脑膜炎奈瑟菌基因分群方法建立与评价

目的建立流脑实验室快速诊断及基因分群方法,进行群别分布研究及分群效率评估。方法应用多重PCR进行同步核酸鉴定及基因分群。平板菌落计数法评估敏感性,基因序列分析确定特异性。结果PCR敏感性为83个菌落形成单位(CFU)/反应;多重PCR分群效率(100%)优于血清凝集法(82.02%),差异有统计学意义。其中带菌者分离株基因分群效率为100%,血清凝集法为65.71%;对与血清凝集分群法结果不同的菌株进行基因测序,分析结果与PCR分群结果相符;分离株菌群分布为A群60.69%,B群24.69%,C群14.62%,未检出其他群别。结论所建立的基因鉴定及分群方法敏感性和特异性均优于血清凝集法,分群效率优于血清凝集法。

mtDNA基因树拓扑距离比较和基因分群

基因树间拓扑距离数据的比较进一步证明 :与分割拓扑距离相比 ,通经拓扑距离是一种更为精确的测度 ,利用相对通经拓扑距离构建了 8个基因的拓扑距离树。基因的拓扑距离树能直观地反映不同基因树的拓扑结构差异大小 ,可用来对基因进行分群。此外 ,发现不同DNA序列用于构建多基因树中其系统发生信息存在“累加”、“合取”、“含盖”、“相斥”等数学关系。这可解释在mtDNA基因组中一些基因比另一些基因更适合用来构建树的结果。结果提示从GenBank中应选择具有累加基因的DNA序列或蛋白质氨基酸序列合并来构建物种。

金黄色葡萄球菌基因分群方法研究

目的建立快速、准确的金黄色葡萄球菌实验室诊断及基因分群方法并进行群别分布研究。方法应用多重PCR技术进行同步核酸鉴定及基因分群。应用平板菌落计数法评估敏感性、基因序列分析确定特异性。结果PCR敏感性为79 CFU/反应;多重PCR分群效率为100%,优于血清凝集法的79.78%(χ2=15.451 4,P<0.01)。其中健康带菌者分离株基因分群效率为100%,优于血清凝集法的65.71%(χ2=8.661 3,P<0.01)。对与血清凝集分群法结果不同的菌株进行基因测序,结果与PCR分群相符。结论多重PCR建立的基因鉴定及分群方法优于血清凝集法。

脑膜炎奈瑟菌检测及基因分群

目的建立多重聚合酶链反应(PCR)技术对脑膜炎奈瑟菌(Neisseria meningitidis,Nm)的快速诊断及分群方法。方法应用多重PCR技术对本实验室保存的70株已鉴定Nm进行核酸鉴定及基因分群,同时检测23例流行性脑脊髓膜炎(简称流脑)疑似病例脑脊液标本中脑膜炎奈瑟菌特异性DNA片段。用传统的细菌培养法和血清凝集法作对照,比较其特异性。结果 68株Nm的多重PCR检测结果与血清分群结果一致,2株血清未能分群的Nm经多重PCR检测为C群;23例流脑疑似病例脑脊液标本经多重PCR检测有5例为C群,1例为A群,1例为B群,阳性检出率为30.43%。传统的病原分离培养、生化鉴定和血清学鉴定出4例为C群,阳性检出率为17.39%。结论多重PCR技术对脑膜炎奈瑟菌检测的特异性优于传统的细菌培养法以及血清凝集法。

基于N基因的小反刍兽疫病毒贵州流行株分子特征与分群研究

为探讨小反刍兽疫病毒(peste des petits ruminants virus,PPRV)贵州流行株N基因分子特征和分群,试验设计了1对特异性引物,应用RT-PCR技术对小反刍兽疫(peste des petits ruminants,PPR)临床样本进行N基因扩增,克隆至pMD19-T载体,对阳性重组质粒进行测序,应用DANStar软件对测序序列和参考序列进行核苷酸同源性、氨基酸同源性、变异位点及系统进化树分析。结果显示:PPRV贵州流行株N基因扩增长度为1 578bp,其相互间核苷酸、氨基酸同源性分别为99.6%~100.0%及99.2%~100.0%,与国内参考株N基因的核苷酸序列(97.7%~99.9%)及氨基酸序列(98.3%~100.0%)同源性较国外参考株(88.5%~97.7%和92.2%~98.5%)高;PPRV贵州流行株N基因编码的氨基酸同疫苗株Nigeria 75-1相比存在26个位点突变,但没有氨基酸的缺失或增加;基于N基因系统进化分析显示,PPRV贵州流行株同国内参考株处于同一个进化分支,但与国外参考株处于不同进化分支;其属于病毒进化的Ⅳ基因群,与国内参考株处于同一系统分群,但与疫苗株Nigeria 75-1(Ⅰ基因群)处于不同基因群。

