The effect of crosslinker, functional monomer and extraction on the long-term performance and degradation of molecularly imprinted polymers was investigated through adsorption studies, NMR, SEM, TGA and BET. l-phenylalanine methyl ester imprinted polymers were studied to reveal performance loss over 100 adsorption–regeneration cycles. Furthermore, crosslinker and functional monomer specific reversible and irreversible decomposition of imprinted polymers as a result of adsorbent regeneration were investigated through adsorption studies, electron microscopy, N2 adsorption and thermogravimetric analysis. A decomposition mechanism was proposed and revealed using NMR spectroscopy. Solutions to avoid or overcome the limitations of the most common crosslinkers, functional monomers and extraction techniques were proposed and experimentally validated.Molecularly imprinted materials are man-made mimics of biological receptors. Their polymer network has recognition sites complementary to a substrate in terms of size, shape and chemical functionality. They have diverse applications in various chemical, biomedical and engineering fields such as solid phase extraction, catalysis, drug delivery, pharmaceutical purification, (bio)sensors, water treatment, membrane separations and proteomics. The stability and reusability of molecularly imprinted polymers (IPs) have crucial roles in developing applications that are reliable, economic and sustainable. In the present article the effect of crosslinkers, functional monomers and conditions for template extraction on the long-term stability and reusability of IPs was systematically investigated. Adsorption capacity, selectivity, morphology and thermal decomposition of eleven different  Building blocks are allowed to self-assemble with the template, and the functional groups are held in position via crosslinking polymerisation. Subsequent removal of the template by solvent extraction or chemical cleavage leaves binding sites that are complementary to the template in terms of both topography and chemical functionality. Numerous applications of IPs have been reported such as catalysis, drug delivery and purification, sensors, water treatment, membranes and proteomics.7 Apart from the single-use analytical devices most of the chemical and engineering applications require regeneration of the IPs to improve efficiency, economics and sustainability.Molecular imprinting is a technique to design robust molecular recognition materials able to mimic natural recognition entities, such as antibodies and biological receptors. The preparation and application of molecularly imprinted polymers (IPs) have been thoroughly reviewed.12 the sole investigation into the stability of IPs was reported by Svenson et al. for a theophylline imprinted methacrylate based copolymer.13 It was demonstrated that exposure to acids (1–10 M HCl), bases (5–25% NH3), autoclave treatment and elevated temperatures up to 150 °C over a period of 24 hours does not result in loss of affinity for the template. Despite its importance, the literature on template extraction itself is also scarce and intended to reduce template leaching.16The reusability of imprinted materials has a crucial role in developing applications that are reliable, economic and sustainable. Nonetheless, reusability studies in the literature are limited to about 10 adsorption cycles and there is no assessment of long-term stability and reusability of imprinted polymers. Although the monomer-template assemblies and their stability have been thoroughly studied both theoretically and experimentally,l-phenylalanine methyl ester (ME) imprinted polymers having different compositions. 18 the application of the prepared IPs is not the focal point of the present study. ME was chosen as the template because of its multifaceted chemical nature having hydrogen donor and acceptor sites as well as an aromatic moiety capable of π–π interactions. Having such a versatile template in hand allowed investigating a wide range of functional monomers such as carboxylic acid, urea, amide, amine and boronic acid derivatives.The present study systematically investigates the effect of crosslinkers, functional monomers and conditions for template extraction on the long-term stability and reusability of imprinted polymers . Twenty-N,N′-methylenebis(acrylamide) (BMA). The crosslinker is important in controlling the morphology of the polymer matrix, serves to stabilize the imprinted binding sites, and imparts mechanical stability to the polymer matrix in order to retain its molecular recognition capability. The crosslinking degree was set at 1/15 and 1/30 functional monomer to crosslinker ratios. The template extraction was carried out with washing solvents having different eluent strength at both room (25 °C) and elevated (65 °C) temperatures. Besides fundamental understanding, the present study aims to provide guidance on adsorbent regeneration for both professionals and academicians working in the field of polymers, in particular, imprinted materials.Acrylate, acrylamide and styrene based polymers were prepared using commonly employed crosslinkers such as ethylene glycol dimethacrylate (EDMA), divinylbenzene (DVB) and et al.,19 and its characterisation is included in the ESI.2SO4 and distilled over CaH2 under reduced pressure before use. A VWR-Hitachi Chromaster DAD-HPLC system equipped with an ACE 5 μm, C18, 300 Å, 250 × 4.6 mm column was used for template analysis (260 nm) with an isocratic elution using 25% acetonitrile and 75% phosphate buffer (pH = 7). Elemental analyses were performed in the Microanalysis Laboratory of the School of Chemistry at The University of Manchester. The nitrogen sorption measurements were performed on a Quantachrome Autosorb 6B automatic adsorption instrument. Thermal gravimetric analysis (TGA) was performed with a Seiko Instrument Inc. TG-DTA 6200 using an alumina pan under a 50 mL min–1 nitrogen flow at a heating rate of 10 °C min–1. NMR spectra were recorded in either CDCl3 or DMSO-d6 on a Bruker DRX-500 Avance spectrometer (at 500 MHz for 1H and at 125 MHz for 13C spectra).Reagents (reagent grade) and solvents  were purchased from Sigma-Aldrich and Fisher Scientific, respectively. The 1-(4-vinylphenyl)-3-phenyl)urea functional monomer was synthesized following the protocol reported by Hall 20 Briefly, in a typical IP fabrication procedure 1 mmol functional monomer, 1 mmol template, 20 mmol crosslinker, 0.1 wt% initiator, 100 mg perfluoro polymeric surfactant (PFPS) emulsifier, 80 mL perfluoro methylcyclohexane (PMC) dispersing phase and 15 mL acetonitrile porogen were stirred at 300 rpm  or two-hours SXE at the boiling point of the solvent (65 °C) were used. The extraction was followed by a 100 mL methanol wash per gram of adsorbent at a flow rate of 1 mL min–1 at room temperature in order to neutralise the polymers. 