Plasmodium falciparum circumsporozoite protein (PfCSP) is one of the most extensively studied malaria vaccine candidates, but the genetic polymorphism of PfCSP within and among the global P. falciparum population raises concerns regarding the efficacy of a PfCSP-based vaccine efficacy. In this study, genetic diversity and natural selection of PfCSP in Myanmar as well as global P. falciparum were comprehensively analysed.P. falciparum infected Myanmar patients. Fifty-one full-length PfCSP genes were amplified from the blood samples through a nested polymerase chain reaction, cloned into a TA cloning vector, and then sequenced. Polymorphic characteristics and natural selection of Myanmar PfCSP were analysed using the DNASTAR, MEGA6, and DnaSP programs. Polymorphic diversity and natural selection in publicly available global PfCSP were also analysed.Blood samples were collected from 51 The N-terminal and C-terminal non-repeat regions of Myanmar PfCSP showed limited genetic variations. A comparative analysis of the two regions in global PfCSP displayed similar patterns of low genetic diversity in global population, but substantial geographic differentiation was also observed. The most notable polymorphisms identified in the N-terminal region of global PfCSP were A98G and 19-amino acid length insertion in global population with different frequencies. Major polymorphic characters in the C-terminal region of Myanmar and global PfCSP were found in the Th2R and Th3R regions, where natural selection and recombination occurred. The central repeat region of Myanmar PfCSP was highly polymorphic, with differing numbers of repetitive repeat sequences NANP and NVDP. The numbers of the NANP repeats varied among global PfCSP, with the highest number of repeats seen in Asian and Oceanian PfCSP. Haplotype network analysis of global PfCSP revealed that global PfCSP clustered into 103 different haplotypes with geographically-separated populations.Myanmar and global PfCSP displayed genetic diversity. N-terminal and C-terminal non-repeat regions were relatively conserved, but the central repeat region displayed high levels of genetic polymorphism in Myanmar and global PfCSP. The observed geographic pattern of genetic differentiation and the points of evidence for natural selection and recombination suggest that the functional consequences of the polymorphism should be considered for developing a vaccine based on PfCSP.The online version of this article (10.1186/s12936-018-2513-0) contains supplementary material, which is available to authorized users. Plasmodium spp. infections, is one of the most significant life-threatening infectious diseases to humans worldwide. It accounted for more than 216 million cases and approximately 450,000 deaths across the globe in 2017 [Malaria, caused by  in 2017 . In spitPlasmodium falciparum have been investigated. Among these, RTS,S, currently the most advanced malaria vaccine candidate [P. falciparum (PfCSP). RTS,S is comprised of a liposome-based adjuvant (AS01) and hepatitis B virus surface antigen (HBsAg) virus-like particles incorporating a portion of the PfCSP genetically fused to HBsAg. PfCSP is a dominant surface protein of sporozoites, and it plays a critical role in the invasion of hepatocytes by sporozoites [To date, there is no licensed vaccine against malaria, though many efforts and studies have been performed in order to develop effective vaccines. Various vaccine constructs based on diverse antigens from sexual and asexual stages of andidate , 6, is brozoites –9. PfCSProzoites –12. The rozoites –15.P. falciparum from different geographic regions have diverse genetic makeup [P. falciparum population. Indeed, most P. falciparum vaccine candidate antigens including PfCSP have been found to show various genetic and antigenic polymorphisms in global isolates [P. falciparum isolates is critical for designing an effective vaccine. In this study, genetic polymorphism and natural selection of PfCSP in P. falciparum Myanmar isolates were analysed. A comparative analysis of global PfCSP was also performed in order to gain an in-depth understanding of the genetic makeup of PfCSP in the global P. falciparum population.Recent genome sequencing studies have demonstrated that c makeup , 17, whiisolates , which cP. falciparum in Myanmar in 2015. The patients were selected in field surveys for malaria, which were conducted in towns and villages in Naung Cho, Pyin Oo Lwin, Tha Beik Kyin townships, and Mandalay in Upper Myanmar. Infections were diagnosed through microscopic examination of thin and thick blood smears. Finger-prick blood samples were taken from P. falciparum infected symptomatic patients prior to drug treatment and spotted on Whatman 3 MM filter paper  for confirmation by polymerase chain reaction (PCR) targeting the 18S ribosomal RNA (rRNA) gene [A total of 51 blood samples used in this study were collected from malaria patients infected with NA) gene , 19, as Taq DNA polymerase  with proof-reading activity was used in all PCR amplification steps to minimize the nucleotide mis-incorporation. The following thermal cycling conditions were used for both amplifications: 94 °C for 5 min; and 30 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min 30 s, followed by a final extension at 72 °C for 10 min. The PCR product was resolved on a 1.2% agarose gel, purified from gel, then ligated into the T&A cloning vector . Each ligation mixture was transformed into Escherichia coli DH5α competent cells and positive clones, with appropriate inserts screened by colony PCR. The nucleotide sequences of the cloned PfCSP were analysed through automatic DNA sequencing with M13 forward and M13 reverse primers by the Sanger method. Plasmids from at least two independent clones from each transformation mixture were sequenced in both directions so as to verify the sequence accuracy. The nucleotide sequences reported in this study have been deposited in the GenBank database under the accession numbers MF350670–MF350720.Genomic DNA was extracted from dried blood spots using the QIAamp DNA Blood Kit  following the manufacturer’s protocol. The full-length region encoding PfCSP was amplified through a nested PCR method. The primers for the first round PCR were 5′-ATG ATG AGA AAA TTA GCT ATT TTA TCT GTT-3′ and 5′-CTA ATT AAG GAA CAA GAA GGA TAA TAC CAT-3′. The primers used for nested PCR were 5′-AGA AAA TTA GCT ATT TTA TCT GTT TCT-3′ and 5′-ACA AGA AGG ATA ATA CCA TTA TTA ATC-3′. Ex P. falciparum strain 3D7 (XM_001351086) was included in the alignment for comparison as a reference sequence. The values of segregating sites (S), the average number of pair-wise nucleotide differences (K), haplotype diversity (Hd), and nucleotide diversity (π) were calculated using DnaSP version 5.10.00 [P < 0.05) in MEGA6 program [The nucleotide and deduced amino acid sequences of PfCSP were analysed using EditSeq and SeqMan in the DNASTAR package . The PfCSP sequence of the laboratory-adapted  5.10.00 . The π w program  using Ne program  with the program , Fu and  program  were per program  in order program .P. falciparum isolates were analysed. The PfCSP sequences used in this study were from Thailand, Philippines, Vietnam, India, Iran, Papua New Guinea, Vanuatu, Solomon Islands, Kenya, Cameroon, Ghana, Tanzania, Senegal, Gambia, Brazil, and Venezuela . In order to investigate the genetic relationships among global PfCSP haplotypes, the haplotype network for 817 full-length sequences of PfCSP from Myanmar and other countries listed above was analysed using NETWORK version 5.0.0.3 with the Median joining algorithm [The genetic diversities of PfCSP among global lgorithm .Fifty-one full-length PfCSP were successfully amplified from the 51 blood samples analysed in this study. As expected, size variations were observed in the amplified PfCSP. The approximate sizes of amplified products varied from 0.9 to 1.3 kb, which was mainly caused by differences in the number of tandem repeats in the central repeat region.84EKLRKPKHKKLKQPADGNPDP104. However, the conserved motif (KLKQP) that was involved