苏云金杆菌Vip蛋白经历正向达尔文选择的证据

营养期杀虫蛋白(Vip)是苏云金杆菌在营养期所产生的一类新型杀虫蛋白,代表了第二代转基因杀虫蛋白,它能在一定程度上克服许多害虫对δ-内毒素低敏感或者不敏感的缺陷。但是,目前和已经深入研究的δ-内毒素相比较,有关Vip蛋白结构和功能关系方面的报道还甚少。本文采用最大似然方法和基于最大简约的滑窗分析对Vip蛋白的分子进化机制进行了评价。结果发现Vip蛋白在进化过程当中经历了正选择,并采用贝叶斯方法确定了16个正选择氨基酸残基。有意思的是所有这些正选择残基都位于Vip蛋白C端从705到809的区域。当把这些正选择残基定位到二级结构和三级结构时,发现绝大部分正选择残基都暴露在Vip蛋白空间结构的表面并且聚集在环的区域。推测Vip蛋白分子进化的机制应该是受到了正选择压力而不是功能约束的松弛。导致Vip蛋白C端多样性的潜在正选择压力可能是Vip蛋白为了在和目标昆虫之间竞争取得优势,或者是为了扩大Vip蛋白的杀虫范围。文中确定的经历了正选择残基很有可能是和昆虫宿主范围有关,因此可以为今后研究Vip蛋白的结构和功能提供相应的靶点。     

Evidence for Positive Selection on a Sexual Reproduction Gene in the Diatom Genus Thalassiosira (Bacillariophyta)

Single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), and several random effects likelihood (REL) methods were utilized to identify positively and negatively selected sites in sexually induced gene 1 (Sig1) of four different Thalassiosira species. The SLAC analysis did not find any sites affected by positive selection but suggested 13 sites influenced by negative selection. The SLAC approach may be too conservative because of low sequence divergence. The FEL and REL analyses revealed over 60 negatively selected sites and two positively selected sites that were unique to each method. The REL method may not be able to reliably identify individual sites under selection when applied to short sequences with low divergence. Instead, we proposed a new alignment-wide test for adaptive evolution based on codon models with variation in synonymous and nonsynonymous substitution rates among sites and found evidence for diversifying evolution without relying on site-by-site testing. The performance of the FEL and REL approaches was evaluated by subjecting the tests to a type I error rate simulation analysis, using the specific characteristics of the Sig1 data set. Simulation results indicated that the FEL test had reasonable Type I errors, while REL might have been too liberal, suggesting that the two positively selected sites identified by FEL (codons 94 and 174) are not likely to be false positives. The evolution of these codon sites, one of which is located in functional domain II, appears to be associated with divergence among the three major Thalassiosira lineages. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Rice Pangenome Genotyping Array: an efficient genotyping solution for pangenome-based accelerated genetic improvement in rice

The advent of the pangenome era has unraveled previously unknown genetic variation existing within diverse crop plants, including rice. This untapped genetic variation is believed to account for a major portion of phenotypic variation existing in crop plants. However, the use of conventional single reference-guided genotyping often fails to capture a large portion of this genetic variation leading to a reference bias. This makes it difficult to identify and utilize novel population/cultivar-specific genes for crop improvement. Thus, we developed a Rice Pangenome Genotyping Array (RPGA) harboring probes assaying 80K single-nucleotide polymorphisms (SNPs) and presence–absence variants spanning the entire 3K rice pangenome. This array provides a simple, user-friendly and cost-effective (60–80 USD per sample) solution for rapid pangenome-based genotyping in rice. The genome-wide association study (GWAS) conducted using RPGA-SNP genotyping data of a rice diversity panel detected a total of 42 loci, including previously known as well as novel genomic loci regulating grain size/weight traits in rice. Eight of these identified trait-associated loci (dispensable loci) could not be detected with conventional single reference genome-based GWAS. A WD repeat-containing PROTEIN 12 gene underlying one of such dispensable locus on chromosome 7 (qLWR7) along with other non-dispensable loci were subsequently detected using high-resolution quantitative trait loci mapping confirming authenticity of RPGA-led GWAS. This demonstrates the potential of RPGA-based genotyping to overcome reference bias. The application of RPGA-based genotyping for population structure analysis, hybridity testing, ultra-high-density genetic map construction and chromosome-level genome assembly, and marker-assisted selection was also demonstrated. A web application ( http://www.rpgaweb.com ) was further developed to provide an easy to use platform for the imputation of RPGA-based genotyping data using 3K rice reference panel and subsequent GWAS.     