0.01 mbar vacuum pressure at room temperature was applied for 12 hours in order to dry the adsorbents. The experiments were carried out in duplicate with independently prepared polymers and the resulting standard deviations are shown in the adsorption diagrams.120 mL of 0.1 M HCl or NaOH in methanol as washing solvents per gram of adsorbent were used for extraction. Either SPE cartridges at a washing flow rate of 1 mL min‡The retention behaviour of imprinted polymers (IP) is often compared with the reference non-imprinted, control polymer (CP), to evaluate the effect of imprinting which is often expressed by the imprinting factor defined as the ratio of the adsorbed template by an IP and its corresponding CP. were found to be between 2.9 and 5.2 proving successful incorporation of the template molecule into the polymer matrix during the polymerisation process. Polymers crosslinked at the same degree with EDMA and DVB showed similar adsorption capacities whilst BMA crosslinking at the lower crosslinking degree resulted in about 26% lower values compared to EDMA and DVB crosslinking. The highest adsorption capacity and imprinting factor was demonstrated by IP3 comprising methacrylic acid (MA) as a functional monomer and DVB as a crosslinker. The polymers prepared with the other functional monomers showed 41–77% lower adsorption capacities  of the analytical method. The initial absolute adsorption values for the first adsorption–regeneration cycle are shown in pacities .21In order to evaluate the long-term stability and reusability of the imprinted polymers adsorption–regeneration cycles were performed up to 100 times, and the results are summarized in Td5) for BMA and EDMA crosslinked polymers decreased by 14% (from 178 °C to 153 °C) and 24% (from 259 °C to 197 °C), respectively, the DVB-based polymer had the highest Td5 value of 350 °C, which remained virtually the same. In contrast, the performance of EDMA and BMA crosslinked polymers deteriorates over time under both acidic  and basith cycle resulted in an irregular surface with defects  demonstrated about 10% higher adsorption than IP1 (1/15 MA/EDMA) at the 100th adsorption–regeneration cycle  was used to investigate the selectivity of the polymers. Selectivity factors were calculated as the ratio of adsorbed ME and BE, and the results are summarized in th cycle and 18% by the 100th cycle. The increase in surface area is unexpected and it is probably due to the molecular level degradation of the polymer without collapse of the polymer network.These observations are in line with the changes in the adsorption capacity shown in 15 They should be exhaustively removed in order to ensure reliable results, in particular if IPs are used for quantitative analytical applications or product purification. Such clean-up is time-consuming and requires substantial amounts of the extraction solvent. Eppler et al. developed an extraction device specifically designed to facilitate the rapid and efficient clean-up of imprinted polymer matrices offering significant advantages: the ability to use solvent mixtures, temperature-controlled extraction conditions, improved kinetics, and continuous control of the extraction process.16 However, both reversible and irreversible degradation of the polymer network can occur under basic and acidic conditions.Leaching of the template and unreacted monomers from IPs is an often observed phenomenon, which is considered as one of the main drawbacks of the technology.via TGA, SEM, adsorption and selectivity studies. Although the resulting esters or sodium salts remain part of the polymer network, the ethylene glycol and methylenediamine leach out from the adsorbents and could have an adverse effect during the application of the imprinted polymer. Boronic acids can also undergo irreversible esterification in methanol in the presence of acids and the reaction can proceed further resulting in the elimination of trimethyl borate . In contrast, the performance of methacrylate- and acrylamide-based polymers deteriorates over time under both acidic and basic extraction conditions. It has been demonstrated that besides the well-known template and monomer leaching, polymer degradation products can also result in contamination of samples during the application of the adsorbents. Performance loss of imprinted polymers having carboxylic acid functional moieties can be recovered by an aqueous acidic wash at the end of the regeneration cycle. However, methacrylate- and acrylamide-based polymers irreversibly degrade under both acidic and basic conditions, which results in decreased degree of crosslinking. Imprinted polymers having a boronic acid functionality should not be regenerated under acidic conditions in alcohol as it results in their irreversible degradation and elimination of trialkyl borates. Further challenges to be resolved include the regeneration of macromolecularly imprinted polymers.26The reusability of imprinted polymers has a crucial role in developing applications that are reliable, economic and sustainable. The effect of crosslinkers, functional monomers and conditions for template extraction was systematically investigated to reveal the long-term stability and reusability of imprinted polymers. Adsorption–regeneration cycles were carried out 100 times for


Bivalve molluscs constitute a ubiquitous taxonomic group playing key functions in virtually all ecosystems, and encompassing critical commercial relevance. Along with a sessile and filter-feeding lifestyle in most cases, these characteristics make bivalves model sentinel organisms routinely used for environmental monitoring studies in aquatic habitats. The study of epigenetic mechanisms linking environmental exposure and specific physiological responses  stands out as a very innovative monitoring strategy, given the role of epigenetic modifications in acclimatization and adaptation. Furthermore, the heritable nature of many of those modifications constitutes a very promising avenue to explore the applicability of epigenetic conditioning and selection in management and restoration strategies. Chromatin provides a framework for the study of environmental epigenetic responses. Unfortunately, chromatin and epigenetic information are very limited in most non-traditional model organisms and even completely lacking in most environmentally and ecologically relevant organisms. The present work aims to provide a comprehensive and reproducible experimental workflow for the study of bivalve chromatin. First, a series of guidelines for the molecular isolation of genes encoding chromatin-associated proteins is provided, including information on primers suitable for conventional PCR, Rapid Amplification of cDNA Ends (RACE), genome walking and quantitative PCR (qPCR) experiments. This section is followed by the description of methods specifically developed for the analysis of histone and SNBP proteins in different bivalve tissues, including protein extraction, purification, separation and immunodetection. Lastly, information about available antibodies, their specificity and performance is also provided. The tools and protocols described here complement current epigenetic analyses  by incorporating the study of structural elements modulating chromatin dynamics. In eukaryotes, DNA is packaged and compacted within the cell nucleus thanks to its association with chromosomal proteins, constituting the chromatin fiber. The fundamental subunit of the chromatin, the nucleosome, consists of approximately 145 bp of DNA wrapped around a protein core formed by small and highly basic proteins known as histones   have evolved additional H2A.X genes (unpublished work), which will have to be considered when performing PCR experiments. In addition, primers for canonical histones as well as primers annealing in coding regions suitable for RACE or genome walking experiments are indicated in Supplementary Tables The design of PCR and qPCR primers for histone variants is quite often limited by the high level of similarity among histones within a given family e.g., H2A). Consequently, it is recommended to incorporate at least part of divergent untranslated regions  into primer design, minimizing unspecific amplifications. On the contrary, the use of primers annealing in coding regions is a good strategy for amplification of histone sequences in unexplored species, either through RACE or genome walking experiments, given the higher conservation of these regions. Accordingly, the design of such primers requires a more careful sequence analysis in order to avoid targeting regions where variants and canonical histones are similar, as the latter are far more abundant and tend to amplify easily in PCR experiments. For primer design we recommend using the Primer-BLAST tool  from the surf clam Spisula solidissima, as well as PL-II/PL-IV (DQ305038) and PL-III (DQ305039) sequences from the mussel M. californianus. In the case of Mytilus, the PL-II/PL-IV precursor is post-translationally cleaved resulting in two different protein byproducts, PL-II* and PL-IV. In addition, primers employed by previous reports in genome walking experiments for the characterization of these