in sporozoite invasion of mosquito salivary gland and in binding to hepatocytes prior to invasion [The N-terminal non-repeat region was highly conserved in Myanmar PfCSP. Only a haplotype with a 57 bp (encoding 19 amino acids of NNGDNGREGKDEDKRDGNN) insertion in the middle portion of the region compared to the 3D7 reference sequence (XM_001351086) was identified  and Th3R (352NKPKDELDYAND363) T-cell epitope regions. Haplotype 3 was the most prevalent haplotype, accounting for 80.4% of the 51 Myanmar PfCSP sequences. The patterns of amino acid polymorphisms in global PfCSP were also assessed. Comparative analysis of polymorphic patterns of the C-terminal region in global PfCSP revealed that the region was relatively conserved in global PfCSP  were identified in the C-terminal non-repeat region of Myanmar PfCSP Fig. a. The seK) in this region was 0.92. The overall haplotype diversity (Hd) and nucleotide diversity (π) in this region were estimated to be 0.329 ± 0.072 and 0.004 ± 0.003, respectively. In order to investigate whether natural selection has contributed to the diversity in C-terminal non-repeat region in Myanmar PfCSP, the value of dN–dS for this region was analysed. The estimated value of dN–dS was found to be 0.005 ± 0.003 (Table P > 0.1) (Table P > 0.1) and 0.992 (P > 0.1), respectively.The nucleotide diversity and genetic differentiation were analysed in the C-terminal non-repeat region in Myanmar PfCSP. The average number of nucleotide differences  was higher than that in South American (3.74), Asian (2.73), and Oceanian (1.09) PfCSP. The greatest nucleotide diversity was observed in African PfCSP (π = 0.105 ± 0.021) followed by South American PfCSP (π = 0.018 ± 0.002), Asian PfCSP (π = 0.013 ± 0.001), and Oceanian PfCSP (π = 0.005 ± 0.0002). The dN–dS values for all global PfCSP were estimated to be positive, suggesting that positive natural selection may occur in the C-terminal non-repeat region of global PfCSP, but this trend was not statistically significant. Negative values of Tajima’s D were identified in the C-terminal non-repeat region of South American PfCSP , African PfCSP , and Asian PfCSP , indicating that they were under purifying selection. Meanwhile, the C-terminal non-repeat region of Oceanian PfCSP  showed positive Tajima’s D values, suggesting the effects of balance selection on the population. Sliding window plot analysis (window length of 10 bp and step size of 5 bp) in global PfCSP showed that global PfCSP shared highly similar patterns of nucleotide diversity across the region. Nucleotide diversity peaked at the Th2R and Th3R T-cell epitopes, although the values of π were slightly different within and between PfCSP populations according to geographical origin , indicating that this region is under host immune responses. The N-terminal region of PfCSP has been largely neglected as a potential vaccine target in spite of being a target of inhibitory antibodies and protective T cell responses. The functional importance of the N-terminal region in protective immunity has been demonstrated. Polypeptides flanking the PfCSP N-terminal region evoked the production of inhibitory antibodies for hepatocyte invasion by sporozoites, and these polypeptides are likely to render partial protective immunity in people residing in malaria-endemic regions [Myanmar PfCSP had a largely well-conserved N-terminal region, which coincided with PfCSP populations from other geographical areas , 27–30. atocytes . The fun regions . A recen regions . These cP. falciparum populations, resulting in the differing number of tetrapeptide repeats in the global PfCSP population. The RTS,S, the current malaria vaccine, is composed of 19 NANP tetrapeptide repeats and C-terminal T cell-epitope that are linked to the Hepatitis B surface antigen [The central repeat region of PfCSP has been recognized to play crucial roles in sporozoite formation and development . It has  antigen . To dateThe C-terminal non-repeat region of Myanmar PfCSP displayed limited diversity with only three differing haplotypes among 51 Myanmar PfCSP sequences, coinciding with the previous reports on PfCSP from different geographical origins . HaplotyConsidering that Vietnam is a hypoendemic country with a low malaria transmission rate, the reason why Vietnamese PfCSP showed high recombination event and haplotype diversity is unclear and it should be elucidated further. Collectively, the results of genetic diversity analysis in the C-terminal region of global PfCSP suggested that global PfCSP showed limited genetic diversity in the region. However, the genetic diversity pattern of the PfCSP C-terminal region differed slightly according to different geographical origins. Complicated natural selection acts on the global PfCSP C-terminal region, which produces genetic diversity of the region in global PfCSP. Recombination may also contribute to the genetic diversity of global PfCSP, although the recombination parameters differed by geographical origins. These genetic polymorphisms in the C-terminal region of global PfCSP suggest that more concern is required for design formulation of PfCSP-based vaccine.P. falciparum NF54/3D7 strain [Haplotype network analysis of 817 global PfCSP sequences indicated that Asian and Oceanian PfCSP formed limited numbers of clusters. Meanwhile, African PfCSP showed highly-branched and complicated patterns of haplotype diversity. No haplotype was identified that fully covers PfCSP from all of the geographic regions analysed in this study. Most singletons were mainly occupied by African PfCSP, supporting the notion that African PfCSP had higher genetic diversity than PfCSP from other geographical regions. The current RTS,S recombinant vaccine was constructed with PfCSP of 7 strain . Haploty7 strain –46, and P. falciparum isolates that collected in restricted areas of Myanmar. Therefore, nation-wide analysis of PfCSP in P. falciparum isolates collected from different regions of Myanmar is needed to clearly understand the overall genetic diversity and population structure of Myanmar PfCSP. Further examination of PfCSP nucleotide and amino acid variations in diverse PfCSP populations with a larger number of global PfCSP sequences would be also necessary to better understand the polymorphic nature of PfCSP.The limitation of this study is that Myanmar PfCSP sequences analysed in this study were from P. falciparum population.PfCSP is a main component of RST,S, the most advanced malaria vaccine currently, but the impact of natural genetic variations in the global PfCSP population on the efficacy of RTS,S remains unclear. Analysis of genetic diversity and natural selection of global PfCSP population suggested that the central repeat region was highly polymorphic in global PfCSP. Meanwhile, the N-terminal and C-terminal non-repeat regions revealed limited polymorphism, but differences were also identified between or among global PfCSP according to the different geographical origins. In particular, a high level of nucleotide diversity was identified in the Th2R and Th3R regions of the PfCSP C-terminal region. Complicated natural selection and recombination event were predicted in the global PfCSP C-terminal region, which may be major driving forces for maintaining and producing genetic diversity of the region in global PfCSP. The geographic patterns of genetic differentiation as well as the points of evidence for natural selection and recombination suggest that the functional consequences of the polymorphism should be considered for a vaccine based on PfCSP. The results of this study not only contribute insight into the genetic nature of Myanmar PfCSP, but also aid the understanding of genetic diversity of global PfCSP, and may provide valuable information for the development of an effective vaccine based on PfCSP. The findings of this study also warrant continuous monitoring of genetic diversity of PfCSP in the global population to better understand the polymorphic nature and evolutionary aspect of PfCSP in global Additional file 1: Table S1. Global PfCSP sequences analysed in this study.


