Demystifying the RAD fad

We are writing in response to the population and phylogenomics meeting review by Andrews & Luikart ( 2014 ) entitled ‘Recent novel approaches for population genomics data analysis’. Restriction‐site‐associated DNA (RAD) sequencing has become a powerful and useful approach in molecular ecology, with several different published methods now available to molecular ecologists, none of which can be considered the best option in all situations. A&L report that the original RAD protocol of Miller et al. ( 2007 ) and Baird et al. ( 2008 ) is superior to all other RAD variants because putative PCR duplicates can be identified (see Baxter et al. 2011 ), thereby reducing the impact of PCR artefacts on allele frequency estimates (Andrews & Luikart 2014 ). In response, we (i) challenge the assertion that the original RAD protocol minimizes the impact of PCR artefacts relative to that of other RAD protocols, (ii) present additional biases in RADseq that are at least as important as PCR artefacts in selecting a RAD protocol and (iii) highlight the strengths and weaknesses of four different approaches to RADseq which are a representative sample of all RAD variants: the original RAD protocol (mbRAD, Miller et al. 2007 ; Baird et al. 2008 ), double digest RAD (ddRAD, Peterson et al. 2012 ), ezRAD (Toonen et al. 2013 ) and 2bRAD (Wang et al. 2012 ). With an understanding of the strengths and weaknesses of different RAD protocols, researchers can make a more informed decision when selecting a RAD protocol.     

Some progress in the study of plant–soil interactions in China

Plants and soil are the base for sustainably surviving human beings on the globe as the role of materials, energy, resources and environment (Shao & Chu 2008; Shao et al. 2008, 2009, 2010, 2012a,b; Liu & Shao, 2010; Ruan et al. 2010; Xu et al. 2010, 2012; Shao 2012; Huang et al. 2013). This topic has been extensively investigated for 100 years with more achievements in many sectors and practical significance in conducting high-efficient agriculture and eco-environmental construction. The plant–soil interaction is the core issue of this topic, which has been given much attention for the past 30 years (Wu et al. 2007, 2010; Zhang et al. 2011, 2013; Xu et al. 2012, 2013).     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Robust generation of hepatocyte-like cells from human embryonic stem cell populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.     

Early human dispersals into the Iberian Peninsula: a comment on Martínez et al.(2010) and Garcia et al. (2011)

Garcia et al. (2011) recently discussed early human dispersals into the Iberian Peninsula, describing several putative lithic artifacts (Martínez et al., 2010) recovered from layer 7 of the Vallpara díssection (Madurell-Malapeira et al., 2010) in Terrassa (Vallès-Penedès Basin, Catalonia, Spain). According to the authors' opinion, such evidence (1) fills a gap in the chronology of early human occupation in Iberia, (2) indicates that these populations had primary and early access to carcasses, and (3) confirms that early human populations were equipped with advanced cultural traits enabling them to survive in unfavourable climatic conditions. We argue below that the record of human activity at Vallparadís (Martínez et al., 2010;Garcia et al., 2011) is doubtful and even that if confirmed, a chronological gap would remain (contra Garcia et al., 2011). Additional remarks on assertions by these authors on the Vallparadís geology, taphonomy and paleonvironment are also provided.     

Human rhinovirus C infection is associated with asthma in children determined by xTAG respiratory viral panel FAST

<正>Dear Editor,Cumulative evidence supports the role of early-life viral infections,especially respiratory syncytial virus(RSV)and human rhinovirus(HRV),as major antecedents of childhood asthma(Lemanske,2002;Jackson et al.,2008).In this study,the x TAG respiratory viral panel FAST(RVP FAST)assay,a multiplex polymerase chain reaction(PCR)-based method(Arens et al.,2010;BaladaLlasat et al.,2011;Gharabaghi et al.,2011;Selvaraju,2012),was used to investigate the association of infec-     