Positive Selection and Horizontal Gene Transfer in the Genome of a Male-Killing Wolbachia

Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome with other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.     

Population-Specific Selection on Standing Variation Generated by Lateral Gene Transfers in a Grass

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Determination of the Selection Capacity of Antibiotics for Gene Selection

Choosing a potent selection antibiotic (SA), is a crucial success factor when creating stably transfected cell lines using an antibiotic selection marker. The selection capacity of this antibiotic is defined as its ability to kill sensitive, untransfected parental cells, while leaving resistant, transfected cells unharmed. Currently, no procedure has been described to determine this selection capacity. Therefore, a protocol to obtain a numerical value, called the “selectivity factor” (SF), that defines the selection capacity of SAs is developed. The SF is determined by using a modified MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐diphenyltetrazolium bromide) assay for both sensitive and resistant cells, and applies to commonly used cell lines. To prove the concept, the SF of the SA G418 and hygromycin B (HmB) on several cell lines is determined. The SF of G418 on BHK‐21 cells is very high, indicating that G418 is an ideal SA for transfected BHK‐21 cells. For HeLa cells, the SF of G418 is very low suggesting G418 is not an optimal SA for selecting transfected HeLa cells. For these cells, HmB would be a better choice. These conclusions are confirmed by an independent cell death assay. The SF identifies the most optimal SA for a certain cell line, reduces the risk of selecting spontaneously resistant cell clones, and streamlines the process of generating stable cell lines. Most importantly, the method is especially time saving when obtaining stable cell lines expressing toxic genes, and reduces culture times for generating large numbers of cell lines from the same parental cell line.     

A Genome-Wide Scan for Evidence of Selection in a Maize Population Under Long-Term Artificial Selection for Ear Number

A genome-wide scan to detect evidence of selection was conducted in the Golden Glow maize long-term selection population. The population had been subjected to selection for increased number of ears per plant for 30 generations, with an empirically estimated effective population size ranging from 384 to 667 individuals and an increase of more than threefold in the number of ears per plant. Allele frequencies at >1.2 million single-nucleotide polymorphism loci were estimated from pooled whole-genome resequencing data, and F_ST values across sliding windows were employed to assess divergence between the population preselection and the population postselection. Twenty-eight highly divergent regions were identified, with half of these regions providing gene-level resolution on potentially selected variants. Approximately 93% of the divergent regions do not demonstrate a significant decrease in heterozygosity, which suggests that they are not approaching fixation. Also, most regions display a pattern consistent with a soft-sweep model as opposed to a hard-sweep model, suggesting that selection mostly operated on standing genetic variation. For at least 25% of the regions, results suggest that selection operated on variants located outside of currently annotated coding regions. These results provide insights into the underlying genetic effects of long-term artificial selection and identification of putative genetic elements underlying number of ears per plant in maize.     

Extreme genome diversity and cryptic speciation in a harmful algal-bloom-forming eukaryote

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Evidence that Natural Selection is the Primary Cause of the Guanine-cytosine Content Variation in Rice Genes

Cereal genes are classified into two distinct classes according to the guanine-cytosine(GC)content at the third codonsites(GC_3).Natural selection and mutation bias have been proposed to affect the GC content.However,there has beencontroversy about the cause of GC variation.Here,we characterized the GC content of 1092 paralogs and other single-copygenes in the duplicated chromosomal regions of the rice genome(ssp.indica)and classified the paralogs into GC_3-richand GC_3-poor groups.By referring to out-group sequences from Arabidopsis and maize,we confirmed that the averagesynonymous substitution rate of the GC_3-rich genes is significantly lower than that of the GC_3-poor genes.Furthermore,we explored the other possible factors corresponding to the GC variation including the length of coding sequences,thenumber of exons in each gene,the number of genes in each family,the location of genes on chromosomes and the proteinfunctions.Consequently,we propose that natural selection rather than mutation bias was the primary cause of the GCvariation.     