Saccharomyces cerevisiae is a widely used model organism, and yeast genetic methods are powerful tools for discovery of novel functions of genes. Recent advancements in chemical-genetics and chemical-genomics have opened new avenues for development of clinically relevant drug treatments. Systematic mapping of genetic networks by high-throughput chemical-genetic screens have given extensive insight in connections between genetic pathways. Here, I review some of the recent developments in chemical-genetic techniques in budding yeast.The budding yeast  S. cerevisiae, also known as brewer’s yeast or baker’s yeast.What is chemical genetics? Chemical genetics is a research approach that makes use of small molecules to explore biological functions and processes; “chemical” indicates the use of biologically active chemicals, whereas “genetics” refers to the fact that this research approach is based on principles often used in the field of genetics. Chemical-genetic screens can be used to determine the mode of action of a given biologically active compound by identifying its cellular target. Alternatively, a drug can be designed to specifically inhibit or activate a protein of interest; this compound can subsequently be used in a high-throughput screen to identify novel functions of the target protein. Typically, a compound is used that affects the function of a single gene product in the context of a complex cellular milieu. This compound may then be employed to screen a large collection of mutants using a specific biological effect as a read-out. Many chemical-genetic studies have made use of protein-targeting drugs. However, chemical-genetic screens are not necessarily restricted to proteins, but can also be expanded to include drugs that target RNA, DNA, or even cellular metabolites. Here I will review some of the recent publications on chemical-genetic studies in the model organism S. cerevisiae genome consists of 16 chromosomes, totaling approximately 12.1 million basepairs. The first eukaryote to be completely sequenced, a map of the yeast genome was released in 1996 [The vast majority of all chemical-genetic screens have made use of budding yeast, because yeast has several important advantages. For example, it is easy and cheap to culture and it grows fast, with a doubling time of 90–100 min under optimal conditions. It is amenable to genetic techniques, and genes can be modified or deleted within just a few days. Yeast cells can exist in a haploid and a diploid life cycle, which makes it easy to combine mutations by intercrossing. The How did budding yeast rise to prominence? The history of budding yeast as a model organism in research basically starts with the founding of the Carlsberg brewery in 1844 in Valby, an outskirt of Copenhagen. At the time, beer brewing was mainly conducted in small, local breweries, and the quality of beer between the different batches was often inconsistent. The Carlsberg Laboratory was founded in 1875 with the aim of developing scientific and industrial methods that would lead to a more consistent quality of beer. Of particular importance to the development of yeast genetics was a microbiologist named Emil Christian Hansen working at the Carlsberg Laboratory. Previous studies by Louis Pasteur on wine fermentation had indicated that different microorganisms could be isolated during the fermentation process, some of which were thought to be responsible for producing ill-tasting byproducts that spoiled the quality of the wine. Inspired by this information, Dr. Hansen discovered how to isolate and culture individual isolates of yeast clones in 1883. The isolation of a pure yeast strain was an important step for beer brewing, but it was also a major advancement for science. A second key development in yeast research came almost 50 years later, when Dr. Øjvind Winge  discovered that yeast cells can alternate between haploid and diploid life cycles; haploid cells can mate to form a diploid cell, which subsequently can undergo meiosis to form four haploid cells, each of which can be isolated for phenotypic analysis. He then showed that many genetic traits, such as colony morphology and fermentation markers, follow simple Mendelian rules. These discoveries laid the foundation for the field of classical yeast genetics.forward genetics. Forward genetic screens start with a given phenotype of interest, and large collections of mutants are then screened for mutations that specifically alter that phenotype. A well-known example is the study of eye color in Drosophila melanogaster by Thomas Hunt in the early 1900s [Classical yeast genetics refers to the genetic techniques commonly used before the dawn of molecular biology in the mid-20th century, when the molecular nature of genes had not yet been identified. Typically, genetic screens were aimed at identifying the genes responsible for a particular phenotype, a method referred to as ly 1900s . Wild-tyly 1900s , even thReverse genetics, as the name implies, is a research approach that follows the opposite direction of forward genetics; a gene is mutated and the effect of that mutation is studied by searching for a phenotype. Thus, forward genetics is phenotype-centered and is aimed at identifying the genes responsible for that specific phenotype, whereas reverse genetics is gene-centered and is aimed at identifying the phenotype associated with a given gene. The advent of molecular biology, which resulted both in a clear definition of the concept of the gene as well as development of techniques to modify gene sequences and to modulate gene expression levels in vivo, has greatly stimulated reverse genetics.Before the era of molecular biology, the classical problem was to identify genes associated with a given phenotype. Identification of these genes was often a laborious and pain-staking affair, which involved creating a library of mutants by mutagenesis, screening for mutants with an altered phenotype, and mapping of the mutations through genetic complementation studies. However, in the post-genomic era, this problem has been reversed; high-throughput genome sequencing projects have identified all the genes of numerous organisms, but the function of many of those genes remains unclear. For instance, the genome of budding yeast, arguably the best understood eukaryotic organism, contains approximately 6,000 genes, but the function of nearly 1,000 of them remains elusive .A major tool in a geneticist’s toolbox to identify the function of a gene is the analysis of its genetic network. Genes  do not work alone in the cellular processes they control, but rather function in intricate biological networks to fine-tune these processes in response to cellular and environmental stimuli. These biological networks tend to be robust, and removing one component often has minor consequences for cell viability because other components of the network can compensate for the lost function, an effect sometimes referred to as ‘genetic buffering’ ,7. HowevVarious forms of genetic interaction can be defined. For instance, a negative genetic interaction occurs when the combination of two mutations results in a worse phenotype than expected based on either single mutation alone. This may happen when two redundant genes function in parallel pathways to control a certain process, and combining mutations in these genes may cripple the process and lead to reduced fitness of the yeast cell, leading to a synthetic growth defect. In the example shown in i.e., gene A can partially compensate for loss of gene B. However, the cell can no longer carry out the biological process when both genes are lost simultaneously, leading to a much more severe phenotype than expected based on either single mutation alone  of the protein of interest to study relations between genes. In the example shown in One of the major consequences of mapping the yeast genome has been a shift in research from a focus on individual genes to a more global view of genetic networks . This wai.e., high enough for genetic interactions to occur, but low enough to permit a level of transcription that allows growth of the tet-repressible single mutants . However, one problem of these high-throughput genetic screens is that essential genes are underrepresented, because they are absent from the yeast deletion collection. This problem may be circumvented by using partial-loss-of-function mutations. For instance, one study made use of a promoter replacement approach, in which the endogenous promoter of 575 essential genes was replaced with the tetracylin-repressible tet promoter ,22. QuerCDC28 , which regulates the cell cycle in budding yeast [cdc28-4, which blocks cell cycle progression at late G1 under restrictive temperature), whereas others act late in the cell cycle . Screening for genetic interactions with these alleles may only give information on very specific functions of CDC28. Therefore, while informative, there are disadvantages to the use of temperature-sensitive alleles in genome-wide screens. Another popular loss-of-function mutant phenotype is temperature sensitivity, in which a mutant is viable at permissive temperature but very sick or inviable at non-permissive temperature. Most temperature-sensitive mutants display a partial loss-of-function phenotype at semi-permissive temperatures, allowing them to be used in SGA screens. Large collections of temperature-sensitive mutants of essential genes have recently been developed, paving the way for further exploration of the genetic interaction networks of essential genes ,24. Howeng yeast , specifiInhibiting essential genes, either by reducing their expression levels or by temperature-sensitive inactivation, can lead to a complete loss of all the functions of the protein. This is a potential disadvantage, because enzymes can have non-catalytic functions in addition to their enzymatic activity. For example, there are numerous reports in the literature of kinase-independent roles of kinases in signal transduction; just to name a few, ERK1/2, aurora, ZAP-70, FAK and Cdk1 all have kinase-independent functions in addition to their catalytic activity ,28,29,30cdc28-as1, in which the gatekeeper