SNBPs  proteins. However, all of them share a common evolutionary origin that can be traced back to histone H1 , melting temperatures of approximately 60°C, 50–60% GC content, and low self-complementarity. However, since primers target gene regions displaying divergence among histones from the same family, it is not always possible to fulfill all requisites mentioned. Still, it is possible to design efficient qPCR primers for bivalve histone variants such as those shown in Table 00 bp, meMytilus as a reference. These protocols have also been successfully applied to other bivalves, including clams , supporting their efficiency across multiple taxonomic groups. Based on previous experience, gill, male gonad and sperm tissues produce good amounts of histone proteins following a standard acidic extraction protocol , using the mussel in vivo by opening a small aperture in the shell using a lime , reaching to the posterior adductor muscle using a hypodermic syringe. It is recommended to add Alsever's solution  to the extraction syringe (500 μL of Alsever per every 10 mL of hemolymph) in order to avoid the hemocytes aggregation. Hemolymph must be collected carefully , immediately transferred into 15 mL tubes and placed on ice until all samples are collected. Samples should be subsequently centrifuged at 250 × g for 5 min at 4°C, obtaining a pellet of bivalve hemocytes that are suitable for the subsequent processes.A starting material of 0.5 g of tissue (fresh or frozen) is enough for most experimental purposes. The collection of solid tissues  does not require special considerations other than the correct identification of such tissues, which can differ substantially among bivalve species. For instance, in the present context, the difference between male gonad and sperm refers to the developmental stage of the gonad. In other words, the sperm constitutes a very mature gonad whose content is mostly sperm cells. Thermal shock treatment  mixed with protease inhibitors 1/100 (v/v) . The homogenate is then placed on ice for 10 min (breaking cellular membranes), and centrifuged at 4,000 × g for 10 min at 4°C. In order to maximize yield and quality, this protocol should be modified depending on the tissue source as follows: In the case of digestive gland and muscle tissue, it is recommended to repeat homogenization steps at least once, as the insoluble materials contained in fat cells, plus the shape and composition of muscle cells, hamper cell membrane break and cell disaggregation, respectively. In the case of hemocytes, these steps should be repeated up to three times to obtain higher protein yields, due to the small size of these cells and their tendency to form aggregates obstructing disruption of cell membranes. Lastly, in the case of female gonad, a double amount of protease inhibitors should be used while skipping the incubation on ice for 10 min, in order to avoid the degradation of histone proteins as a result of the high content in proteolytic enzymes of this tissue.2), adding a mix of protease inhibitors 1/100 (v/v). The homogenate is then incubated on ice for 10 min and centrifuged at 4,000 × g for 10 min at 4°C. Once more, specific modifications are required for different tissue types. For digestive gland and muscle, prior to the homogenization in Buffer B, it is recommended to remove insoluble materials (present in high amounts in both tissues) by filtering nuclear extracts through a sterilized cheesecloth soaked in Buffer B. In the case of female gonad, doubling the amount of protease inhibitors and skip the incubation on ice for 10 min is recommended, in order to avoid the degradation of histone proteins by proteases. Overall, the nuclear fraction obtained at this point can be used in downstream experiments such as Micrococcal Nuclease (MNase) chromatin fractionation , and subsequently centrifuged at 8,200 × g for 10 min at 4°C. The resulting supernatant will be then transferred to fresh tube with 6 volumes of acetone, mixing the solutions by inverting the tubes 6-8 times, and incubating the samples at −20°C for at least 1 h. The solution will turn cloudy after a few minutes due to the precipitation of histones and SNBPs in acetone. In the case of tissues producing low protein yields , it is recommended that acetone incubation is extended overnight. Upon protein precipitation, samples are pelleted by centrifuging at 10,000 × g for 10 min at 4°C, discarding the supernatant and carefully washing the pellet with acetone in order to remove rests of acid. Samples are then subject to an additional centrifugation, carefully discarding the supernatant and air-drying the histone pellet for 20–30 min. Alternatively, a vacuum concentrator can be employed for 3–5 min . Lastly, proteins are dissolved in a variable volume of ultrapure water , being ready to be used immediately or stored at −80°C. The integrity and concentration of histones and SNBPs can be assessed through protein separation in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and/or by using western blot experiments (see sections below).* in Figure The protein yield resulting from extractions will vary depending on the source tissue Figure . For insChromosomal proteins can be easily purified using chromatographic approaches. More specifically, histones and SNBP extractions can be further purified using Reverse Phase High Performance Liquid Chromatography (RP-HPLC) , AU-PAGE , triton AU(AUT)-PAGE  or two-dimensional (2D)-PAGE . Standard protocols for these gels can be used for the analysis of histones, although some modifications are recommended (see protocols in Box Box 1– Set up the plates and prepare the gels as indicated in the tables. APS and TEMED should be added immediately before pouring the gels (the addition of these compounds initiates the polymerization).– Add APS and TEMED to the separating gel and pour it into the plate letting a 2–3 cm gap in the upper part. Equilibrate it with water or isopropanol.– Once the separating is solidified, remove the water or isopropanol, add APS and TEMED to the stacking gel and pour it introducing the comb immediately after.– Dissolve the histone extract in the same volume of 2X SDS Sample Buffer as indicated in the table and boil the samples at 100°C for 2–3 min.– Run the gel at 100 V for 1 h 30 min or until the dye band reaches the end of the gel.Running buffer (10X): 30.3 g of Tris Base, 144 g of glycine, 10 g of SDS, up to 1 L of dH2O.2O2.– Set up the plates and prepare the gel as indicated in the tables. Pour the gel into the plate and insert the comb very quickly as these gels polymerize very quickly once you add 30% H2X Urea Sample Buffer .– Dissolve the histone/SNBP extract in the same volume of Running Buffer (1X): 5% acetic acid.– Run the gel at 100 V for 3 h 30 min to visualize histones in AU or AUT gels (or until the dye band reaches 2/3 of the gel) and for 2 h for the visualization of PLs in AU gels (for Mytilus spp.). *Note that these gels run toward the negative pole, unlike SDS gels.– Gel stainingStaining Solution  for 1 h with constant shacking.– Dissemble the gel and incubate it in Distaining Solution  for 15 min with constant shacking and overnight with fresh Distaining solution.