条斑紫菜EST-SSR引物种间转移扩增真实性研究

当条斑紫菜EST-SSR引物在坛紫菜几个品系上进行种间扩增时,微卫星引物的扩增结果中也出现了多带形式。为了研究的假阳性问题,扩增产物被从胶上回收克隆测序。在回收的坛紫菜扩增产物中,同阳性对照条斑紫菜扩增产物大小相同的产物中有微卫星序列重复,但在大分子量的条带中却没有,显现出典型的假阳性问题。在坛紫菜扩增中产生的假阳性带不适合进行建立在微卫星突变基础上的遗传研究,然而,在确定相关的遗传性状后,这些引物也可以用来进行品系鉴定等遗传研究。     

植物微卫星分子标记中无效等位基因检测方法的比较与应用

微卫星分子标记因其开发便捷、突变率高、成本较低等优势一直被广泛应用于群体遗传学、保护生物学和分子生态学研究中。近年来二代测序技术、多重PCR方法以及毛细管电泳等新技术的发展和完善,极大地提高了微卫星分子标记的开发和使用效率并降低了使用成本。但是在开展微卫星实验过程中普遍存在的无效等位基因(或称为哑等位基因,null alleles）会对研究结果造成偏差,是微卫星分子标记应用中的最大缺陷之一。然而,长期以来无效等位基因的检测问题并未受到研究者的足够重视。本文通过对国内外相关文献查阅,在对无效等位基因有一个较为深入和全面认识的基础上,对目前无效等位基因的主要检测方法进行全面的介绍和深入的比较。最后,结合研究实例总结出植物微卫星分子标记研究中无效等位基因检测的有效办法。     

Maintenance of genetic diversity in cyclic populations-a longitudinal analysis in Myodes glareolus

Conspicuous cyclic changes in population density characterize many populations of small northern rodents. The extreme crashes in individual number are expected to reduce the amount of genetic variation within a population during the crash phases of the population cycle. By long-term monitoring of a bank vole (Myodes glareolus) population, we show that despite the substantial and repetitive crashes in the population size, high heterozygosity is maintained throughout the population cycle. The striking population density fluctuation in fact only slightly reduced the allelic richness of the population during the crash phases. Effective population sizes of vole populations remained also relatively high even during the crash phases. We further evaluated potential mechanisms contributing to the genetic diversity of the population and found that the peak phases are characterized by both a change in spatial pattern of individuals and a rapid accession of new alleles probably due to migration. We propose that these events act together in maintaining the high genetic diversity within cyclical populations.     

A comparison of microsatellite polymorphism and heterozygosity among field and laboratory populations of Schistosoma mansoni

The genetic diversity of a field population (recently collected in Melquiades, Brazil) and two laboratory strains (LE and NMRI) of a human blood fluke, Schistosoma mansoni, were analysed using microsatellite markers. Data from the three groups showed an extreme and consistent discrepancy in the level of polymorphism at all microsatellite loci between the field population and laboratory populations. The numbers of alleles detected in LE and NMRI populations averaged only 14 and 10% of those found in the field population, respectively. Especially apparent was the abundance of rare alleles in the Melquiades population when compared with the laboratory strains. The reduction in allelic diversity in the laboratory strains is most likely due to the founder effect and potential bottlenecks that may have occurred during decades of laboratory maintenance. Surprisingly, a much less drastic difference was found when comparing the average heterozygosity of the field population with the laboratory strains. This apparent anomaly may be explained by observed population substructuring (and a potential resultant Wahlund effect) in the natural population. Our comparison of genetic diversity between laboratory and field populations of S. mansoni emphasizes the need for studies of representative populations in schistosome vaccine development.     

TECHNICAL ADVANCES: Effects of genotyping protocols on success and errors in identifying individual river otters (Lontra canadensis) from their faeces

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method.     

Effect of the slow (K) or rapid (k+) feathering gene on body and feather growth and fatness according to ambient temperature in a Leghorn × brown egg type cross

Chicks of both sexes issued from the cross of heterozygous K/k⁺ cocks for the slow-feathering sex linked K allele with k⁺ (rapid feathering) hens, were compared from the age of 4 to 10 weeks at two ambient temperatures. In individual cages, 30 male chicks of each genotype (K/k⁺ and k⁺/k⁺) were raised at 21°C, and 60 others, distributed in the same way, were raised at 31°C. 71 K/W females and 69 k⁺/W females were raised in a floor pen at 31°C till 10 weeks of age. In the males, the body weight, feed consumption and feed efficiency at different ages were influenced only by temperature (lower growth rate and feed intake at 31°C); no significant effects of the genotype at locus K nor genotype × temperature interaction were observed. In females, all at 31°C, the genotype (K/W or k⁺/W) had no significant effect on growth rate. Plumage weight and weight of abdominal fat (absolute or related to body weight) were measured on half of the males of each group in individual cages, at 10 weeks of age. Moreover, on 36 males and 48 females of the two genotypes, in a group battery at 31°C, the absolute and relative weight of plumage were measured on a sample every two weeks between 4 and 10 weeks. In the first case, no significant effect of genotype appeared. In the second case, an interaction between age and genotype was suggested from plumage weight: its growth, especially in male chicks, appears to be temporarily and unexpectedly faster from 4 to 6 weeks of age for the K/k⁺ and K/W genotypes.     