residue  has been replaced with a glycine, such that the Cdc28-as1 protein can be inhibited with the bulky ATP analog 1-NM-PP1 [A powerful chemical-genetic tool has been developed to specifically inhibit the catalytic activity of kinases without affecting their expression levels . This st1-NM-PP1 .CDC28 is an essential gene, and therefore it cannot simply be deleted to study its genetic network. However, titration of 1-NM-PP1 gradually inhibits growth of cdc28-as1 mutants, and sub-lethal doses of 1-NM-PP1 that reduce the growth rate of cdc28-as1 mutants have been used to screen for mutations that aggravate the phenotype of the cdc28-as1 allele [1 allele . This sc1 allele ,33,34. TBy far most of the high-throughput genetic screens have been performed under conditions of steady-state loss-of-function . While informative, the cells may adapt to the cellular conditions under which these query genes are inactive. One advantage of a chemical-genetic approach is that the consequence of just briefly inhibiting the kinase of interest can now be investigated. This will be particularly interesting when combined with specific cellular challenges, like nutrient starvation and DNA damage.Of course, chemical-genetic methods are not restricted to just mapping of genetic networks, but can also be used to characterize the biochemical functions of a given protein. Numerous studies have used chemical-genetic methods to identify novel protein functions, and here I will only highlight a few recent reports.in vitro kinase assays with Cdc28-as1 on an arrayed library of affinity tag-coupled yeast proteins, numerous potential Cdc28 targets were identified [in vivo [CDC28 was previously found to have extensive genetic interactions with DNA damage checkpoint genes [bona fide Cdc28 target, and that phosphorylation of Rad9 promotes checkpoint activity [Gatekeeper mutations are particularly suitable for identification of kinase targets. For example, by performing entified . Many of[in vivo . These snt genes , indicatactivity . Thus, ckin28-as1 gatekeeper allele to probe the function of Kin28 in this process. Kin28 is homologous to human CDK7, a cyclin dependent kinase that phosphorylates the C-terminal domain (CTD) of RNA polymerase II [1S2P3T4S5P6S7, and Kin28 preferentially targets Serine at position 5. Most studies have used temperature-sensitive alleles of KIN28 and found that inactivation of Kin28 has major consequences for transcription [kin28-as1 mutants have shown that the kinase activity of Kin28 plays a much more subtle role in transcription than expected based on results from studies that used temperature-sensitive kin28 alleles, showing that only a subset of genes is affected by Kin28 kinase activity [Gatekeeper mutations have also been used to study regulation of transcription. For example, several studies have used the erase II . The CTDcription ,40,41. Hactivity ,43,44. Ractivity .cdc7-as allele) revealed that Cdc7 kinase activity also promotes meiotic progression by enabling transcription of NDT80, a meiosis-specific transcription factor, and that Cdc7 is required for proper orientation of sister kinetochores [Another example of the identification of an unexpected function of a protein comes from a chemical-genetic study of Cdc7. Cdc7 is a kinase best known for its role in initiation of DNA replication . Howevertochores . As a final example, a recent variant of a chemical-genetic screen aimed to characterize the pathways that control cell-surface localization of the arginine permease Can1 . Can1 noTaken together, these examples illustrate the power of chemical-genetics in characterization of very diverse cellular processes.Streptomyces hygroscopicus, and is currently used as an immunosuppressive after organ transplantation [i.e., FPR1, TOR1 and TOR2 [Many compounds are known to have biological effects even though the molecular targets of these compounds often remain unknown. Identification of the cellular targets of bioactive compounds is a major bottleneck in the development of novel therapeutic agents . Buddingantation . Rapamycantation ,52, and and TOR2 ,54. Fpr1and TOR2 . Thus, bThe research field of chemical-genomics aims to identify functional relationships between specific genes and chemical compounds through systematic analysis of all genes in a genome . The devChemical profiling can be performed in several ways. Yeast collections can be spotted on plates harboring the drug of interest, and numerous drugs have been profiled using this method. For instance, pathways that mediate resistance to the DNA methylating agent MMS have been mapped this way . An alteSeveral bioinformatics tools have been developed to facilitate the identification of the mode of action of a given bioactive compound. A major tool in determining a drug’s mechanism of action is clustering analysis. First, the entire set of haploid yeast deletion mutants is treated with a compound of interest and the response of all deletion mutants is documented . Then, all mutants that display similar behavior in response to the compound are assigned into clusters, thereby generating a specific chemical-genetic profile; this profile is predictive of the drug’s mechanism of action. The search for a molecular target can be further narrowed by comparing the chemical-genetic profile of the drug to genetic interaction profiles of loss-of-function mutants. The reasoning behind this is that a loss-of-function mutation in a gene encoding the target of an inhibitory compound models the primary effect of the compound , and therefore the genetic interaction profile for the target gene resembles the chemical-genetic interaction profile of its inhibitory compound . Thus, oin vitro determination of a drug’s biochemical activity [PDR1, which is involved in transcription of multidrug resistance genes such as the pleiotropic drug pump PDR5. Other multidrug resistance genes were involved in regulation of membrane lipid composition and vesicular transport of proteins. However, these high-throughput drug profiling screens also show that drugs often display a much more complex behavior in a living cell than expected from activity . One comactivity . Furtheractivity , althougactivity . Finallyactivity . This stDue to these issues, not all high-throughput screens in haploid deletion collections may reveal the mechanism of action of a drug. However, these studies still provide insight in cellular strategies against chemical stress , and intFrom the initial isolation of pure budding yeast strains from wort in the late 1800s to modern high-throughput automated genetic screens, yeast genetics has provided insight in numerous biological processes, including regulation of the cell cycle, autophagy, cell polarity, and repair of DNA damage. Recent developments in the field of chemical-genetics have provided tools to further dissect the genetic network of the cell, and yeast will be the first eukaryotic organism in which all genetic pathways have been mapped. Furthermore, studies in budding yeast will have clinical relevance, because chemical-genetic screens and chemical-genomic profiling are becoming increasingly important tools for drug discovery.HSP90, which encodes the major molecular chaperone in yeast, genetically interacts with very sets of genes under normal growth conditions than when cells are challenged with environmental stress [By far most of the genetic interaction studies have been performed under optimal growth conditions. However, under conditions of cell stress, the cell may rely on very different genetic pathways to promote cell viability, and it may rewire its biological networks to deal with the altered conditions. For example, l stress . This isl stress ,76,77. U


Karst collapse column is a serious geological disaster in China’s coal mines. There are various karst collapse columns in coal mine areas, such as Huainan, Huaibei, Xingtai, Lu’an. And they have seriously affected mining safety and geological environment. The present research is focused on analyzing subsidence mechanism and dynamic collapse process. Based on mechanical analysis of thin plate theory, a detailed model of collapse column slipping and bending fracture is constructed to gather the critical conditions of the collapse column roof. The sensitivity parameters analysis shows that both the radius and roof thickness of cave have a great influence on the sliding instability and bending fracture. Meanwhile, the buried depth also affects bending failure. The discontinuous deformation analysis (DDA) method is used to simulate and analyze the collapse process. The numerical results indicate that the stability of inverted funnel collapse column is dominated by the bending stress of roof strata. The movement of columnar collapse column is mainly caused by sliding instability. However, the funnel collapse column is relatively stable, and does not change in the same condition. The displacement field analysis shows that the collapse range of inverted funnel collapse column is obviously larger than that of columnar collapse column, in which its maximum displacement is approximately 1.5 times that of the columnar collapse column. There is no large area collapse on the upper part of the funnel collapse column, and the block system is relatively stable. The principal stress field analysis proves the above results. There are many collapse columns in mining areas in China. And their morphologies are greatly diverse . Due to The collapse columns cause damage not only to the mining industry, but also to railways, highways and karst reservoirs. Therefore, the karst collapse columns have been widely investigated –8. ScholThe existing researches mainly focus on the forming mechanism, water inrush and numerical simulation of the inverted funnel collapse column. However, most of them are based on the theoretical or simplified model. In fact, the collapse mechanism of collapse column can be well studied by numerical calculation. At present, the simulation is mostly based on finite element method or finite difference method, and it is difficult to obtain the deformation field and subsidence mechanisms in the different morphology collapse column. The DDA can simulate