– Incubate in After the run, the staining procedure is the same for all the gels described above:Histones are separated based on their molecular weights in SDS gels. Consequently, while canonical histones H1, H3, and H4 can be easily isolated, it is more difficult to differentiate H2A and H2B due to their similar size Figure . Overall*, PL-III, and PL-IV) can be perfectly separated using AU gels as depicted in Figure Spisula solidissima is composed by a single PL type (PL-I), running in the upper part of AU gels due to its big size (>50 KDa). Similarly, additional separation of histone variants is possible using two-dimensional AUT-SDS gel electrophoresis. This approach provides higher resolution than any other gel type, allowing to differentiate H2A.X, H2A.Z, H3, and H2B variants  for 1 h at room temperature. That is ensued by the incubation of the membrane with a primary antibody at 4°C overnight, followed by three washing cycles in a PBS and 0.1% Tween solution at room temperature with constant shaking for 10 min. The membrane can be then incubated with a secondary antibody  for 1 h at room temperature. This antibody is usually employed at 1:5,000 dilution in blocking buffer. The process is completed with three additional washing cycles as indicated above, proceeding to develop the blot. For that purpose, an enhanced chemiluminescent (ECL) system  and visualization in X-ray films is recommended.The evolutionary conservation of histone proteins enables, in some instances, the use of commercial antibodies  on invertebrates including bivalve molluscs. The present work provides information about the validity of commercial antibodies specific for different histone variants and PTMs in bivalves  antibody, as it was the case of the recently developed anti-macroH2A antibody suitable to detect this variant in a wide range of invertebrates including molluscs . One group of gonads is kept uncut for light/fluorescence microscopy. Another group is cut into long 2-mm thick strips (it is important to create strips with different orientations). Fixation of both whole gonads and gonad strips is conducted for 1 h at room temperature.Dissect the gonads of mussel specimens and immerse them in primary fixative  using a volume of dehydrating solution of approximately 10-fold the volume of the tissue. Gonad strips are subsequently infiltrated with 50% LR-White (London Resin Co.) in 100% ethanol for 3 h, followed by pure LR-White resin overnight at room temperature. The whole gonads are treated with two steps of xylene for 1 h each at room temperature, followed by 6 steps of melted pure paraffin kept at 60°C for 20 min each. Gonads are then infiltrated in pure melted paraffin overnight.Next day, LR-White embedded specimens are solidified in block molds at 60°C in a vacuum oven, as oxygen inhibits the polymerization of the LR-White resin. Paraffin-embedded specimens are then solidified in molds at room temperature. Paraffin blocks are kept at 4°C until sectioning.Sectioning and mounting the sectionsLR-White blocks are sectioned into 0.3–0.5 μm-thick sections in an ultramicrotome; while paraffin blocks are sectioned into ribbons of approximately 5 μm-thick sections in a histological microtome. Sections are subsequently mounted on glass slides, air dried and baked for 10 min at 50°C. Paraffin sections are then de-paraffinized in Copling jars using the following step series: xylene for 5 min (2 times)—100% ethanol for 5 min (2 times)—70% ethanol for 5 min (2 times)—Millonig's buffer for 5 min.Immunohistochemical staining* and H4 were diluted 1:400 and 1:200, respectively, in Millonig's phosphate buffer with 2% BSA; whereas commercial goat anti-rabbit IgG tagged with Rhodamine red (R6394) and Oregon green (O6381) from Molecular Probes  were diluted 1:200 in the same buffer.Immunolabeling steps are performed at room temperature in Coplin jars, with the exception of antibody incubations, for which small volumes  are used to cover the sections and the slides are kept in a humid chamber to prevent drying of the antibody solution during incubation. Briefly, sections are blocked in 1% bovine serum albumin −1% casein in Millonig's phosphate buffer for 1 h, washed once for 10 min in the same buffer, incubated with the primary antibody for 2 h, washed 3 times as before, incubated with the secondary antibody for 1 h, washed 3 times as before, blotted and mounted with No. 1 coverslips in gelvatol mounting medium  for observation. In this work, primary antibodies, house-made PL-IIHistological stainingAfter immunolabeling, sister sections are histologically stained to be used as reference for structural features of the immunolabeled sections. LR-White sections baked on glass slides (as above) are stained in a single-step procedure, covering the sections with freshly filtered Richardson's stain , evaporating the stain to near dryness on a heating block at 50°C, and rinsing in deionized water to remove excess stain. De-paraffinized hydrated sections on glass slides (as above) are conventionally stained with Hematoxylin-Eosin. Histologically stained sections are permanently mounted with No. 1 coverslips in Permount™, before observation.The study of the mechanisms mediating physiological responses to environmental changes constitutes a key discipline to understand how climate change will affect organisms. Environmental epigenetics is at the center stage of such efforts, given the role of epigenetic modifications during the regulation of gene activity, and their implications for acclimatization and adaptation under ever-changing environments. Unfortunately, epigenetic information for most non-model and ecologically relevant organisms is very limited, with environmental epigenetic studies almost exclusively focused on DNA methylation (leaving other mechanisms largely unexplored). However, it is now clear that chromatin-associated proteins participate in organism-environment interactions in different capacities . By providing a description of experimental methods for studying chromatin-associated proteins, the present work aims to provide a reference for researchers interested in studying how DNA is organized and regulated in molluscs, a ubiquitous taxonomic group playing critical functions in virtually all ecosystems. By doing so, this work fosters a more holistic approach to study the epigenetic mechanisms underlying environmental responses in bivalve molluscs, ultimately improving our understanding of their physiological responses to climate change, their application as environmental sentinel organisms as well as optimizing their management.The concept of the work was conceived by JE and developed in collaboration with CR, RG-R and JA. All authors were involved in the design and performance of the experiments as well as in the analyses of the data. CR and JE wrote the article, with contribution from RG-R and JA. All authors reviewed the manuscript draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PI and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Quercus rubra, with an approach optimized to produce a high-quality map. Our study design also enabled us to model the results we would have obtained with less depth of coverage.Restriction site associated DNA sequencing (RADseq) has the potential to be a broadly applicable, low-cost approach for high-quality genetic linkage mapping in forest trees lacking a reference genome. The statistical inference of linear order must be as accurate as possible for the correct ordering of sequence scaffolds and contigs to chromosomal locations. Accurate maps also facilitate the discovery of chromosome segments containing allelic variants conferring resistance to the biotic and abiotic stresses that threaten forest trees worldwide. We used ddRADseq for genetic mapping in the tree de novo reference and the SAMtools SNP variant caller yielded the most SNP calls . The major drivers of map inflation were multiple SNPs located within the same sequence . The highest quality map was generated with a low level of missing data (5%) and a genome-wide threshold of 0.025 for deviation from Mendelian expectation. The final map included 849 SNP markers . Downsampling the individual FASTQ files to model lower depth of coverage revealed that sequencing the progeny using 96 samples per lane would have yielded too few SNP markers to generate a map, even if we had sequenced the parents at depth 248×.Our sequencing design produced a high sequencing depth in the parents (248×) and a moderate sequencing depth (15×) in the progeny. The digital normalization method of generating a The ddRADseq technology produced enough high-quality SNP markers to make a moderately dense, high-quality map. The success of this project was due to high depth of coverage of the parents, moderate depth of coverage of the progeny, a good framework map, an optimized bioinformatics