Zooplankton community analysis in the Changjiang River estuary by single-gene-targeted metagenomics

DNA barcoding provides accurate stages. Single-gene-targeted metagenomic analysis identification of zooplankton species through all life based on DNA barcode databases can facilitate long- term monitoring of zooplankton communities. With the help of the available zooplankton databases, the zooplankton community of the Changjiang （Yangtze） River estuary was studied using a single-gene-targeted metagenomic method to estimate the species richness of this community. A total of 856 mitocbondrial cytochrome oxidase subunit 1 （coxl） gene sequences were determined. The environmental barcodes were clustered into 70 molecular operational taxonomic units （MOTUs）. Forty-two MOTUs matched barcoded marine organisms with more than 90% similarity and were assigned to either the species （similarity〉96%） or genus level （similarity〈96%）. Sibling species could also be distinguished. Many species that were overlooked by morphological methods were identified by molecular methods, especially gelatinous zooplankton and merozooplankton that were likely sampled at different life history phases. Zooplankton community structures differed significantly among all of the samples. The MOTU spatial distributions were influenced by the ecological habits of the corresponding species. In conclusion, single-gene-targeted metagenomic analysis is a useful tool for zooplankton studies, with which specimens from all life history stages can be identified quickly and effectively with a comprehensive database.

Development of Three Multiplex PCR Primer Sets for Ark Shell(Scapharca broughtonii)and Their Validation in Parentage Assignment

Scapharca broughtonii is a commercially important and over-exploited species.In order to investigate its genetic diversity and population structure,43 novel polymorphic microsatellites were isolated and characterized.The number of alleles per locus ranged from 3 to 22 with an average of 6.93,and the observed and expected heterozygosities varied between 0.233 and 1.000,and 0.250 and 0.953,with an average of 0.614 and 0.707,respectively.Three highly informative multiplex PCRs were developed from nine of those microsatellites for S.broughtonii.We evaluated and validated these multiplex PCRs in 8 full-sib families.The average polymorphism information content(PIC) was 0.539.The frequency of null alleles was estimated as 3.13% of all the alleles segregation based on a within-family analysis of Mendelian segregation patterns.Parentage analysis of real offspring demonstrated that 100% of all offspring were unambiguously allocated to a pair of parents based on 3 multiplex sets.Those 43 microsatellite loci with high variability will be helpful for the analysis of population genetics and conservation of wild stock of S.broughtonii.The 3 sets of multiplex PCRs could be an important tool of pedigree reconstruction,population genetic analysis and brood stock management.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Torr. & Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction (PCR) assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045.

Metagenome of microorganisms associated with the toxic Cyanobacteria Microcystis aeruginosa analyzed using the 454 sequencing platform

In this study, the 454 pyrosequencing technology was used to analyze the DNA of the Microcystis aeruginosa symbiosis system from cyanobacterial algal blooms in Taihu Lake, China. We generated 183 228 reads with an average length of 248 bp. Running the 454 assembly algorithm over our sequences yielded 22 239 significant contigs. After excluding the M. aeruginosa sequences, we obtained 1 322 assembled contigs longer than 1 000 bp. Taxonomic analysis indicated that four kingdoms were represented in the community: Archaea (n = 9; 0.01%), Bacteria (n = 98 921; 99.6%), Eukaryota (n = 373; 3.7%), and Viruses (n = 18; 0.02%). The bacterial sequences were predominantly Alphaproteobacteria (n = 41 805; 83.3%), Betaproteobacteria (n = 5 254; 10.5%) and Gammaproteobacteria (n = 1 180; 2.4%). Gene annotations and assignment of COG (clusters of orthologous groups) functional categories indicate that a large number of the predicted genes are involved in metabolic, genetic, and environmental information processes. Our results demonstrate the extraordinary diversity of a microbial community in an ectosymbiotic system and further establish the tremendous utility of pyrosequencing.

