沼泽红假单胞菌偶氮还原酶相关基因的克隆及其生物信息学分析

脱色实验证明沼泽红假单胞菌(Rhdopsedomonas palustris)对偶氮染料有较强的降解能力,作者通过Gen Bank搜索,对所获得的所有偶氨还原酶基因在NCBI进行比对并设计引物,从沼泽红假单胞菌质粒中扩增获得了一条含471bp完整开放阅读框架的序列 GenBank搜索表明该基因为未登录的新基因。通过互联同数据库及生物信息学分析工具进行初步分析表明：该基因编码的蛋白是一种等电点为9.65的不稳定蛋白,定位于细胞质;由α螺旋、延伸带和随机卷曲三种形式组成,具有蛋白激酶C磷酸化等多个位点,并具有一个强跨膜疏水区。     

Cosmopolitanism of microbial eukaryotes in the global deep seas

Deep sea environments cover more than 65% of the earth’s surface and fulfil a range of ecosystem functions, yet they are also amongst the least known habitats on earth. Whilst the discovery of key geological processes, combined with technological developments, has focused interest onto geologically active areas such as hydrothermal vents, most abyssal biodiversity remains to be discovered ( Danovaro et al. 2010 ). However, as for terrestrial reservoirs of biodiversity, the world’s largest biome is under threat from anthropogenic activities ranging from environmental change to the exploitation of minerals and rare‐earth elements ( Kato et al. 2011 ). It is therefore important to understand the magnitude, nature and composition of deep sea biological communities to inform us of levels of local adaptation, functionality and resilience with respect to future environmental perturbation. In this issue of Molecular Ecology, Bik et al. utilize 454 Roche metagenetic environmental sequencing to assess microbial metazoan community composition and phylogenetic identity across deep sea depth gradients and between ocean basins. The analyses suggest that although the majority of microbial eukaryotic taxa are regionally restricted, a small percentage might maintain cosmopolitan deep sea distributions, and an even smaller fraction appear to be eurybathic (live across depth gradients).     

Stacking up RADSeq assembly programs: From complete hit to completely abysmal

Decreasing sequencing costs have driven a rapid expansion of novel genotyping methods. One of these methods is the exploitation of restriction enzyme cut sites to generate genome‐wide but reduced representation sequencing libraries (RRLs), alternatively termed genotyping by sequencing or restriction‐site associated DNA sequencing. Without a reference genome, the resulting short sequence reads must be assembled de novo. There are many possible assembly programs, most not explicitly developed for RRL data, and we know little of their effectiveness. In this issue of Molecular Ecology Resources, LaCava et al. (2020) systematically evaluate six commonly used programs and two commonly varied parameters for complete and accurate assembly of RRLs, using simulated double digests of Homo sapiens and Arabidopsis thaliana genomes with varied mutation rates and types. The authors find substantial variation in performance across assembly programs. The most consistently high‐performing assembler is infrequently used in their literature survey (CD‐HIT; Li and Godzik, 2006), while several others fail to produce complete, accurate assemblies under many conditions. LaCava et al. additionally recommend best practices in parameter choice and evaluation of future assembly programs—advice that molecular ecologists working to assemble sequences of all kinds should take to heart.     

New gene-derived simple sequence repeat markers for common bean (Phaseolus vulgaris L.)

Common bean is an important and diverse crop legume with several wild relatives that are all part of the Phaseoleae tribe of tropical crop legumes. Sequence databases have been a good source of sequences to mine for simple sequence repeats (SSRs). The objective of this research was to evaluate 14 sequence collections from common bean for SSRs and to evaluate the diversity of the polymorphic microsatellites derived from these collections. SSRs were found in 10 of the GenBank sequence collections with an average of 11.3% of sequences containing microsatellite motifs. The most common motifs were based on tri- and dinucleotides. In a marker development programme, primers were designed for 125 microsatellites which were tested on a panel of 18 common bean genotypes. The markers were named as part of the bean microsatellite-database (BMd) series, and the average polymorphism information content was 0.404 for polymorphic markers and predicted well the genepool structure of common beans and the status of the wild and cultivated accessions that were included in the study. Therefore, the BMd series of microsatellites is useful for multiple studies of genetic relatedness and as anchor markers in future mapping of wide crosses in the species.     

Don't throw the baby out with the bathwater: identifying and mapping paralogs in salmonids

Many eukaryotic genomes contain a large fraction of gene duplicates (or paralogs) as a result of ancient or recent whole‐genome duplications (Ohno 1970 ; Jaillon et al. 2004 ; Kellis et al. 2004 ). Identifying paralogs with NGS data is a pervasive problem in both ancient polyploids and neopolyploids. Likewise, paralogs are often treated as a nuisance that has to be detected and removed (Everett et al. 2012 ). In this issue of Molecular Ecology Resources, Waples et al. ( 2015 ) show that exclusion might not be necessary and how we may miss out on important genomic information in doing so. They present a novel statistical approach to detect paralogs based on the segregation of RAD loci in haploid offspring and test their method by constructing linkage maps with and without these duplicated loci in chum salmon, Oncorhynchus keta (Fig.  1 ). Their linkage map including the resolved paralogs shows that these are mostly located in the distal regions of several linkage groups. Particularly intriguing is their finding that these homoeologous regions appear impoverished in transposable elements (TE). Given the role that TE play in genome remodelling, it is noteworthy that these elements are of low abundance in regions showing residual tetrasomic inheritance. This raises the question whether re‐diploidization is constrained in these regions and whether they might have a role to play in salmonid speciation. This study provides an original approach to identifying duplicated loci in species with a pedigree, as well as providing a dense linkage map for chum salmon, and interesting insights into the retention of gene duplicates in an ancient polyploid.     