pipeline, and rigorous premapping filters. The ddRADseq approach is useful for the construction of high-quality genetic maps in organisms lacking a reference genome if the parents and progeny are sequenced at sufficient depth. Technical improvements in reduced representation sequencing (RRS) approaches are needed to reduce the amount of missing data.The online version of this article (doi:10.1186/s12864-017-3765-8) contains supplementary material, which is available to authorized users. Quercus rubra L.), a highly heterozygous, outcrossing angiosperm forest tree lacking a reference genome. None of the ecologically dominant and economically valuable oaks of eastern North America have reference genomes and studies of population dynamics remain limited. RRS techniques have the potential to provide an affordable technology for dense genetic mapping and gene discovery in oaks and the other long-lived angiosperm forest trees of eastern North America.The low cost and broad applicability of reduced representation sequencing (RRS) technologies have enabled a burst of genetic architecture and gene discovery studies in natural populations. A widely used RRS technique, restriction site associated DNA sequencing (RADseq), inexpensively generates tens of thousands of SNP calls, a seemingly sufficient number for detecting fine-scale population substructure, constructing phylogenies, and generating densely populated genetic maps –4. TechnQ. rubra. We used a high coverage design, devoting one lane of sequencing on an Illumina HiSeq for the two parents and five lanes for the progeny (50/lane), considerably fewer than the 96 individuals typically loaded in each lane for RADseq involving non-model organisms lacking reference genomes [de novo reference for Q. rubra and two SNP variant callers. We generated a statistically robust framework map with gSSR and EST-SSR markers, and then used ddRADseq to discover SNP markers for the same individuals. This study design enabled us to test the performance of ddRADseq for genetic mapping under optimized conditions and to model the results we would have received had we used an experimental design with less coverage per individual.Our approach to mapping with ddRADseq included a full-sib population from one seed parent and one pollen parent, a pedigree that provides more information for genetic mapping than a half-sib family of the same size. The outcrossing parents were expected to have many SNP loci heterozygous for the same SNP, enabling the construction of one map rather than separate male and female maps. Both parents have seed, bud and leaf morphologies consistent with those expected for  genomes . The infQuercus rubra is the most dominant and wide-ranging species of the Lobatae, a section of the Quercus genus containing nearly 100 species ranging from California to the Atlantic coast, north to northern Ontario and south across Mexico and Central America to northern Columbia [Lobatae are ecologically significant in many forest communities of eastern North America, occurring across a wide range of ecosystems, including dry savannahs, mesic bottomlands and upland forests [Quercus section Quercus), which are native in North America, Europe, and Asia the red oaks are native only in the Americas [Oaks are outcrossing, diploid forest trees with relatively tractable genome sizes (~600–800 Mb) , 13 and Columbia . The Lob forests . Unlike Americas . The accAmericas . The devPopulus [Populus [Frangula alnus) [Eucalyptus tricocarpa [Quercus genus [Cedrus atlantica) [Ziziphus Mill. [Pyrus communis L.) and the Chinese pear (Pyrus bretschneideri Rehd.) [Citrus grandis Osbeck) [Actinidia chinensis Pl.) [Rubus idaeus L.) [Pinus balfouriana Grev. & Balf.) [Populus deltoides Marsh x P. simonii [Populus species are undomesticated.Prior studies on woody perennials have used RADseq to examine gene flow among ecologically divergent species of Populus , adaptiv[Populus , signatua alnus) , adaptaticocarpa  and phylus genus . Use of lantica) . Howeverus Mill. , a crossi Rehd.) , pomelo  Osbeck) , kiwifrusis Pl.) , red rasaeus L.) , foxtail& Balf.)  and the  simonii . Of thesQuercus robur L.) [Q. robur x European sessile oak (Q. petraea (Matt.) Liebl.) [Castanea sativa Mill.) [C. mollissima Blume) [C. dentata (Marsh.) Borkh.) [Quercus robur and Quercus petraea [Lobatae.In the Fagaceae , genetic maps are reported for the European pedunculate oak (obur L.) , 33, the Liebl.) , Europeaa Mill.) , Chinesea Blume) , 37, and Borkh.) . In the  petraea . Prior tQuercus and Lobatae sections of the genus Quercus diverged between 15 and 40 Mya [Lobatae [Paleobotanical data suggest that d 40 Mya . Even th[Lobatae . The exo[Lobatae . In cont[Lobatae . Thus weQ. rubra has a 2-year acorn), parentage-verified with gSSR and outplanted in 2011. The progeny and parents were propagated as replicated clones at the Horticulture and Agroforestry Research Center (HARC) in New Franklin, MO in 2013. Co-author Coggeshall collected voucher specimens for SM1 and SM2 in 2017 and deposited sun leaves and shade leaves specimens for each parent in the Greene-Nieuwland Herbarium (herbarium code NDG) at the University of Notre Dame. The voucher specimen codes are ND145625, ND145626, ND145627, and ND145628.SM1 and SM2 are the labels given to the two parents of the mapping population. The parent trees are located on the campus of Purdue University, approximately in the middle of the native range for this species. The species identity was verified by co-author Dr. Mark Coggeshall. Parentage analysis identified SM2 as the predominant pollen parent for our selected seed tree SM1 . The fulQ. rubra library enriched for CA repeats . Primers were designed using Primer3 v. 0. 4.0 [Q. rubra EST-SSRs detected in the northern red oak tissue above ground (ROA) and northern red oak roots below ground (ROB) (http://hardwoodgenomics.org/content/de-novo-northern-red-oak-quercus-rubra-ro454v2). We tested all CA and GA repeat gSSRs previously reported for Q. rubra [Quercus robur L. [Castanea mollissima [1 progeny of outcrossing parents [DNA was initially extracted from the parents and the 2010 sibship using a previously reported modified CTAB protocol . For RAD. 0. 4.0 . We alsoQ. rubra –48, EST-robur L.  and EST-llissima . Markers parents .2, 10 mM dNTP, 1 μl of 10× Mg free PCR reaction buffer, 1 μl of 4% BSA, 0.25 U/μl TaKaRa Taq™  and 3.5 μl of double distilled H2O. The PCR amplification profile consisted of initial denaturation at 94 °C for 2 min, 35 cycles of 94 °C for 30 s, annealing at a marker specific temperature for 30 s, then 72 °C for 45 s followed by 60 °C for 45 min and ending with a final extension at 72 °C for 10 min. Fluorescently labeled amplicons were size fractionated on an ABI 3730 XL genetic analyzer  using GeneScan™ 400 HD ROX™ (Applied Biosystems) as internal size standard. Fragment length polymorphisms were scored using GeneMapper® v 4.0 (Applied Biosystems). Of the 379 SSR markers tested , 116 markers were informative , 4 pmol of each forward and reverse primers, 25 mM MgClEcoRI and MseI [EcoRI adapter and an MseI adaptor [We chose 225 full-sibs, a subset of the 399 full-sibs used for the framework map, for ddRADseq. Each DNA sample was diluted to a final concentration of 150 ng/μl and plated into a 96-well plate with each well containing 900 ng of DNA in a final volume of 6 μl. Library construction was done in the Genomics and Bioinformatics Core Facility at the University of Notre Dame. Libraries were prepared using a ddRADseq approach  modifiedand MseI . At the  adaptor  modifiedQ. rubra individuals not included in this project, so every progeny lane did have 50 individuals. Sequencing was done on an Illumina HiSeq 2000 using 101 bp paired-end reads.Pooled libraries were sent to BGI International  for sequencing. We pooled libraries of 50 full-sib samples per lane and to ensure accuracy of the SNP variant calls, used one lane for the two parent libraries with the expectation of obtaining better sequence coverage than the standard 96 