediction and BayesB. Here, we discuss a method that uses a genomic relationship matrix to estimate the genomic breeding value of animals directly, without regressors. A one‐step procedure evaluates both genotyped and ungenotyped animals at the same time, and produces one common ranking of all animals in a whole population. An augmented pedigree–genomic relationship matrix and the removal of prerequisites produce more accurate evaluations of all connected animals. Copyright © 2010 Society of Chemical Industry     

Association study of single nucleotide polymorphisms in IL-10 and IL-17 genes with the severity of microbial keratitis

PurposeExploratory analysis to assess the association of single nucleotide polymorphisms (SNPs) in the interleukin (IL) 10 and IL-17 genes with severity of contact lens keratitis.MethodsThis was a retrospective case control study of 88 contact lens keratitis cases (25 severe) and 185 healthy contact lens wearers recruited from studies conducted at Moorfields Eye Hospital and in Australia-wide during 2003–2005. Buccal swab samples were collected on Whatman FTA cards and mailed by post for DNA extraction and SNP genotyping. IL-10 (rs1800871; rs1800896; rs1800872) and IL-17 (rs1800871; rs1800896; rs1800872) SNPs were screened by pyrosequencing. Genetic association analyses were performed via Cochran-Armitage trend tests and logistic regression models using PLINK software.ResultsNone of the SNPs tested showed evidence of association with severity of contact lens keratitis at P < 0.05. Nevertheless, minor allele G in SNP rs2397084 of the IL-17F gene was associated with increased risk of severe MK, with OR=2.1 (95% CI=0.9-4.8, P = 0.066).ConclusionOur study cannot exclude with confidence that genetic variation in the IL-17 F proinflammatory cytokine is associated with more severe outcomes of MK. However, there is general body of information that the IL-17 pathway is important in the mechanisms of MK. Studies with larger power and the expanded array of laboratory tools will elucidate the exact role of IL-17 in MK.     

基于SLAF-seq技术的烟草抗CMV主效QTL定位

烟草花叶病（CMV）是烟草主要病害之一,筛选与CMV抗病QTL紧密连锁的分子标记是烟草抗病分子育种的重要部分。本研究以抗病材料台烟8号和感病材料NC82为亲本,获得BC1群体,构建抗、感池。采用BSA和SLAF-seq技术开发CMV抗病相关SNP分子标记,共获得180 370个SLAF标签,经分析后获得6156个多态性SNP标记。利用SNP-index分析方法,发现两个与CMV抗病高度关联的区域。利用这些分子标记成功定位到了两个CMV抗病相关QTL,其中一个QTL定位于Chr01上的55.72~60.44 Mb区间内,区间大小为4.72 Mb;另一个则定位于烟草Chr18上的44.21~48.70 Mb区间内,区间大小为4.49 Mb。在关联到的QTL区间内设计分子标记可以用于烟草抗CMV育种的辅助选择。     

High-resolution melting analysis: a promising molecular method for meat traceability

To find a promising molecular method for meat traceability, three methods of single nucleotide polymorphism (SNP) detection: RFLP-PCR analysis, high-resolution melting (HRM) analysis, and TaqMan probe analysis, have been compared in terms of accuracy, ease of use, throughput capability, and cost. We genotyped ten pork DNA samples across three SNPs. The results showed that the HRM genotyping method was the most accurate and easiest to use with the lowest cost, while TaqMan probe analysis provided similar results, but its cost was much higher.     

Characterisation of chicken Campylobacter jejuni isolates using resolution optimised single nucleotide polymorphisms and binary gene markers

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.     

烤烟烟碱含量的全基因组关联分析

烟碱是烟草属特有的一类生物碱,为加深对烟碱遗传调控机制的解析,充分挖掘高烟碱优异等位。本研究选取219份烤烟品种开展了RAD重测序研究,获得了384,904个高质量SNP位点的群体分型数据。同时在四川西昌和山东诸城2个试验点,于2012-2015年相同栽培管理条件下对供试品种的烟叶烟碱含量进行了表型鉴定。利用全基因组关联分析的策略,共发掘到2个与烟碱含量显著关联的SNP位点,分别位于1号染色体的69,912,063bp处和2号染色体的81,115,794bp处。基于上述SNP位点预测到2个候选基因,并将目标SNP位点转化3对可通过PCR检测的功能标记。通过以上研究,加深了烟碱遗传调控机制的解析,为高烟碱优异等位基因型的发掘和高烟碱新品种的分子标记辅助选择提供了可用的标记和基因资源。      

Technical note: detection of the C allele of beta-casein (CSN2) in Czech Dairy goat breeds using LightCycler analysis

A protocol was developed for rapid genotyping of A and C variants at the CSN2 locus in goat species (White Shorthaired and Brown Shorthaired goat) by PCR and LightCycler analysis. The LightCycler technique combines rapid and efficient in vitro amplification of DNA in glass capillaries, with melting curve analysis based on fluorescence resonance energy transfer, for the sensitive detection of point mutation. Analysis of the CSN2 variability in the 2 goat breeds reared in the Czech Republic validated the genotyping test. Monitoring of CSN2 variability in the goat breeds indicates the predominance of the C allele. In both breeds, CSN2*A and CSN2*C showed almost similar frequencies. Variant CSN2*C occurred with a frequency of 0.699 in White Shorthaired goats and 0.570 in Brown Short-haired goats.     