the formation mechanism of collapse columns –20.However, one of the most prominent features of DDA in the calculation process is that it needs open and close iterations, that is, repeatedly falling to find the locking position. As a result, DDA is very difficult to complete excavation calculation. Therefore, in this paper, the high efficiency excavation calculation of DDA is realized by changing the calculation method of DDA. In order to analyze the collapse mechanism of different morphologies of collapse columns, this paper establishes a model of collapse column slipping and bending fracture. Meanwhile, based on DDA simulation, the dynamic evolution mechanism of various collapse columns is investigated in detail.The collapse column is a special morphology of collapse in coal mine fields. Due to the concealed characteristics of collapse columns and their complexity, it is important to understand the existing morphologies and study the failure law. The collapse columns in northern China are mostly distributed in the development zone of karst spring. It is mainly caused by the collapse of overlying strata in the Ordovician karst cave. Some collapse columns show an inverted funnel shape, others present cylindrical morphology and funnel shape. Their horizontal sections are mostly circular or oval –22. UndePeng et al.  uses theThe key performance measures mainly include the critical conditions of slip instability and bending failure of collapse columns. In order to analyze the collapse parameters in a more comprehensive way, the cave radius, cave roof thickness and the cave depth are selected as the sensitive parameters to analyze the unstable failure of the collapse column.After the dissolution of rock mass, the cave roof will be destroyed. Since the horizontal sections of the three morphologies of collapse columns are mostly round or oval, it is assumed that the top plate of the cave is a peripherally fixed thin plate, as shown in Vertical stress of karst cave roof :σh=γH,,3), the In order to obtain the sliding force around the entire roof model, integral for Eq :T=∫0L∫HThat is:The weight of the cave roof and its overlying rock is:2 is cross-sectional area, m2; h is thin plate thickness, m; L = 2πr is cave circumference, m; r is cave radius, m.Where s = πrWhen the load on the karst cave roof exceeds the limit slip force on the whole periphery, the failure will occur:The slip instability condition of the collapse column can be obtained:From Eq , it can H = 0~1000m, h = 20m, φ = 30°, c = 2.4MPa, γ = 25kN/m3 into Eq , and the Eqs  and 13)13) are oFrom the elastic mechanics , it is kAssuming φ is 0, the equations of bending moment and transverse shear force of thin plate in polar coordinates are obtained from elastic mechanics:By further transformation, the internal force equation is obtained:Because the roof is assumed to be boundary clamped, the upper load is γ H, and the radius is r, thus the boundary conditions can be obtained:The deflection equation is:The moment equation is obtained as follows:R = r into Eq  of a specific point . And the displacement  of any point  is as follows:The DDA method is characterized by inherent discontinuity, measured displacement, strain or stress as input data. Numerically, the method is to use a set of inequality constraints to minimize the least squares. The input consists of measured data and the output is the block sliding, and joint opening, which are real measures of safety and failure. In the DDA method, rock systems can be regarded as aggregate blocks cut from discontinuous surfaces. The strain of each block is constant, and the deformation of each block is described by the rigid body translation displacement , (εxεyγxy) are the strains of the block; i represents the i-th block.Where Formula (27) can be given in the following form:The simultaneous equation is obtained by minimizing the square differences or the errors Ψ. The single block is connected to each other by the contact between the blocks and the displacement constraints on the single block and forms a block system. These kinematic requirements and constraints have the form:p>0 is a large positive number:The penalty term(28) is added to Ψ to impose the kinematic constraints, where Suppose there are n blocks in the defined block system, and the form of the simultaneous equilibrium equation is:The i-th row of Eq  consistsi is a 6×1 vector representing the loading on block i. The elements of 6×6 submatrix Kij are determined by:where FThe application of Eq  in numer3D is widely used in analyzing break, so it is compared with DDA in detail as follows:At present, FLAC(a) Comparative analysis of stress fieldThe overall distribution of the stress field obtained by the DDA simulation is uniform, and the stress concentration is in the local area, the whole distribution of stress field simulated by FLAC is very smooth and uniform. In the whole simulation process, the stress release of the block system calculated by the DDA method is larger due to the large area collapse, so that the overall stress value is reduced and the stress concentration is reduced. During the FLAC simulation, there is only stress release near the roof and floor of the mining face. However, because the calculation unit has not collapsed, the gravity of this part of the element and its own stress aggravates the stress concentration of the deep roof and the two sides of the working face, making the overall analysis too conservative. It shows that the simulation results obtained by DDA method are more accurate.(b) Comparative analysis of displacement fieldThe maximum displacement of the DDA method varies greatly, which is consistent with the roof caving characteristics. However, the maximum displacement of FLAC does not change much compared with the displacement of other monitoring points. During the whole calculation process, DDA method give the three-zone distribution of overlying strata caused by face mining, the displacement field is not continuous. FLAC cannot give the distribution of three-zone the calculated displacement field is continuous, and there is no sudden change, the simulation results are quite different from the actual strata collapse.3D. The FLAC and UDEC can solve geotechnical and mining problems, such as analysis of large deformation of soft rock roadway and roof collapse in mining engineering, etc, and the roof failure of collapsed column is the problem of collapse, so we chose the above method for comparison. Although the above method can assume that the object of study is independent block element, these block elements have the shape limitation, their dynamic analysis is not very complete, therefore they cannot solve the structural plane failure problem. DDA can also be used in mining engineering to simulate the roof separation, surrounding rock failure, top coal caving and dynamic response of mining through coal seam, so the DDA method is also selected. However, one of the most prominent characteristics of DDA in the calculation process is that it needs open-closed iteration, that is, repeatedly falling to find the locked position. Therefore, DDA is very difficult to complete the excavation calculation, and still has the advantage of improvement.At present, the state-of-the-art methods to study collapse of collapsed columns are DDA, UDEC and FLACIn order to realize excavation simulation by two-dimensional DDA, it is necessary to know the whole calculation process. One of the most prominent features of DDA in the calculation process is that it needs open and close iterations, that is, repeatedly falling to find the locking position. As a result, DDA does not have excavation function. To analyze the settlement mechanism of karst collapse column with numerical method, the excavation method of DDA program is put forward. Because of the discreteness of DDA calculation unit, it can also be deleted directly in the calculation process, so as to realize excavation simulation in the true hypothetical sense.The first problem to consider is the judgment of contact. If the calculation unit is suddenly reduced, there should not be an error in the contact judgment. However, because the geometric information of the computing unit is stored in the main program, the geometric information of the unit is an indicator matrix. Therefore, the indicator matrix needs to be modified when deleting the corresponding block. The physical parameters are also stored correspondingly in the program, and the corresponding storage matrix of physical parameters should also be modified. In the last step, the contact displacement is also passed to the time step, the corresponding modification store information and all contacts of the deleted unit will be deleted. The stress matrix, displacement matrix, velocity matrix passed to the time step will also be modified to adjust block number and delete record information to delete block unit. After modification, the program enters contact judgment. Then the contact judgment is finished, the contact transformation is carried out. At this point, the computing unit of the whole program is no longer n, but n-1 or a lot of cuts. At this time step, the main program will do the calculation for the unit that has not been deleted, until the next time it is necessary to delete the unit to make a new change. This process is written as a separate subroutine and added to the front end of the main program, which can complete the deletion of the computational block elements, and realize the excavation simulation.To verify the accuracy of the proposed method for computing excavation, taking the centroid of three blocks as observation points, the variation of displacement component is analyzed. The stability time of the three blocks is set to 2s, then the block 2 is suddenly removed after 2s. The time removing block is considered 0s, that is, there is no force on the other two blocks in the process of removing the block, and there is no friction to do the work. Then the displacement of block 3 is observed. Using the modified DDA program calculation, the calculation results are depicted in After the block system is stable, the block 2 is suddenly removed, and block 3 