samples per lane design. One of the progeny lanes contained libraries of 25 We checked raw sequences from the six Illumina HiSeq lanes for initial quality using FastQC . As the To minimize the computational requirements of deriving a reference assembly, we tested two non-alignment methods, a digital normalization and a center star method Fig. . For theFor the center star method, progeny reads derived from either parent were clustered based on BWA alignments to a parental allele used to build a center star tree, such that the distance of each sequence is computed to all other sequences. The resulting reference set is all alleles that have the minimum distance to fellow progeny alleles, presumably because they have fewer sequencing errors. As the digital normalization strategy identified more hk x hk markers than the center star method, the digital normalization strategy was used for all subsequent steps . These alignments were used to create the intermediate files for variant detection. We tested the SNP calling methods implemented in SAMtools and in the GATK HaplotypeCaller Walker annotated default [The reads of the individual quality-controlled FASTQ files were aligned to the generated reference sequences using default parameters of BWA, SAMtools  and Pica default , on both1 progeny of two outcrossing parents [The informative SNP called by SAMtools were transformed into the JoinMap® format required for mapping with the F parents . The pre parents . Our secWe generated the framework map first, using an independence LOD threshold of 20 for grouping markers and the Kosambi mapping function for regression mapping. For the final map, the initial data consisted of the 116 framework markers and 1413 SNP markers (see results for how our filters produced this number). Prior to mapping with the full dataset, we removed eight individuals with > 90% missing data (most of which were SNP markers), leaving a mapping population of 217 individuals. Finally, we excluded SNP markers with similarity value ≥ 0.945, leaving 1344 unique SNP markers and all of the framework markers. The data were grouped using an independence LOD threshold of 30. Framework markers were specified using the fixed order function in JoinMap® 4.1 before mapping. The fixed order function specifies only a fixed order, not a fixed distance. We generated maps using both the approximate maximum likelihood and regression mapping algorithms with default settings. The final map was generated using round two regression mapping with the Kosambi mapping function and charted using MapChart 2.30 .We tested the effectiveness of our conservative use of sequencing capacity (one lane for the two parents and only 50 progeny per lane) by comparing our results with those we may have obtained if we had used a less conservative design. We conducted two progeny downsampling experiments (Exp1 and Exp2). For Exp1, we downsampled the trimmed and demultiplexed FASTQ files for the 225 progeny used for mapping while keeping the parent data intact, to simulate 96 progeny per lane, but reserving one lane for the two parents. For Exp2, we downsampled both progeny and parents to simulate the condition in which all of the progeny and the parent libraries were run in three lanes: 96 progeny samples in two lanes, 33 progeny in the third lane with 31 replicates for one parent and 32 for the other.We implemented the downsampling approach by randomly selecting 52% (50/96 *100) of the FASTQ data from each progeny for the downstream analysis. For Exp2, progeny downsampling was the same as in Exp1. For the parents, our design has the effect of utilizing ~32% of the sequencing lane capacity for each parent, as opposed to 50% based on our initial sequencing approach. We see this as a choice an investigator is likely to make, to save the cost of using another lane, while at the same time getting more parent reads. We implemented parent downsampling by randomly selecting 64% (32/50*100) of the data for each parent for downstream analysis. In all experiments, we used the DNR approach, with SAMtools as the SNP caller, the same as we did with the full data set.The number of reads for parents and progeny totaled 877,796,304. The parent lane yielded 88,788,165 reads for SM1 and 61,994,649 reads for SM2. The mean number of reads per progeny was 2,908,053, the median 3,056,516. Using the DNR reference sequence, SAMtools called more than six times as many SNPs  as Haplotype Caller   for matches to the 21,526 sequences with > 1 SNP resulted in only 46 matches at an E-value ≤ 9.91E-07, 34 of which had best hits to Gypsy or Copia LTR-retrotransposons. Our query of P-mite, a database for plant miniature inverted-repeat transposable elements [Q. rubra may have diverged in sequence significantly from the repeats of the model plants represented in the two databases. Removal of these multi-SNP loci left 18,038 SNP markers. Finally, previous experience with SNP chips suggests that SNPs located in the first 9 bp of the marker sequence are more likely to generate artifacts. After removal of loci with SNPs in the first 9 bp (2263 markers), 15,775 SNP markers remained for use in mapping. This filtered SNP marker number is greater than the number of unfiltered SNP calls we obtained with GATK-HaplotypeCaller .Our initial filters  resulted in severely inflated maps, even at the strict criteria of 5% missing data and a chi-square value < 10. The maximum likelihood map for linkage group 3 exceeded 1000 cM, nearly ten times the distance inferred with the round two regression map. All other linkage groups were inflated as well. We found that sequences with > 1 SNP call were driving this result. Of the 78,725 ddRADseq markers in which informative SNPs were detected, 60,687 (77%) had > 1 SNP. These 60,687 SNPs occurred on 21,526 ddRADseq sequences, indicating that some sequences had more than two SNPs. Our query of Repbase , the order is the same . The GO annotation suggests similarity to a family of proteins involved in response to cadmium stress [Quercus petraea cDNA clone WZ0AQPAI7YG19FM1 (NCBI FN736994). The GO annotation suggests similarity to Arabidopsis genes involved in response to colder temperatures [Quercus robur cDNA clone LG0AAA8YO09RM1 (NCBI FP025018). The GO annotation suggest similarity to Calcineurin B-like protein 9, a protein involved in the regulation of early stress-related CBF/DREB transcription factors [GO annotations suggested stress resistance functions for three of the 73 EST-SSRs located on the final map. The PIE_126 sequence matches the m stress . The WAGeratures . The FIR factors .To generate the final map we used the 217 individuals that had both framework markers and high-quality SNP marker genotypes. Our round two final regression map contains 957 markers distributed over 1014.47 cM  on the nsus map  were alss et al.  for the  and LG8 Q. robur .de novo reference and our goal of generating a dense map, the sequencing design we actually used  proved to be an effective one.In Exp1, the trimmed and demultiplexed FASTQ files for the progeny were downsampled, while the parent FASTQ files were not downsampled. This yielded 5090 SNP calls using DNR and SAMtools. When both the parents and the progeny were downsampled in Exp2, the yield was even smaller   a rigorous test of the ddRADseq approach for constructing dense genetic maps in outcrossing, undomesticated woody perennials lacking a reference genome and 2) the production of a high-quality linkage map for Genetic mapping projects have the advantage of two of the strongest priors in all of biology: Mendelian expectation and the linear information storage system of DNA. The first prior enables a rigorous test of the performance of a RRS technology and associated informatics pipelines for accurate and consistent detection of alleles, i.e. alleles present in the parents, if correctly called, must be present in the progeny of these parents and will occur with an expected frequency in the progeny population. Next, the probability of recombination between any two loci