Short communication: characterization of a kappa-casein genetic variant in the Chinese yak, Bos grunniens

A PCR-single strand conformation polymorphism protocol has been developed for rapid genotyping of the yak κ-casein gene. A total of 307 yaks from the Tianzhu White, Jiulong, Maiwa, and Datong breeds in China were genotyped at the κ-casein locus using the protocol developed in the present study. A polymorphism of κ-casein gene exon 4 has been identified in Tianzhu White breed by evaluating genomic DNA. The polymorphic site consists of a single nucleotide substitution G→C at position 362 of the exon 4, resulting in an AA substitution from Arg to Pro at position 121 of the AA sequence and in 2 alleles named, respectively, G and C based on nucleotide 362. The occurrence of allele C in the Tianzhu White breed was high with an allele frequency of 0.15. However, allele C appears to be absent in the yaks from Jiulong, Maiwa, and Datong breeds.     

Short communication: characterization of a new genetic variant in the caprine kappa-casein gene

A new polymorphism has been identified in the goat kappa-casein gene by evaluating genomic DNA from the Montefalcone breed in Italy. The polymorphic site consists of a single nucleotide substitution A to G at position 242 of the exon 4 and produces an amino acid substitution Asp/Gly. A polymerase chain reaction-restriction fragment length polymorphism protocol for rapid genotyping of the variant has been developed, using the HaeIII enzyme. Animals from Italian, Spanish, and French breeds have been analyzed to investigate the occurrence of the allele in other populations. The allele appears to be exclusive to the Montefalcone breed.     

利用GBS技术开发烟草SNP标记及遗传多样性分析

研究烟草种质资源的遗传背景,为烟草种质资源的高效利用提供依据。采用GBS （genotyping-by-sequencing）技术,挖掘92份烟草种质资源的SNP位点,并进行遗传多样性和遗传结构分析。结果表明,测序共获得了147.165 Gb数据,平均每个样本1.599 Gb,筛选后共获得93 685个高质量的SNP位点;不同地理来源烟草群体等位基因数（Na）为1.27~1.93,观测杂合度（Ho）变化范围为0.22~0.76,期望杂合度（He）为0.32~0.59,多态性位点数（PLN）范围为15 877~44 249,多态性位点比率（PPL）为26.61%~74.17%,遗传多样性指数（H）为0.19~0.52,遗传距离（GD）为-0.0148~0.3849。基于遗传距离的NJ聚类将材料划分为两个类群;群体遗传结构分析表明,本研究所用材料基于模型可划分为4个亚群。通过GBS技术得到的测序数据量较大且质量较高,群体多态性较高,整体遗传多样性偏低,群体结构划分与种质来源不完全相关。     

Effects of variation in porcine MYOD1 gene on muscle fiber characteristics, lean meat production, and meat quality traits

Three single nucleotide polymorphisms (SNPs) in the porcine MYOD1 gene were used for association analysis and haplotype construction to evaluate the effects of their substitution. Four hundred and three pigs of Yorkshire and Berkshire breeds were used. The mRNA expression levels of MYOD1 were examined. The g.489C>T and g.1264C>A SNPs were significantly associated with several muscle fiber characteristics, the loin eye area, and lightness. Particularly, animals having hetero-genotypes of both sites showed good performance both in lean meat production and meat quality traits. The results of haplotype substitution were similar to the associations of individual SNPs. Moreover, the 2 SNPs had significant effects on mRNA expression. Therefore, the g.489C>T and g.1264C>A SNPs in MYOD1 may be meaningful DNA markers that can be used for improving important porcine economic traits.     

Genetic polymorphism of the caprine kappa casein gene

Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction-restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.     

Short communication: Simultaneous identification of five kappa-casein (CSN3) alleles in domestic goat by polymerase chain reaction-single strand conformation polymorphism

Until now, a total of nine polymorphic sites corresponding to six different alleles have been described at the κ-casein (CSN3) locus in the domestic goat (Capra hircus). A protocol for the rapid and simultaneous genotyping of five goat CSN3 alleles by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was developed. Moreover, the developed test was validated by screening the CSN3 variability in four Italian breeds, Garganica, Jonica, Maltese, and Camosciata. Seven different patterns were readily identifiable. These corresponded to five known alleles and two newly identified variants. The G/A substitution at nucleotide position 471, which is not identifiable at the protein level but was found to be very frequent in the typed breeds, is easily detectable by the protocol developed. The PCR-SSCP analysis is a powerful tool for the genetic study of CSN3 variability in domestic goats, allowing both the simultaneous identification of different alleles, and the detection of new variants.