多重PCR法用于脑膜炎奈瑟菌血清群分群鉴定的适用性

目的应用多重PCR法鉴定脑膜炎奈瑟菌(Neisseria meningitidis,Nm)A、B、C、W135、Y 5个血清群,验证在基因水平鉴定疫苗用菌种血清群的适用性,并与实验室经典细菌培养法和血清凝集试验进行比对。方法提取标准Nm血清群A、B、C、W135、Y的基因组DNA,针对5个血清群Nm荚膜多糖合成基因上的特异序列,应用多重PCR方法对目的片段进行扩增,根据PCR扩增产物的碱基片段大小不同区分这5个血清群;采用PCR方法鉴定7株Nm分离菌种的血清群;并与实验室传统细菌培养法和血清玻片凝集试验比对。结果多重PCR方法能够鉴定Nm,并同时完成血清分群鉴定。该方法特异性和灵敏度(最低检测限为基因组DNA 0.1 ng)均较高。7株Nm分离菌种PCR检测结果与实验室经典培养法和血清玻片凝集试验结果一致,其中1株为血清群A群,1株B群,2株C群,1株W135群,2株Y群。结论多重PCR方法对Nm检测的特异性和灵敏度均优于传统细菌培养法和血清玻片凝集试验,可同时检测Nm菌种的种特异基因和群特异基因,提高了分群鉴定的准确性。此外,试验结果客观,且可长期保存和追溯性查询,有利于疫苗用菌种的质量控制。

Identification of the novel recessive gene pi55(t) conferring resistance to Magnaporthe oryzae

The elite rice cultivar Yuejingsimiao 2 （YJ2） is characterized by a high level of grain quality and yield, and resistance against Magnaporthe oryzae. YJ2 showed 100% resistance to four fungal populations collected from Guangdong, Sichuan, Liaoning, and Heilongjiang Provinces, which is a higher frequency than that shown by the well-known resistance （R） gene donor culti- vats such as Sanhuangzhan 2 and 28zhan. Segregation analysis for resistance with F2 and F4 populations indicated the re- sistance of YJ2 was controlled by multiple genes that are dominant or recessive. The putative R genes of YJ2 were roughly tagged by SSR markers, located on chromosomes 2, 6, 8, and 12, in a hulked-segregant analysis using genome-wide selected SSR markers with F4 lines that segregated into 3 resistant （R）： 1 susceptible （S） or 1R：3S. The recessive R gene on chromosome 8 was further mapped to an interval ~1.9 cM/152 kb in length by linkage analysis with genomic position-ready markers in the mapping population derived from an F4 line that segregated into 1R：3S. Given that no major R gene was mapped to this inter- val, the novel R gene was designated as pi55（t）. Out of 26 candidate genes predicted in the region based on the reference genomic sequence of the cultivar Nipponbare, two genes that encode a leucine-rich repeat-containing protein and heavy-metalassociated domain-containing protein, respectively, were suggested as the most likely candidates for pi55（t）.

Elevational divergence in the great tit complex revealed by major hemoglobin genes

Gene flow and demographic history can play important roles in the adaptive genetic differentiation of species, which is rarely understood in the high-altitude adaptive evolution of birds. To elucidate genetic divergence of populations in the great tit complex （Parus major, P. minor and P. cinereus） at different elevations, we compared the genetic structure and gene flow in hemoglobin genes with neutral loci. Our results revealed the elevationally divergent structure of αA-globin gene, distinctive from that of the/βA-globin gene and neutral loci. We further investigated gene flow patterns among the populations in the central-northern （〉 1,000 m a.s.l.）, south-eastern （〈 1,000 m a.s.l.） regions and the Southwest Mountains （〉2,000 m a.s.l.） in China. The high-altitude （〉 1,000 m a.s.l.） diverged aA-globin genetic structure coincidαed with higher αA-globin gene flow between highland populations, in contrast to restricted neutral gene flow concordant with the phylogeny. The higher aA-globin gene flow suggests the possibility of adaptive evolution during population divergence, contrary to the lower aA-globin gene flow homogenized by neutral loci during population expantion. In concordance with patterns of historical gene flow, genotypic and allelic profiles provide distinctive patterns of fixation in different high-altitude populations. The fixation of alleles at contrasting elevations may primarily due to highland standing variants aA49Asn/72Asn/108Ala originating from the south-western population. Our findings demonstrate a pattern of genetic divergence with gene flow in major hemoglobin genes depending on population demographic history.