in the linear DNA array is a function of the distance between them. Finally, if the variant calls are correct and the recombination estimates are accurate, then the inferred linear order of the markers will be the actual linear order if the inference algorithm is appropriate.in silico digests of a related genome, but at the time this project was designed there were no genomes released for any oak species. With the Q. robur and Q. lobata (Quercus section Quercus) genomes now released [In genotyping by sequencing, the requirement for accurate calls is likely to be met by high coverage, but the depth of sequencing coverage for parent and for progeny need not be equal, as we have shown. Our downsampling experiment indicated that multiplex sequencing 96 samples per lane would not have produced enough high-quality data for genetic mapping, even if the parents were sequenced to a high depth of coverage. Thus the answer to the design problem of “large numbers of individuals at low depth vs. a small number of individuals at greater depth” has different solutions depending on the intended use. When no reference genome is available, generating good sequencing depth in the progeny  and higher sequencing depth in the parents  is prudent, regardless of other conditions. The values of “good” and “higher” can be approximated by released , 70 the The primary purpose of our work was to generate a high quality genetic map and, by using sequenced markers, provide a tool for correctly ordering sequence scaffolds and contigs to chromosomal locations. However, a given progeny population contains a fixed amount of information regardless of the marker system used for detection, whereas mapping algorithms have no limit on map length. Thus the measure of map quality must not be how many of the SNPs called were mapped. If the LOD criteria used for grouping and mapping are low and very similar SNP genotypes are included, longer linkage groups may result, but the relationship of this inference to actual order may be weak. The “ground truth” test of comparing the inferred linear order with the actual linear order is rarely available for non-model organisms. A useful indirect test is a comparison of the map length produced by a regression approach with that produced by approximate maximum likelihood. This approach is well described by others , 63, butde novo from the RADseq data itself. Our initial tests indicated that our digital normalization approach, with the SAMtools variant caller, yielded the most SNP calls . A recent investigation of the accuracy of variant calling pipelines across different technology platforms showed that a variation of the BWA alignment tool (BWA-MEM) with the SAMtools SNP variant caller, performed better on Illumina data than the BWA-MEM with the GATK-HC pipeline [When a reference genome is lacking, one must be generated pipeline . This sucalls 78,5. A receEven with the aid of a good framework map and using only those SNPs remaining after application of the premapping filters, we found that map inflation was best minimized using the strict criterion of 5% missing data, a lower value than the 10–25% typically reported for genetic maps constructed with RRS technologies , 73, 74.1 progeny of outcrossing, highly heterozygous parents is an exacting and tedious process. In conifers, genetic maps can be constructed using the haploid megagametophyte seed storage tissue from the seeds of a single tree [Callitris glaucophylla [Citrus tamurana) [Regardless of the technology employed, the construction of a high-quality genetic map using the Fgle tree –77, as wcophylla . The teccophylla  and was amurana) , but theQ. rubra complements the dense map produced for the European oaks Q. robur and Q. petraea [Quercus genus. These dense maps, together with the data from the Q. robur genome, the Q. lobata  genome [Castanea mollissima (Chinese chestnut) genome [Quercus and of the Fagaceae , a major family of forest trees in the temperate and subtropical regions of the world. Finally, we anticipate that improved, low-cost RRS technologies and more accessible informatics pipelines will enable the solution of a fundamental puzzle in evolutionary biology, one for which oaks are justifiably famous: rapid sympatric speciation, in the presence of persistent gene flow, within and across a wide array of ecological niches, on all of the continents in which the oaks are native.Using ddRADseq in combination with an SSR-based framework map, we have constructed an oak genetic map that will enable testing of explicit hypotheses about the organization of loci contributing to adaptive evolution in oaks and provide a tool for the detection of allelic variants contributing to stress tolerance. Although mapping stress tolerance genes was not the main focus of this study, three of the 73 EST-SSR markers located on the final map have annotations suggestive of involvement in stress tolerance. The generation of a moderately dense genetic map in  petraea , confirm) genome  and the ) genome , 81, 82,Additional file 1:Q. rubra. These include 37 gSSRs, 38 EST-SSRs from Q. rubra, 1 C. mollissima EST-SSR and 40 Q. robur EST-SSRs. The table shows the marker name, the corresponding sequence accession numbers from NCBI, forward and reverse primers used for amplification and base pair sizes in Q. rubra mapping parents. (XLSX 78 kb)All informative microsatellite markers and associated data. In total 116 informative markers were developed for Additional file 2:de novo reference construction. Tabular data shows how the number of SNP markers generated by SAMtools and HaplotypeCaller changed after elimination of the SNPs with 30% or more missing genotypes for each of the three marker categories possible with SNPs and informative for mapping in F1 of outcrossing parents. (XLSX 9 kb)Performance of SNP variant callers with two methods of Additional file 3:Q. rubra mapped markers and associated data. The table shows all of the mapped framework and SNP markers, the cM distances between them, marker category, position on the Q. rubra linkage group, the sequence in which the marker occurs and the genotypes for the 217 full-sib progeny used for mapping. (XLSX 890 kb)


Background: Current levels of traffic-related air pollution (TRAP) are associated with the development of childhood asthma, although some inconsistencies and heterogeneity remain. An important part of the uncertainty in studies of TRAP-associated asthma originates from uncertainties in the TRAP exposure assessment and assignment methods. In this work, we aim to systematically review the exposure assessment methods used in the epidemiology of TRAP and childhood asthma, highlight recent advances, remaining research gaps and make suggestions for further research. Methods: We systematically reviewed epidemiological studies published up until 8 September 2016 and available in Embase, Ovid MEDLINE (R), and “Transport database”. We included studies which examined the association between children’s exposure to TRAP metrics and their risk of “asthma” incidence or lifetime prevalence, from birth to the age of 18 years old. Results: We found 42 studies which examined the associations between TRAP and subsequent childhood asthma incidence or lifetime prevalence, published since 1999. Land-use regression modelling was the most commonly used method and nitrogen dioxide (NO2) was the most commonly used pollutant in the exposure assessments. Most studies estimated TRAP exposure at the residential address and only a few considered the participants’ mobility. TRAP exposure was mostly assessed at the birth year and only a few studies considered different and/or multiple exposure time windows. We recommend that further work is needed including e.g., the use of new exposure metrics such as the composition of particulate matter, oxidative potential and ultra-fine particles, improved modelling e.g., by combining different exposure assessment models, including mobility of the participants, and systematically investigating different exposure