Immune plexins and semaphorins: old proteins,new immune functions

Plexins and semaphorins are a large family of proteins that are involved in cell movement and response. The importance of plexins and semaphorins has been emphasized by their discovery in many organ systems including the nervous (Nkyimbeng-Takwi and Chapoval, 2011; McCormick and Leipzig, 2012; Yaron and Sprinzak, 2012), epithelial (Miao et al., 1999; Fujii et al., 2002), and immune systems (Takamatsu and Kumanogoh, 2012) as well as diverse cell processes including angiogenesis (Serini et al., 2009; Sakurai et al., 2012), embryogenesis (Perala et al., 2012), and cancer (Potiron et al., 2009; Micucci et al., 2010). Plexins and semaphorins are transmembrane proteins that share a conserved extracellular semaphorin domain (Hota and Buck, 2012). The plexins and semaphorins are divided into four and eight subfamilies respectively based on their structural homology. Semaphorins are relatively small proteins containing the extracellular semaphorin domain and short intracellular tails. Plexins contain the semaphorin domain and long intracellular tails (Hota and Buck, 2012). The majority of plexin and semaphorin research has focused on the nervous system, particularly the developing nervous system, where these proteins are found to mediate many common neuronal cell processes including cell movement, cytoskeletal rearrangement, and signal transduction (Choi et al., 2008; Takamatsu et al., 2010). Their roles in the immune system are the focus of this review.     

187 Plucking the high hanging fruit: a systematic approach for targeting protein interfaces

Development of specific ligands for protein targets that help decode the complexities of protein–protein interaction networks is a key goal for the field of chemical biology. Despite the emergence of powerful in silico and experimental high-throughput screening strategies, the discovery of synthetic ligands that selectively modulate protein–protein interactions remains a challenge for the chemical biologists. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these domains, we can modulate the function of a particular protein with metabolically-stable synthetic molecules (Raj et al., 2013). This presentation will discuss computational approaches (Bullock et al., 2011; Jochim & Arora, 2010) to identify targetable interfaces along with synthetic methods (Patgiri et al., 2008; Tosovska & Arora, 2010) to develop protein domain mimics (PDMs) as modulators of intracellular protein–protein interactions (Henchey et al., 2010; Patgiri et al., 2011).     

Epigenetic Variations in Plant Hybrids and Their Potential Roles in Heterosis

Heterosis,one of the most important biological phenomena,refers to the phenotypic superiority of a hybrid over its genetically diverse parents with respect to many traits such as biomass,growth rate and yield.Despite its successful application in breeding and agronomic production of many crop and animal varieties,the molecular basis of heterosis remains elusive.The classic genetic explanations for heterosis centered on three hypotheses:dominance (Davenport,1908;Bruce,1910;Keeble and Pellew,1910;Jones,1917),overdominance (East,1908;Shull,1908) and epistasis (Powers,1944;Yu et al.,1997).However,these hypotheses are largely conceptual and not connected to molecular principles,and are therefore insufficient to explain the molecular basis of heterosis (Birchler et al.,2003).Recently,many studies have explored the molecular mechanism of heterosis in plants at a genome-wide level.These studies suggest that global differential gene expression between hybrids and parental lines potentially contributes to heterosis in plants (e.g.,Swanson-Wagner et al.,2006;Zhang et al.,2008;Wei et al.,2009;Song et al.,2010).Research suggests that genetic components,including cis-acting elements and trans-acting factors,are critical regulators of differential gene expression in hybrids (Hochholdinger and Hoecker,2007;Springer and Stupar,2007;Zhang et al.,2008).However,other research indicates that epigenetic components,the regulators of chromatin states and genome activity,also have the potential to impact heterosis (e.g.,Ha et al.,2009;He et al.,2010;Groszmann et al.,2011;Barber et al.,2012;Chodavarapu et al.,2012;Greaves et al.,2012a;Shen et al.,2012).     