should be in free falling motion. The formula for calculating the displacement of free falling body is as follows:During the program calculation, the step was 353 steps at the end, and the block 2 was deleted in 2.004s, when 732 steps are calculated, block 3 collides with block 1. The numerical calculation and theoretical calculation of displacement at No.3 observation point are shown in From the To investigate the subsidence mechanisms and the dynamic collapse process, a discrete numerical method, discontinuous deformation analysis (DDA) is used to study three different morphological collapse columns.A representative area of the Xieqiao coal mine is used as the simulation object and simplified into a two-dimensional plane strain block model along the roadway. The model length is 1000m and the height is 400m. Both the bottom and the side of the model are fixed. In order to simulate the gravity of overlying strata, the top surface of the model is approximately uniformly distributed, and the collapse column diagram is shown in According to the geological data, the average dip angle of rock joint surface is 4°, so the random joint inclination angle is 86°, the average spacing of joint is 11m, and the average length is 200m. Except for the different morphologies of the collapse columns, the model establishment parameters and calculation parameters are the same, and the model hollowing length is designed to be 150m. The mechanical parameters of rock strata is shown in Tables The collapse column model found in coal mines is shown in The calculation time step is set to 4000 steps, allowing maximum deformation rate of 0.1%. The simulation results are shown in Figs The internal structure of the columnar collapse column does not change much before it becomes the separation strata. As the dissolution time approaches, the karst caves continue to expand, resulting in stress concentration effects, accompanied by separation strata. When the columnar collapse column is separated, the immediate roof of the cave column loses its supporting capacity by long-term erosion. While the overlying rock mass rupture degree increases, the stability of adjacent strata gradually decreases. The roof of the karst cave is also affected by the internal joints and faults. The rock mass is prone to collapse in the direction of easy sliding. With the sharp increase of roof load, the instability failure will occur when the load of the cave roof exceeds the maximum sliding force around the surrounding rock . At the When the funnel collapse column is in the same operation step, there is no separation strata. The internal structure of the collapse column does not change, and there is no large-scale collapse deformation from beginning to end. Only the rock mass in the upper part of the cavity has small displacement deformation, and basically no deformation occurs, as shown in The displacement field results are shown in Figs According to the above analysis, the collapse of the inverted funnel collapse column shows the following characteristics: the collapse column mass is affected by fault space position to have a large area of collapse. The instability collapse block basically occurs in the collapse column, and few occurs near the collapse column. The left side of the collapse column block is more unstable than the right side, because of the influence of 86 degree random joints. At the same time, the overall collapse shows a tendency to tilt to the left.As shown in From the displacement curve of the bottom row of monitoring points in the direction of the y axis, it can be known that the inverted funnel collapse column has a large degree of collapse with the exception of a few monitoring points outside the cavity. The maximum subsidence value of inverted funnel collapse columns and columnar collapse columns is 9.5 times and 6.0 times greater than that of funnel collapse columns . The disThe principal stress line of the block in the model is shown in At the initial stage of collapse, the immediate roof above the karst cave can be regarded as the key stratum, and the blocks are interlocked with each other, which weakens the instability and deformation ability between the blocks. With the extension of dissolution, the karst cave becomes larger, the degree of rupture of the overlying rock mass increases, but the stability of the collapse column decreases. At the same time, due to the influence of spatial joints, rock mass collapse occurs in a direction conducive to sliding. When the collapse column fault presents a central axis symmetry, the roof arch collapse position appears in the center of the roof arch. During the failure stage of the collapse column, the energy caused by stress is suddenly released, and it leads instantaneous collapse and causes the formation of collapse columns. When the load on the roof of the cave exceeds the limit slip force on the whole periphery, that is, Δ<1, it will be destroyed. The simulation results of the columnar indicate that the final collapse failure degree is small, and the collapse failure process is similar to slip failure.σmax≥σt, the roof will be broken, presenting a bending deformation. As shown in Figs In the initial stage of the collapse column, the immediate roof plays a supporting role due to the existence of the direct roof of the inner hollow area. Therefore, the top of karst cave is in a stable state. As the erosion of groundwater increases, the collapse column rocks are gradually washed away. Then the stress concentration is formed, which accelerates the occurrence of the separation strata. The collapse occurs inside the collapse column, and the upper strata presents bending deformation. When the roof of the karst cave reaches the critical failure condition, that is, The failure law is different due to different morphologies of collapse columns. The reasons are listed as follows: the columnar collapse column is similar to the cylinder; in the geological tectonic movement, the stress concentration area is reduced. Compared with the inverted funnel collapse column, the collapse height decreases, the collapse value is obviously less than the inverted funnel and the pressure is weakened. The stress concentration distribution area and stress value are weaker than those of the inverted funnel collapse column.Although the morphology of the funnel collapse column is irregular, its damage is the least. The possible reason is that the stress of this form of collapse columns is approximately parallel to the stress direction of tectonic stress or that the angle is small, which is least influenced by the tectonic stress. The stability of the block system is the greatest.In the article, different morphologies of collapse columns are investigated. The collapse model of collapse columns is established, and the dynamic collapse process of collapse column with different morphologies is simulated by DDA. The main conclusions are obtained as follows:(1) Based on the existing detection results, the collapse columns are classified into three morphologies: inverted funnel, columnar and funnel. The critical conditions of sliding instability and bending fracture of the collapse column roof are established by analyzing the model of the collapse column.(2) Combined with the slip and bending failure instability conditions, the parameter sensitivity analysis was carried out. The main sensitive parameters of bending fracture instability are different from those of slip instability conditions. Besides the cave radius and the cave roof thickness, the parameters which have a significant influence on the instability are also affected by the buried depth.(3) The discontinuous deformation analysis (DDA) results indicate that the collapse process of the inverted funnel is mainly caused by bending fracture; the columnar collapse column mainly shows slip instability; and the stability of funnel collapse column is the best.(4) The displacement field analysis shows that: the subsidence displacement of inverted funnel is the largest. The monitoring subsidence displacement is more than 1.5 times that of the columnar, and its stability is the worst. The cave upper of funnel does not show subsidence, and the upper strata forms an arch structure, so the whole tends to be stable. The above results prove the rationality of DDA computing method.


Didemnum vexillum. We analysed single zooids by whole genome amplification and genotyping-by-sequencing and obtained genotypic information for more than 2,000 loci per individual. In the prevalence study, we analysed nine colonies and identified that 44% of them were chimeric, composed of 2–3 different genotypes. In the fusion experiment 15 intra- and 30 intercolony pairs were assayed but one or both fragments regressed and died in ~45% of the pairs. Among those that survived for the length of the experiment (30 d), 100% isogeneic and 31% allogeneic pairs fused. Fusion was unlinked to global genetic relatedness since the genetic distance between fused or non-fused intercolony pairs did not differ significantly. We could not detect any locus directly involved in allorecognition, but we cannot preclude the existence of a histocompatibility mechanism. We conclude that chimerism occurs frequently in D. vexillum and may be an important factor to enhance genetic diversity and promote its successful expansion.The formation of chimeric entities through colony fusion has been hypothesized to favour colonisation success and resilience in modular organisms. In particular, it can play an important role in promoting the invasiveness of introduced species. We studied prevalence of chimerism and performed fusion experiments in Mediterranean populations of the worldwide invasive colonial ascidian  Genetic heterogeneity within organisms represents an evolutionary challenge, as several potential risks and advantages varying among taxa have been suggested but rarely tested4. Among marine invertebrates, chimerism and allorecognition have been studied in the main groups with colonial or modular species: Cnidaria6, Tunicata7, Porifera9 and Bryozoa10. These organisms show highly polymorphic histocompatibility systems which determine the output of conspecific