Low genotyping error rates in non-invasively collected samples from roe deer of the Bavarian Forest National Park

Genetic wildlife monitoring is increasingly carried out on the basis of non-invasively collected samples, whereby the most commonly used DNA sources are skin appendages (hairs, feathers) and faeces. In order to guide decisions regarding future adequate ways to monitor the roe deer (Capreolus capreolus) population of the Bavarian Forest National Park in Germany, we tested these two different types of DNA source materials to compare their suitability for genetic monitoring. We determined the haplotypes (d-loop) of 19 roe deer and genotyped each individual (tissue, hairs, faeces) across 12 microsatellite loci. The amount of missing and erroneous microsatellite alleles obtained from hair and faeces samples, respectively, was estimated based on comparisons with the corresponding tissue sample control. We observed no missing alleles in hair samples, but in fecal samples PCR failed in 30 out of 228 instances (19 individuals x 12 loci), corresponding to a frequency of missing alleles of 13.2% across all loci and individuals. In genotypes generated from hairs erroneous alleles were detected in 2 out of 228 instances (0.9%), while genotypes retrieved from fecal samples displayed erroneous alleles in 6 out of 198 remaining instances (3%). We conclude that both hair and fecal samples are generally well suited for genetic roe deer monitoring, but that fecal sample based analyses require a larger sample size to account for higher PCR failure rates.     

The Benjamini-Hochberg method with infinitely many contrasts in linear models

Benjamini and Hochberg's method for controlling the false discoveryrate is applied to the problem of testing infinitely many contrastsin linear models. Exact, easily calculated critical values arederived, defining a new multiple comparisons method for testingcontrasts in linear models. The method is adaptive, dependingon the data through the F-statistic, like the Waller–DuncanBayesian multiple comparisons method. Comparisons with Scheffé'smethod are given, and the method is extended to the simultaneousconfidence intervals of Benjamini and Yekutieli.     

Non-Mendelian transmission of alleles at microsatellite loci: an example in Ixodes ricinus, the vector of Lyme disease

Microsatellite loci are usually considered to be neutral co-dominant and Mendelian markers. We undertook to study the inheritance of five microsatellite loci in the European Lyme disease vector, the tick Ixodes ricinus. Only two loci appeared fully Mendelian while the three others displayed non-Mendelian patterns that highly frequent null alleles could not fully explain. At one locus, IR27, some phenomenon seems to hinder the PCR amplification of one allele, depending on its origin (maternal imprinting) and/or its size (short allele dominance). DNA methylation, which appeared to be a possible explanation of this amplification bias, was rejected by a specific test comparing the amplification efficiency that did not differ between unmethylated and experimentally methylated DNA. The role of allele size in heterozygous individuals was then revealed from the data available on field collected ticks and consistent with the results of a theoretical approach. These observations highlight the need for prudence while inferring reproductive systems (selfing rates), parentage or even allelic frequencies from microsatellite markers, in particular for parasitic organisms for which molecular approaches often represent the only way for population biology inferences.     

Calibration plot for proteomics: A graphical tool to visually check the assumptions underlying FDR control in quantitative experiments

In MS‐based quantitative proteomics, the FDR control (i.e. the limitation of the number of proteins that are wrongly claimed as differentially abundant between several conditions) is a major postanalysis step. It is classically achieved thanks to a specific statistical procedure that computes the adjusted p‐values of the putative differentially abundant proteins. Unfortunately, such adjustment is conservative only if the p‐values are well‐calibrated; the false discovery control being spuriously underestimated otherwise. However, well‐calibration is a property that can be violated in some practical cases. To overcome this limitation, we propose a graphical method to straightforwardly and visually assess the p‐value well‐calibration, as well as the R codes to embed it in any pipeline. All MS data have been deposited in the ProteomeXchange with identifier PXD002370 ( http://proteomecentral.proteomexchange.org/dataset/PXD002370 ).     

Proteome-wide prediction of PKA phosphorylation sites in eukaryotic kingdom

Protein phosphorylation is one of the most essential post-translational modifications (PTMs), and orchestrates a variety of cellular functions and processes. Besides experimental studies, numerous computational predictors implemented in various algorithms have been developed for phosphorylation sites prediction. However, large-scale predictions of kinase-specific phosphorylation sites have not been successfully pursued and remained to be a great challenge. In this work, we raised a “kiss farewell” model and conducted a high-throughput prediction of cAMP-dependent kinase (PKA) phosphorylation sites. Since a protein kinase (PK) should at least “kiss” its substrates and then run away, we proposed a PKA-binding protein to be a potential PKA substrate if at least one PKA site was predicted. To improve the prediction specificity, we reduced false positive rate (FPR) less than 1% when the cut-off value was set as 4. Successfully, we predicted 1387, 630, 568 and 912 potential PKA sites from 410, 217, 173 and 260 PKA-interacting proteins in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens, respectively. Most of these potential phosphorylation sites remained to be experimentally verified. In addition, we detected two sites in one of PKA regulatory subunits to be conserved in eukaryotes as potentially ancient regulatory signals. Our prediction results provide an excellent resource for delineating PKA-mediated signaling pathways and their system integration underlying cellular dynamics and plasticity.     

种群微卫星DNA分析中样本量对各种遗传多样性度量指标的影响

我们以中国飞蝗种群的微卫星遗传分析数据为例 ,评估了取样对种群遗传多样性指标的影响 ,结果显示 :样本大小与所观测到的每位点等位基因数、平均等位基因数及基因丰富度指数均呈显著正相关 ,而与期望杂合度无显著相关 ;微卫星位点多态性的高低直接影响所观测到的种群基因丰富度及其检测所需的样本量 ;对大多数种群遗传和分子生态学研究而言 ,30 - 5 0个个体是微卫星DNA分析所需要的最小样本量。基因丰富度经过稀疏法或多次随机抽样法校正后 ,可适用于瓶颈效应等种群历史数量变动的检测。另外 ,在研究中 ,还应避免采集时间的不同及样本的性比构成所可能造成的对种群遗传结构的影响