FEN nucleases: bind, bend, fray, cut

In this issue, Orans et al. (2011) and Tsutakawa et al. (2011) report exciting insights into the molecular principles governing diverse endo- and exonucleolytic cleavage specificities of members of the RAD2/FEN superfamily of nucleases, which have critical roles in DNA replication and maintenance.     

A mechanistic understanding of ageing revealed by studying the young

A main focus within biomedical research is to understand how adverse environmental conditions experienced during early development affects lifelong health (Barker 1992). Within this context, extensive research in rodent models and humans has shown that intrauterine growth retardation (IUGR) caused by nutrient restriction during early development is often followed by post-natal 'catch-up' growth when access to food resources improves. However, this accelerated growth rate seems to come at a cost, as metabolic and endocrine processes that are programmed during this time cause later-life onset of diseases such as obesity, insulin resistance and cardiovascular disease (reviewed in Crespi & Denver 2005). In this issue Molecular Ecology, Geiger et al. (2012) asked what are the costs of catch-up growth in nutrient-restricted king penguin chicks (Fig. 1) by measuring lengths of telomeres, the protective DNA sequences at the end of chromosomes, before and after catch-up growth, as the amount and rate of telomere sequence loss over time has been associated with reduced lifespan in both model and nonmodel organisms (see reviews of Costantini et al. 2010; Haussmann & Marchetto 2010). Geiger et al. (2011) found that chicks entering the post-winter growth season at a smaller size exhibited increased growth rates (i.e. catch-up growth) at the cost of increased oxidative stress and reduced telomere lengths compared with the chicks entering the growth period at a larger size. Furthermore, chicks that did not survive had drastically shorter telomere lengths and reduced antioxidant capacities at the beginning of the growth period than all other chicks, thereby directly associating telomere length to mortality. These results suggest that while catch-up growth allows smaller chicks to head off into the world on equal footing with chicks that hatched at a larger size, it likely comes at the cost of a shortened lifespan. Thus, this study provides a mechanism that supports the antagonistic pleiotropy theory of senescence (Promislow 2004).     

Testing the level of ant activity associated with quorum sensing: an empirical approach leading to the establishment and test of a null-model (response to the comment of Richardson et al.)

In a paper in this journal (Nouvellet et al., 2010), we presented results from experiments on the behaviour of the Pharaoh's ant, Monomorium pharaonis, along with a substantial statistical and theoretical analysis of the results. In a minor part of our paper, we compared our results with the related work of Richardson et al. (2010a). These authors have subsequently commented on our interpretation of their work (Richardson et al., 2011). In this Letter we respond to the comments of Richardson et al. (2011), and give detailed arguments why we stand by our original conclusions.     

Remarkable new evidence for island radiation in birds

If island biogeography has a sweet spot, it's where islands generate their own species diversity rather than merely taking on mainland immigrants. In birds and other highly dispersive taxa, however, this 'zone of radiation', may be vanishingly small. Darwin's finches and Hawaiian Honeycreepers are among only a handful of examples of island radiation in birds (Price 2008), suggesting that winged powers of dispersal make sufficient isolation from mainland colonists unlikely, while also hindering speciation within and among isolated islands. Nevertheless, two studies in this issue of Molecular Ecology join a string of other recent analyses suggesting that island radiation in birds remains under-appreciated (see also Moyle et al. 2009; Kisel & Barraclough 2010; Rosindell & Phillimore 2011). Melo et al. (2011) use a phylogenetic analysis of white-eyes on islands in the Gulf of Guinea to identify two previously overlooked island radiations, and reveal replicated adaptive divergence on islands where species occur in pairs. Sly et al. (2011), meanwhile, consider possible explanations for speciation and geographic differentiation within a large island, and find the same type of oceanic barriers that are critical to bird speciation across archipelagos may also contribute to divergence that appears to have occurred within a single island.     

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.     