EST‐SSR markers from five sequenced cDNA libraries of common bean (Phaseolus vulgaris L.) comparing three bioinformatic algorithms

Expressed sequence tags (ESTs) are a rich source of SSR sequences, but the proportion of long Class I microsatellites with many repeats vs. short Class II microsatellites with few repeats is an important factor to consider. Class I microsatellites, with more than 20 bp of repeats, tend to make better markers with higher polymorphism. The goal of this study was to determine the frequency of Class I and Class II microsatellites in a collection of over 21 000 ESTs from a single study of five different tissues of common bean: two types of leaves, nodules, pods and roots. For this objective, we used three different bioinformatics pipelines: Automated Microsatellite Marker Development (AMMD), Batchprimer3 and SSRLocator. In addition, we determined the frequency of single or multiple SSRs in the assembled ESTs, the frequency of perfect and compound repeats and whether Class I microsatellites were mainly di‐nucleotide or tri‐nucleotide motifs with each of the search engines. Primers were designed for a total of 175 microsatellites concentrating on class I microsatellites identified with SSR locator. A few other microsatellites were included from the other search engines, AMMD and Batchprimer3 programs so as to have a representative set of class II markers for comparison sake. The comparison of 95 class I vs. 80 class II markers confirmed that the Class I were more polymorphic and therefore more useful.     

Critical review of NGS analyses for de novo genotyping multigene families

The genotyping of highly polymorphic multigene families across many individuals used to be a particularly challenging task because of methodological limitations associated with traditional approaches. Next‐generation sequencing (NGS) can overcome most of these limitations, and it is increasingly being applied in population genetic studies of multigene families. Here, we critically review NGS bioinformatic approaches that have been used to genotype the major histocompatibility complex (MHC) immune genes, and we discuss how the significant advances made in this field are applicable to population genetic studies of gene families. Increasingly, approaches are introduced that apply thresholds of sequencing depth and sequence similarity to separate alleles from methodological artefacts. We explain why these approaches are particularly sensitive to methodological biases by violating fundamental genotyping assumptions. An alternative strategy that utilizes ultra‐deep sequencing (hundreds to thousands of sequences per amplicon) to reconstruct genotypes and applies statistical methods on the sequencing depth to separate alleles from artefacts appears to be more robust. Importantly, the ‘degree of change’ (DOC) method avoids using arbitrary cut‐off thresholds by looking for statistical boundaries between the sequencing depth for alleles and artefacts, and hence, it is entirely repeatable across studies. Although the advances made in generating NGS data are still far ahead of our ability to perform reliable processing, analysis and interpretation, the community is developing statistically rigorous protocols that will allow us to address novel questions in evolution, ecology and genetics of multigene families. Future developments in third‐generation single molecule sequencing may potentially help overcome problems that still persist in de novo multigene amplicon genotyping when using current second‐generation sequencing approaches.     

The discovery of Antarctic RNA viruses: a new game changer

Antarctic ecosystems are dominated by micro‐organisms, and viruses play particularly important roles in the food webs. Since the first report in 2009 (López‐Bueno et al. 2009 ), ‘omic’‐based studies have greatly enlightened our understanding of Antarctic aquatic microbial diversity and ecosystem function (Wilkins et al. 2013 ; Cavicchioli 2015 ). This has included the discovery of many new eukaryotic viruses (López‐Bueno et al. 2009 ), virophage predators of algal viruses (Yau et al. 2011 ), bacteria with resistance to phage (Lauro et al. 2011 ) and mechanisms of haloarchaeal evasion, defence and adaptation to viruses (Tschitschko et al. 2015 ). In this issue of Molecular Ecology, López‐Bueno et al. ( 2015 ) report the first discovery of RNA viruses from an Antarctic aquatic environment. High sequence coverage enabled genome variation to be assessed for four positive‐sense single‐stranded RNA viruses from the order Picornavirales. By examining the populations present in the water column and in the lake's catchment area, populations of ‘quasispecies’ were able to be linked to local environmental factors. In view of the importance of viruses in Antarctic ecosystems but lack of data describing them, this study represents a significant advance in the field.     

Exploring linkage disequilibrium

Linkage disequilibrium (LD, association of allelic states across loci) is poorly understood by many evolutionary biologists, but as technology for multilocus sampling improves, we ignore LD at our peril. If we sample variation at 10 loci in an organism with 20 chromosomes, we can reasonably treat them as 10 ‘independent witnesses’ of the evolutionary process. If instead, we sample variation at 1000 loci, many are bound to be close together on a chromosome. With only one or two crossovers per meiosis, associations between close neighbours decay so slowly that even LD created far in the past will not have dissipated, so we cannot treat the 1000 loci as independent witnesses (Barton 2011 ). This means that as marker density on genomes increases classic analyses assuming independent loci become mired in the problem of overconfidence: if 1000 independent witnesses are assumed, and that number should be much lower, any conclusion will be overconfident. This is of special concern because our literature suffers from a strong publication bias towards confident answers, even when they turn out to be wrong (Knowles 2008 ). In contrast, analyses that take into account associations across loci both control for overconfidence and can inform us about LD generating events far in the past, for example human/Neanderthal admixture (Fu et al. 2014 ). With increased marker density, biologists must increase their awareness of LD and, in this issue of Molecular Ecology Resources, Kemppainen et al. ( 2015 ) make software available that can only help in this process: LDna allows patterns of LD in a data set to be explored using tools borrowed from network analysis. This has great potential, but realizing that potential requires understanding LD.