interactions12. Chimeras may last for the lifetime of the colony13 or only for a few days, depending on the compatibility of the contacted colonies11. Besides the increase of genetic variability, chimerism in marine invertebrates can provide multiple benefits  but also significant disadvantages 14.Natural chimerism is a widely documented phenomenon occurring in multiple phyla of protists, fungi, plants and animals, including chordates such as ascidians and mammalsBotryllus schlosseri15. In this system, a single gene locus with multiple alleles determines the outcome of the colony contacts. Colonies are usually heterozygous at this locus and they fuse when sharing at least one allele16, which means fusion between colonies occurs when they are genetically similar. Following fusion, the somatic and germ-line components of the composite unit may compete with variable outcomes20. In B. schlosseri there is no evidence of an improvement in growth rates, reproduction or survivorship associated to chimerism, so other ecological or evolutionary advantages should favour chimerism in this species21. Botryllid ascidians possess a common vascular system that mediates the fusion/rejection outcomes of intercolony contacts. Other colonial ascidians , however, lack colonial blood vessels. Without vascular connections, the scope for exchange of stem cells and cell lineage competition is greatly reduced, which seemingly reduces the potential for strict colony specificity and favours more indiscriminate fusion between colonies23.Within tunicates, chimera formation in botryllid ascidians is the most studied system, particularly in 24, chimerism may be boosted. Increased fusion rates among different colonies could result in higher genetic diversity and richer gene expression patterns, promoting the invasiveness of the species and turning the disadvantage into an advantage for the founder population13. Moreover, each genotype from a chimeric colonial individual may adapt better to different conditions in changing environments, enhancing colony survival19.In invasive populations where the low genetic diversity caused by the founder effect is initially a disadvantageDidemnum vexillum is a worldwide invasive species that has colonized most temperate regions . It can form large colonies on either natural or artificial substrates, and it can overgrow other invertebrate species such as commercial bivalves in aquaculture facilities, causing important ecological and economic loses28. D. vexillum can form chimeric colonies29 and it can also reproduce asexually by natural or human-mediated fragmentation, which is probably a major enhancer of its spread26. Its reattachment capability and fragment viability contribute to its invasive success31. Chimerism has also been suggested as a driving mechanism of the species’ remarkable invasiveness33.The colonial ascidian 36. Chimeras can also be induced experimentally by putting in contact colonies, either growing edges or cut surfaces37 and examining the outcomes in the laboratory. Field and laboratory studies should ideally be complemented with genetic analyses to demonstrate intracolony heterogeneity or to characterize interacting partners. Chimeric colonies can be detected by analysing different fragments of the same colony using several genetic techniques. One of the most used methods is microsatellite genotyping, applied to ascidians42, cnidarians43 and sponges9. This kind of studies may underestimate the prevalence of chimerism, as it can only be detected when more than 2 alleles are found at a given locus38. Other genetic markers used include Cytochrome Oxidase subunit I sequence data45 or randomly amplified polymorphic DNA–PCR (RAPD–PCR) band patterns46. The detection of chimeric individuals may be improved with more markers, and whole-genome scanning techniques such as genotyping-by-sequencing (GBS) generate large amounts of genetic markers which can be applied to non model organisms47. In samples with scarce DNA material, a whole genome amplification (WGA) step is needed to obtain enough DNA48. The combination of WGA and GBS has been shown to reliably estimate multilocus genotypes in D. vexillum for clone detection and population genomics25 and can be an efficient and precise tool to assess chimerism.The study of chimerism in ascidians has relied on different techniques. Monitoring of colonies in the field allows the assessment of natural fusion ratesD. vexillum and combine field surveys and experimental fusion tests with WGA-GBS genomic analyses from single zooids. Our objectives are (i) to report the prevalence of chimeric colonies in an introduced locality, (ii) describe the fusion/rejection behaviour between colony pairs, (iii) analyse the relation between colony fusion capability and genetic distance genomewide, and (iv) scan the dataset for candidate loci mediating colony fusion.In this study, we assess chimerism in Didemnum vexillum: (a) the identification of chimeric individuals in the wild, and (b) fusion experiments.Two different approaches were followed to study the chimerism in D. vexillum in an enclosed area49,50. Nine colonies growing on commercial oysters were sampled. One central fragment and four peripheral fragments of 1 cm2, separated each other by at least 5 cm, were cut from each colony to determine the prevalence of chimeric colonies in this population . This system represents a favourable environment for 2 was cut into 7 pieces of 1–2 cm2 with a scalpel. One fragment was preserved in 96% ethanol for DNA extraction, two were paired with each other (intracolony pair), and the other 4 were paired to another colony fragment of the same set (intercolony pairs). The fragments used in this experiment were peripheral whenever possible, as it is described that they reattach faster than central fragments29. In total, there were 45 pairs, corresponding to 5 intracolony and 10 intercolony pairs for each set. All manipulation was done in the laboratory within hours of collection, and taking care to keep the colonies submerged in freshly collected lagoon water at all times.In the second approach, a colony fusion experiment was carried out at the Venetian Lagoon with 3 sets of 5 colonies each Fig. . This loD. vexillum in the region51. The slides were photographed twice a week to track colonies’ growth and fusion behaviour.Each colony pair was fixed on a glass slide using cotton threads, with contacting cut edges to trigger a fast fusion/non-fusion outcome. The slides were submerged in vertical position at 2 meters depth at the ISMAR docks located in the ancient harbour of the Arsenal of Venice  where a genotyping-by-sequencing (GBS) protocol was carried out. DNA was digested by PstI restriction enzyme and a paired-end sequencing of 2 × 125 bp fragments was performed in an Illumina HiSeq2500 platform.A thorax of a single zooid from each sample was dissected under the binocular and used for DNA extraction. We used an individual zooid to make sure we had a genetically homogeneous unit, and selected the thorax to avoid contaminating DNA from gut contents and to prevent a mixture of somatic and reproductive tissues. To get enough DNA from single thoraxes, a whole genome amplification (WGA) procedure was carried out with the REPLI-g® Single Cell kit (Qiagen) following manufacturer’s protocol except for the use of 1.5 µL of polymerase instead of the recommended 2 µL52 and following the same pipeline described in a previous study25. In short, the sequence quality filtering included the elimination of lower quality last bases by truncation and removal of reads with a low average Phred score threshold. The loci identification was divided in two steps: per sample and globally. First, sequences were analysed separately by sample, grouping identical reads into sequence variants and then into loci by pairwise comparisons. Second, a global locus and allele identification was performed to construct the loci dataset. The last main stage was the loci filtering where loci with alleles that could be indel variants, deeply sequenced loci and loci with more than two alleles per sample were removed. After these filters, loci shared by less than 70% of the samples were also deleted. The samples of Ebro Delta and Venice were analyzed separately to get two final loci datasets since they are significantly genetically differentiated populations25.Sequence quality filtering and loci identification and selection was carried out using the GIbPSs toolkit25. The PSG was calculated in R53, plotted using ‘ggplot2’54 and used to identify unique multilocus genotypes. Pairs of samples with PSG higher than 90% were considered the same genotype25. The genepop file was read into R using the ‘adegenet’ package56 and used to calculate the pairwise genetic Prevosti distance in ‘poppr’58 corrected by the exact number of loci shared by each pair.For each loci dataset, a table of haplotypic genotypes  and a genepop file were exported from GIbPSs. The table of genotypes was used to get the Percentage of Shared Genotypes (PSG), defined as the percentage of identical genotypes among shared loci53.The corrected Prevosti genetic distances between fused genotypes from the prevalence study and fused genotypes from the fusion experiment were compared with a Mann-Whitney-Wilcoxon test. For the fusion experiment, the corrected Prevosti distances between fused and non-fused intercolony pairs were compared with a Mann-Whitney-Wilcoxon test. The time to fuse between fused intercolony pairs and fused intracolony pairs was also compared with a Mann-Whitney-Wilcoxon test. A Pearson correlation coefficient between time to fuse and genetic distance was calculated for all fused pairs. All the analyses were run using RCiona intestinalis  and C. savignyi  available on the Ensembl genome database59 (http://www.ensembl.org/index.html). Only hits with an E-value below 10–2 were considered.For each locus of the fusion experiment dataset with less than 20% of missing data, the number of shared alleles (ranging from 0 to 2) of each surviving intercolony pair was calculated. Among them, we selected 1% of the loci with the highest absolute difference in shared alleles between fused and non-fused