Evidence that Natural Selection is the Primary Cause of the Guanine-cytosine Content Variation in Rice Genes

Cereal genes are classified into two distinct classes according to the guanine-cytosine （GC） content at the third codon sites （GC3）. Natural selection and mutation bias have been proposed to affect the GC content. However, there has been controversy about the cause of GC variation. Here, we characterized the GC content of 1 092 paralogs and other single-copy genes in the duplicated chromosomal regions of the rice genome （ssp. indica） and classified the paralogs into GC3-rich and GC3-poor groups. By referring to out-group sequences from Arabidopsis and maize, we confirmed that the average synonymous substitution rate of the GC3-rich genes is significantly lower than that of the GC3-poor genes. Furthermore, we explored the other possible factors corresponding to the GC variation including the length of coding sequences, the number of exons in each gene, the number of genes in each family, the location of genes on chromosomes and the protein functions. Consequently, we propose that natural selection rather than mutation bias was the primary cause of the GC variation.     

Hundreds of Out-of-Frame Remodeled Gene Families in the Escherichia coli Pangenome

All genomes include gene families with very limited taxonomic distributions that potentially represent new genes and innovations in protein-coding sequence, raising questions on the origins of such genes. Some of these genes are hypothesized to have formed de novo, from noncoding sequences, and recent work has begun to elucidate the processes by which de novo gene formation can occur. A special case of de novo gene formation, overprinting, describes the origin of new genes from noncoding alternative reading frames of existing open reading frames (ORFs). We argue that additionally, out-of-frame gene fission/fusion events of alternative reading frames of ORFs and out-of-frame lateral gene transfers could contribute to the origin of new gene families. To demonstrate this, we developed an original pattern-search in sequence similarity networks, enhancing the use of these graphs, commonly used to detect in-frame remodeled genes. We applied this approach to gene families in 524 complete genomes of Escherichia coli. We identified 767 gene families whose evolutionary history likely included at least one out-of-frame remodeling event. These genes with out-of-frame components represent ∼2.5% of all genes in the E. coli pangenome, suggesting that alternative reading frames of existing ORFs can contribute to a significant proportion of de novo genes in bacteria.     

南海北部表层沉积物中原核微生物多样性

[目的]为研究南海北部沉积物原核微生物的多样性和群落结构.[方法]从南海北部XSCS13站位表层沉积物中扩增古菌和细菌的16S rDNA并构建文库,随机挑出阳性克隆子进行测序,选出所有的OTU构建系统进化树,进行系统发育学分析.[结果]多数克隆子来自于未培养原核微生物,沉积物中的古菌分属3大门类:泉古菌(Crenarchaeota)、奇古菌(Thaumarchaeota)和广古菌(Euryarchaeota),其中泉古菌(Crenarchaeota)为主要门类,占71％;广古菌(Euryarchaeota)最少,只有3个克隆子.泉古菌(Crenarchaeota)又以MG Ⅰ为主要类群,占61％.细菌多样性明显高于古菌,共9个门类:变形杆菌(Proteobacteria)(32.6％)、疣微菌(Verrucomicrobia)(3.0％)、拟杆菌门(Bacteroidete)(5.2％)、酸杆菌(Acidobacteria)(4.4％)、绿弯菌(Chloroflexi)(6.0％)、厚壁菌(Firmicute)(3.7％)、浮霉菌(Planctomycete)(5.2％)、芽单胞菌(Gemmatimonadete)(11.1％)、放线细菌(Actinobacteria)(4.4％).变形杆菌为优势类群(包括α-Proteobacteria、γy-Proteobacteria和δ-Proteobacteria 3个亚群),其中γ-Proteobacteria是Proteobacteria中的优势种群,占54.5％.另外所有原核微生物总共有超过50％的克隆子与硫酸盐的还原以及甲烷的形成相关.[结论]结果表明南海北部XSCS13站位表层沉积物中原核微生物的多样性非常丰富,其中蕴含大量未知的微生物资源;另外古菌和细菌群落结构表明该位点可能处于富含甲烷的冷泉活动区.     