A novel role for Greatwall kinase in recovery from DNA damage

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression (Halazonetis et al., 2008; Motoyama and Naka, 2004; Zhou and Elledge, 2000). The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G2/M transition (Archambault et al., 2007; Jackson, 2006; Zhao et al., 2008; Yu et al., 2004; Yu et al., 2006; Zhao et al., 2006) and helps maintain M phase through inhibition of PP2A/B55δ (Burgess et al., 2010; Castilho et al., 2009; Goldberg, 2010; Lorca et al., 2010; Vigneron et al., 2009), the principal phosphatase for Cdk-phosphorylated substrates. Here we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild type, but not kinase dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl over-expression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA     

浙江天台盆地晚白垩世恐龙蛋新类型(英文)

浙江天台盆地上白垩统赖家组和赤城山组是我国最重要的恐龙蛋化石产出地层之一。近年来,我们对天台盆地陆相红层中的恐龙蛋化石层位进行了详细厘定,对恐龙蛋类型进行了系统描述,并对前人报道的一些属种进行了分类订正。研究显示,天台恐龙蛋化石群基本上可分为7蛋科、12蛋属和15蛋种,代表了我国晚白垩世早期的恐龙蛋化石组合。本文简要报道了主要产自天台盆地赤城山组的双塘似蜂窝蛋(新蛋属、新修订种)、木鱼山半蜂窝蛋(新蛋属、新蛋种)、国清寺副蜂窝蛋(新修订种)、天台棱柱形蛋(新修订种)和张头槽马赛克蛋(新蛋属、新修订种)等3新蛋属、5新蛋种和修订种的主要鉴定特征,并建立一新蛋科——似蜂窝蛋科。     

Increasing evidence of the role of gene flow in animal evolution: hybrid speciation in the yellow-rumped warbler complex

In this issue of Molecular Ecology, Brelsford et al. (2011) present strong evidence for a case of hybrid speciation within the yellow-rumped warbler complex. Although homoploid hybrid speciation has now been documented in many animals (Mallet 2007), it seems rare in tetrapods (Mavárez & Linares 2008) and it has barely even been mentioned in birds (Price 2008). Brelsford and colleagues thus present the first detailed molecular evidence suggesting that hybrid speciation can occur in birds. Brelsford et al. (2011) posit that Audubon's warbler (Dendroica auduboni) constitutes a hybrid species originating from the admixture of two distinct parental lineages, represented today by myrtle warbler (D. coronata) and black-fronted warbler (D. nigrifrons). The authors present three major lines of molecular evidence suggesting that this is not simply a case of a hybrid swarm or limited introgression.     

Linkage mapping and comparative genomics using next-generation RAD sequencing of a non-model organism

Restriction-site associated DNA (RAD) sequencing is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis of non-model organisms including genotype-phenotype association mapping, phylogeography, population genetics and scaffolding genome assemblies through linkage mapping. We constructed a RAD library using genomic DNA from a Plutella xylostella (diamondback moth) backcross that segregated for resistance to the insecticide spinosad. Sequencing of 24 individuals was performed on a single Illumina GAIIx lane (51 base paired-end reads). Taking advantage of the lack of crossing over in homologous chromosomes in female Lepidoptera, 3,177 maternally inherited RAD alleles were assigned to the 31 chromosomes, enabling identification of the spinosad resistance and W/Z sex chromosomes. Paired-end reads for each RAD allele were assembled into contigs and compared to the genome of Bombyx mori (n = 28) using BLAST, revealing 28 homologous matches plus 3 expected fusion/breakage events which account for the difference in chromosome number. A genome-wide linkage map (1292 cM) was inferred with 2,878 segregating RAD alleles inherited from the backcross father, producing chromosome and location specific sequenced RAD markers. Here we have used RAD sequencing to construct a genetic linkage map de novo for an organism that has no previous genome data. Comparative analysis of P. xyloxtella linkage groups with B. mori chromosomes shows for the first time, genetic synteny appears common beyond the Macrolepidoptera. RAD sequencing is a powerful system capable of rapidly generating chromosome specific data for non-model organisms.