pairs. Consensus sequences of each selected locus were aligned using Blastn searches to the genomes of An average of 2,510,418 raw reads per sample were obtained in the prevalence study at the Ebro Delta and 79,1% remained after the sequence quality filtering stage. A total of 69,600 loci were found among the 45 samples of which 2,145 polymorphic loci with 6,602 alleles were shared by at least 70% of the samples  of the loci, and pairs of samples with different genotypes (48.07 ± 0.07%) , presented a non-fusion front all along the contacted region Fig. . The othThe comparison between the mean corrected Prevosti distances of fused intercolony pairs (0.231 ± 0.002) and non-fused intercolony pairs (0.239 ± 0.003) showed no significant difference  Fig. . The timFrom the total 2,597 loci of the fusion experiment dataset, 1,456 showed less than 20% of missing data. A total of 15 loci (1%) were considered as possible candidate loci for mediating colony fusion Fig.  based onCiona intestinalis genome, we found 3 hits although with low query cover and identity values. One was aligned to an intron of a predicted gene corresponding to an uncharacterized protein from chromosome 2. Another was aligned to an exon of a predicted gene coding for an uncharacterized protein from chromosome 8. The third locus was aligned to an intron of the gonadotropin releasing hormone receptor 1 (gnrhr1) gene from chromosome 3  technologies such as GBS47 provide a tool to obtain large number of loci from species lacking a reference genome, producing reliable data to identify chimeras. In samples with scarce genetic material or species with reduced individual size, the combination of a WGA method with GBS proved robust and effective25 to obtain a large panel of loci that can be used to assess chimerism. In previous studies, the Percentage of Shared Genotypes (PSG) has been used to identify clones25 and in the present study it proved to be a fast and easy tool to detect chimeras. When zooids of the same genotype were compared, ca. 97% of the loci had the same genotypes and the 3% discrepancies can be explained by amplification and sequencing artifacts which could not be filtered during the bioinformatic analysis61. PSG dropped to less than 50% when colonies with different genotypes were compared. Note here that the relevant point is the existence of such a marked gap in the percentage of shared genotypes, as the precise mean PSG values may differ between studies depending on technical aspects 61.Most of the genetic methods used for chimera detection underestimate the actual prevalence of chimerism due to the polymorphism of the markers usedBotryllus schlosseri42, 1.9% in Botrylloides nigrum45, 1% in Perophora japonica44, from 3 to 61% in Diplosoma listerianum23, and from 17 to 48% in New Zealand populations of D. vexillum62. Our 44% prevalence of chimerism in the invasive population of the Ebro Delta is in the upper range of values found in other species, but low within those found in introduced populations of D. vexillum. In cut surface fusion experiments, an 80% of chimerism has been reported using colonies from the introduced area in New Zealand against only 27% in the native range32. Our experimental results in the Venetian Lagoon (31% fusion) are closer to the values found in the native range and far from those reported in New Zealand. This discrepancy may be explained by the different origin and genetic composition of the introduced populations in Europe and New Zealand25. More populations of this species should be studied to assess the extent of chimerism associated to introduced populations.Natural chimerism has been found in several compound ascidians, but its prevalence varies considerably between and within species, from 0.5 to 39% in 63. Didemnid ascidians such as D. listerianum and D. vexillum lack a colony-wide vascular system and zooids are only connected by the tunic, greatly decreasing the exchange of cells and hence the costs of chimerism. This type of colonial species may have a reduced or even absent allorecognition system, favouring more indiscriminate fusion between different colonies and making chimerism a more common condition46. Similarly, the relatedness of fused colonies also varies among species. In Botryllus spp. fusion can occur only between kin colonies39, while in D. listerianum46 and D.vexillum (present study) no genetic control mechanism has been detected and fusion takes place between non-related colonies. However, a study on D. vexillum described accumulation of diverse cell types in the tunic adjacent to allogeneic fusion areas, mostly phagocytes and morula cells64. Thus, it is likely that these cell types mediate the recognition reaction in this species, and that some limited exchange of tunic cells occurs between interacting colonies.In species where a vascular system allowing an exchange of cells within colonies exists, a well-developed allorecognition system may reduce fitness costs of chimerism associated with somatic and germ-cell parasitismHydractinia symbiolongicarpus four types of allorecognition phenotypes can be observed: fusion, rejection and two types of transitory fusion11. The stony coral Stylophora pistillata shows eight types of allorecognition reactions between kin colonies65. In the ascidians Botryllus schlosseri and Diplosoma listerianum, many different possible outcomes have been described66. In colonies of the ascidian Trididemnum solidum observed in natural environments, fusion has been observed after weeks or months of non-fused contacted margins from different colonies34. In a previous study on D. vexillum, highly dynamic interactions were found when allogeneic colonies came into contact33 and in many cases, fusion was followed by active growth away from fusion zones. The fragments resulting from this retreat showed predominantly segregated genotypes and chimeras were therefore transient in a scale of a few (10–12) days. In the present study, D. vexillum showed four different allorecognition phenotypes: complete fusion, partial fusion with some non-fusion front formation, rejection with stable non-fusion front formation, and rejection with regression. Some interactions were highly dynamic showing a combination of outcomes as the colonies contacted multiple times with a different outcome each time. However, fused colonies remained so until the end of the observation (30 days). Similarly, the finding of large chimeric colonies in the field (Ebro Delta) indicated that chimeras were not just transient, but stable entities. The external appearance of the colonies of D. vexillum and their behaviour in fusion experiments are different when comparing the photographic material from the present and previous studies33. We kept our experimental colonies in the natural environment of the lagoon while these previous studies were made under laboratory conditions. In the present work, colonies looked thicker and less transparent, suggesting that fragments may grow healthier under natural conditions.The most common outcomes described in fusion experiments among fragments of colonial species are fusion or rejection. However, more complex patterns have also been reported in most species. For instance, in the hydrozoan 47. Depending on the enzyme and species used, the studied loci may be differentially distributed among coding and non-coding regions67. For D. vexillum and using the PstI restriction enzyme, only 20% of the searched loci had a blast hit in Ciona genomes. Moreover, the query covers were small (~30%) and the E-value large suggesting that most of the analysed loci in this species will be located in non-coding regions and thus hard to identify in distant genomes. Thus, although we could not apparently detect any locus either directly involved in histocompatibility or linked to the relevant ones that could reveal a genetic control of the fusion/non fusion mechanism, we cannot discard that some of the identified loci are associated to highly specific regions mediating allorecognition. Moreover, the contrasting allorecognition phenotypes in fused isogeneic and allogeneic colonies  might be indicative of a histocompatibility mechanism mediating fusibility in this species.Among our large loci dataset, none of the loci that we considered as possible candidates to mediate fusibility blasted to known genes with functions relevant for the allorecognition mechanisms. GBS produces huge numbers of loci randomly distributed through the genome of the target speciesD. vexillum shows multiple advantageous biological traits that make it an aggressive invasive species that has colonized temperate regions worldwide. It has a rapid growth rate, produces large numbers of short-lived planktonic larvae and lacks significant predators (but see69). The importance of chimerism in adaptation and invasiveness can differ between species13. In the non-invasive but worldwide distributed populations of the branching coral Pocillopora damicornis, high levels of chimerism were found in extremely variable and highly impacted habitats70, suggesting chimerism may be involved in the success of adaptation of the species. In corals, chimerism has been suggested as an evolutionary mechanism of resilience and adaptation to global climate change impacts6. Similarly, chimerism has also been proposed as a driving factor in invasion success44. In colonial species with high growth-shrinkage dynamism and high fusion rates, chimerism may be a key aspect of the fragment dynamics and needs to be assessed for an efficient management62. Moreover, in the ascidian B. schlosseri, invasive populations seem to show higher levels of chimerism than in their native range13, and the same has been suggested for D. vexillum32. Chimeric colonies may show the ability to shift to advantageous genotypes in changing environments4. The invasive populations of D. vexillum assessed in the present study, the Ebro Delta and the Venetian Lagoon, show high levels of prevalence of chimerism and fusion rates, respectively. Thus, the role of chimerism in the success of the worldwide expansion of D. vexillum cannot be underestimated.Supplementary dataset S1Supplementary dataset S2Supplementary dataset S3