Functional genomic analysis of Hawaii marine metagenomes

Using high-throughput sequencing on metagenome to analyze marine microbial community, it is one of current main issues in the field of environmental microbe research. In this paper, we conducted the functional analysis on seven samples of metagenomic data from different depth seawater in Hawaii. The results of gene prediction and function annotation indicate that there are large amounts of potential novel genes of which functions remain unknown at present. Based on the gene annotation, codon usage bias is studied on ribosomal protein-related genes and shows an evident influence by the marine extreme environment. Furthermore, focusing on the marine environmental differences such as light intensity, dissolved oxygen, temperature and pressure among various depths, comparative analysis is carried out on related genes and metabolic pathways. Thus, the understanding as well as new insights into the correlation between marine environment and microbes are proposed at molecular level. Therefore, the studies herein afford a clue to reveal the special living strategies of microbial community from sea surface to deep sea.

Navigating the currents of seascape genomics： how spatial analyses can augment population genomic studies

Population genomic approaches are making rapid inroads in the study of non-model organisms, including marine taxa. To date, these marine studies have predominantly focused on rudimentary metrics describing the spatial and environmental context of their study region （e.g., geographical distance, average sea surface temperature, average salinity）. We contend that a more nuanced and considered approach to quantifying seascape dynamics and patterns can strengthen population genomic investigations and help identify spatial, temporal, and environmental factors associated with differing selective regimes or demographic histories. Nevertheless, approaches for quantifying marine landscapes are complicated. Characteristic features of the marine environment, including pelagic living in flowing water （experienced by most marine taxa at some point in their life cycle）, require a well-designed spatial-temporal sampling strategy and analysis. Many genetic summary statistics used to describe populations may be inappropriate for marine species with large population sizes, large species ranges, stochastic recruitment, and asymmetrical gene flow. Finally, statistical approaches for testing associations between seascapes and population genomic patterns are still maturing with no single approach able to capture all relevant considerations. None of these issues are completely unique to marine systems and therefore similar issues and solutions will be shared for many organisms regardless of habitat. Here, we outline goals and spatial approaches for land- scape genomics with an emphasis on marine systems and review the growing empirical literature on seascape genomics. We review established tools and approaches and highlight promising new strategies to overcome select issues including a strategy to spatially optimize sampling. Despite the many challenges, we argue that marine systems may be especially well suited for identifying candidate genomic regions under environmentally mediated selection and that seascape genomic approaches are especially useful for identifying robust locus-by-environment associations.

ASSOCIATION ANALYSIS OF POLYMORPHISMS IN SIX GENES WITHIN THE GH/IGF-1 AXIS IN PATIENTS WITH IDIOPATHIC SHORT STATURE IN THE CHINESE HAN POPULATION

Objective To investigate relationships of polymorphisms in six genes （ GHR, IGF-1, IGF-1R, IGFBP-3, JAK2, and STAT5b） in the growth hormone （GH）/insulin-like growth factor-1 （IGF-1） axis with idiopathic short stature （ISS） in the Chinese Han population. Methods A case- control study was carried out on a cohort of 198 ISS patients and 306 healthy controls. A total of 106 tagging single nucleotide polymorphisms （tagSNPs） from the six genes were selected from the HapMap （haplotype map of the human genome ） Han Chinese in the Beijing subset. Results of genotyping conducted by high- throughput lllumina GoldenGateTM Assay were analyzed by statistical software. Results Both individual tagSNPs and haplotypes showed an association with 1SS in the Han Chinese population （ P 〈 O. 05 ）. For each single test, both allele and genotype were tested. By allele frequency analysis, six positive SNP sites （ rsNo. 1, rsNo. 2, rsNo. 3, rsNo. 4, rsNo. 5, and rsNo. 6） of 3 genes （ JAK2, 1GF-1R, and GHR） were found having associations with ISS. By genotype frequency analysis, there were significant differences between the patient and control groups in the following SNP sites： 4 sites in JAK2 gene （ rsNo. 1, rsNo. 2, rsNo. 3, and rsNo. 4） and 1 site in GHR gene （ rsNo. 6）. The risk which affected ISS was found related to the JAK2 gene in 4 sites （ increase in rsNo. 1 and decrease in rsNo. 2, rsNo. 3, and rsNo. 4 ） and to the GHR gene in 1 site （decrease in rsNo. 6）. They were four haplotypes in gene of IGF-1R as ＂TGC＂, ＂CGCT＂, ＂TA＂, and ＂ CA＂, one haplotype in IGFBP-3 as ＂TA＂, and one haplotype in JAK2 as ＂CTG＂, which revealed high significance for risks of affecting ISS. At last, multivariate logistic regression analysis of specific site rsNo. 6 of the GHR gene revealed that the serum IGF-1 was related to genotypes AA and AC, with genotype CC as the reference （ P =0. 015）. Conclusion Genetic variances in six genes within the GH/IGF-1 axis may be important etiological factors for ISS in the Chinese Han population.