Sequence Variation in the Gene Encoding the Nonstructural 3 Protein of Hepatitis C Virus: Evidence for Immune Selection

To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3) gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star topology suggesting that a wild-type HCV sequence is maintained, unlike HIV. Received: 2 November 2000 / Accepted: 1 October 2001     

Low Contents of Carbon and Nitrogen in Highly Abundant Proteins: Evidence of Selection for the Economy of Atomic Composition

Proteins that assimilate particular elements were found to avoid using amino acids containing the element, which indicates that the metabolic constraints of amino acids may influence the evolution of proteins. We suspected that low contents of carbon, nitrogen, and sulfur may also be selected for economy in highly abundant proteins that consume large amounts of the resources of cells. By analyzing recently available proteomic data in Escherichia coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we found that at least the carbon and nitrogen contents in amino acid side chains are negatively correlated with protein abundance. An amino acid with a high number of carbon atoms in its side chain generally requires relatively more energy for its synthesis. Thus, it may be selected against in highly abundant proteins either because of economy in building blocks or because of economy in energy. Previous studies showed that highly abundant proteins preferentially use cheap (in terms of energy) amino acids. We found that the carbon content is still negatively correlated with protein abundance after controlling for the energetic cost of the amino acids. However, the negative correlation between protein abundance and energetic cost disappeared after controlling for carbon content. Building blocks seem to be more restricted than energy. It seems that the amino acid sequences of highly abundant proteins have to compromise between optimization for their biological functions and reducing the consumption of limiting resources. By contrast, the amino acid sequences of weakly expressed proteins are more likely to be optimized for their biological functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence of Positive Darwinian Selection in Omp85, a Highly ConservedBacterial Outer Membrane Protein Essential for Cell Viability

Omp85 is a highly conserved outer membrane protein found in all gram-negative bacteria. It is essential for bacterial cell viability and plays an integral function in the positioning and folding of other outer membrane proteins into the bacterial outer membrane. We have employed a maximum likelihood and a maximum parsimony approach to detect evidence of positive Darwinian selection in Omp85 homologues from 10 -proteobacteria and have identified 14 amino acid sites that show evidence of being under the influence of adaptive evolution. Interestingly all sites bar one are concentrated within surface loops of the protein that most likely interact with host immune response or the surrounding environment. Alternatively amino acids within membrane-spanning regions of the protein are found to be under purifying selection most likely as a result of structural constraints.Reviewing Editor: Dr. Siv Anderson     

Ecological Load and Balancing Selection in Circumboreal Barnacles

Acorn barnacle adults experience environmental heterogeneity at various spatial scales of their circumboreal habitat, raising the question of how adaptation to high environmental variability is maintained in the face of strong juvenile dispersal and mortality. Here, we show that 4% of genes in the barnacle genome experience balancing selection across the entire range of the species. Many of these genes harbor mutations maintained across 2 My of evolution between the Pacific and Atlantic oceans. These genes are involved in ion regulation, pain reception, and heat tolerance, functions which are essential in highly variable ecosystems. The data also reveal complex population structure within and between basins, driven by the trans-Arctic interchange and the last glaciation. Divergence between Atlantic and Pacific populations is high, foreshadowing the onset of allopatric speciation, and suggesting that balancing selection is strong enough to maintain functional variation for millions of years in the face of complex demography.     

以木糖异构酶基因为筛选标记的玉米遗传转化

利用木糖异构酶基因作为筛选标记可以在含有不同浓度木糖的培养基上筛选出玉米再生植株,其中50％-100％木糖浓度的总体筛选效果较好,但不同玉米基因型之间筛选的最佳浓度差异很大。通过DNA点杂交、PCR及PCR．Southern印记法检测表明,木糖异构酶基因已经整合到转基因植株中。以木糖作为筛选剂,可以减小潜在的生物安全隐患。     

基因表达研究中内参基因的选择与应用

管家基因是一类无组织特异性的,在物种的所有组织细胞中都表达的基因,被广泛用作内参基因来检测目标基因在不同的组织器官、一定的发育阶段或胁迫的环境条件下的表达规律变化。这些管家基因并不是在所有生理条件下都能作为理想内参基因稳定表达。在基因表达转录分析中,大多数普遍使用的内参基因已不能满足准确定量的要求。基于统计学分析软件,如geNorm、BestKeeper和NormFinder三种分析软件,可以筛选出稳定性较好的内参基因。本文综述了内参基因的选择条件、方法及应用。