time windows. Conclusions: Although our previous meta-analysis found statistically significant associations for various TRAP exposures and subsequent childhood asthma, further refinement of the exposure assessment may improve the risk estimates, and shed light on critical exposure time windows, putative agents, underlying mechanisms and drivers of heterogeneity. Asthma is a chronic inflammatory disease of the airway which has a large impact on quality of life and poses a great burden on health services . In chil2) can be up to 80%, and ranges between 9% and 53% for urban particulate matter less than 10 micrometres in diameter (PM10), and 9%–66% for urban particulate matter less than 2.5 micrometres in diameter (PM2.5) [TRAP is a particularly important and challenging exposure to study given its ubiquity, its dominance in present urban areas, its proximity to human receptors, and its high spatial and temporal variability ,9,10,11.2 on birth weight [2 from within-community studies that used LUR models  = 1.14, 95% confidence interval (CI) 1.06, 1.23) and those from studies that used dispersion models  [10 and NO2 differ substantially to cases of asthma attributable to black carbon [In the epidemiological studies included in the most recent meta-analyses of TRAP and the development of childhood asthma, different exposure assessment methods and indices have been used to characterise the exposure to TRAP, including distance to roads, active measurement of air pollutants, use of routinely measured air pollution data, land-use regression (LUR) modelling, air dispersion modelling and remote sensing . These vh weight . In a pr7, 1.07) . Furtherk carbon .In this paper, we aim to describe and discuss the exposure assessments conducted in studies of TRAP and childhood asthma development, including the methods used in the different regions, the pollutants and exposure assignment and time windows studied. We then highlight research gaps and make suggestions for further research in this rapidly growing area. Our focus is on the exposure assessments and not the effects of TRAP on asthma development per se; which we reviewed in depth elsewhere . Our resWe conducted a systematic review to synthesize the literature on TRAP exposures and the subsequent risk of childhood asthma development defined as incidence or lifetime prevalence . We follhttp://ovidsp.ovid.com/). We searched the following databases for relevant studies: Embase , Ovid MEDLINE (R) (1996 to August 2016), and “Transport Database” (1988 to August 2016). We identified relevant studies by entering four sets of combined keywords in the “Multi-Field Search” option in OvidSP. We searched for the selected keyword combinations in “All Fields”. The keyword combinations were:“Child*” AND “air pollution” AND “asthma”;“Child*” AND “air quality” AND “asthma”;“Child*” AND “vehicle emissions” AND “asthma”; and“Child*” AND “ultra-fine particles” AND “asthma”.We performed the searches on 8 September 2016 via the database search interface OvidSP , nitric oxide (NO), NO2, hydrocarbons, particles of different aerodynamic diameters  or PM2.5 absorbance as a marker for black carbon (BC) concentrations [Examined the childhood exposure from birth until 18 years old  to any dtrations ; andExamined and reported associations between preceding exposure to TRAP and subsequent risk of asthma reported as incidence or lifetime prevalence from birth until 18 years old.We applied no limits on the initial publication date and no limits on language although we eventually excluded three foreign language studies due to translation difficulties ,21,22. WStudy reference and setting;Study design;Age group;Number of participants;Exposure assessment method(s);Pollutant(s) studied;Exposure assessment place;Exposure assessment time; andAir pollution estimates validation, if any.All titles and abstracts were reviewed against the inclusion criteria by one researcher (Haneen Khreis) with a random 20% independently reviewed by another researcher. All potentially relevant studies were then retrieved and the available full-papers reviewed against the inclusion criteria by one researcher (Haneen Khreis) with a random 50% independently reviewed by another researcher (Mark J. Nieuwenhuijsen). Screening was undertaken manually and differences were resolved by consensus. The following data items were extracted from each included study:Data was primarily extracted from the main papers of the included studies. Where necessary, data items were missing from the main papers, data was extracted from the The databases searches yielded 4276 unique articles, from which 95 were selected for detailed assessment of the full text, one of which was identified by a peer reviewer. A summary of the included studies’ key characteristics is shown in N = 22) used LUR models, 16 studies used TRAP surrogates , 11 studies used traffic-related air pollutant concentrations measured at fixed-site monitoring stations, 8 studies used air dispersion modelling, 1 study used remote sensing and 1 study used diffusion tubes at the residence to measure NO2. These methods vary substantially in terms of their spatial and temporal resolution, specificity to traffic, data and effort/expertise required, transferability and information provided on the actual pollutants , 7 of which were from Japan, Taiwan, Korea and China. Also, 12 out of the 22 studies using LUR model used this exposure method only, 9 of which were from Europe (predominantly from the PIAMA cohort in The Netherlands), while the remaining 3 were from Canada and the USA. The remaining USA studies showed the most variability in the exposure assessment methods choice and used residential diffusion tube monitoring, dispersion modelling, fixed-site monitoring stations, proximity measures and multiple novel TRAP surrogates .In the literature, it was also apparent that the use of the different exposure assessment methods varied by region . For exa2 was the pollutant most studied (31 studies), followed by PM2.5 (18 studies), BC or PM2.5 absorbance (15 studies), and PM10 (14 studies). Other pollutants including NOx (7 studies), EC (4 studies), CO (3 studies), PMcoarse (3 studies), NO (2 studies) were less frequently studied. Only two studies assessed particulate matter composition elements, considered as non-exhaust road traffic emissions, including copper (Cu), iron (Fe), zinc (Zn), nickel (Ni), sulfur (S), and vanadium (V). These studies exclusively originated from the Dutch PIAMA cohort [NOA cohort ,75. One  studied  studies,The exposure assignment was generally static; i.e., not taking children’s mobility into account. In many cases, this could be argued as reasonable as participants were in their infancy or early life (birth–3 years old), and residential exposure is then thought to be most relevant. Only 10 studies, mostly recent, considered children’s mobility in the exposure assessment and assigned time-weighted exposures at day cares and/or schools ,50,83,84In terms of the exposure time window investigated, studies differed, but birth year was the most explored time window . Very feStudies using LUR or dispersion modelling validated their modelled exposure estimates against measured concentration using different methods including leave-one-out cross validation procedure (mainly for LUR models) and independent cross validation against fixed-site monitoring stations measurements (mainly for dispersion models). Generally, the validation of the LUR model estimates were not conducted using a separate test validation dataset which significantly limits the comprehensiveness of the validation. No study reported validation against personal exposures.2 exposure at the individual residential level also showed statistically significant associations between the exposure and asthma [Studies using different TRAP surrogates were the least consistent to show an increased asthma risk associated with TRAP. Studies using dispersion model were more consistent in showing associations. For example, out of 8 studies using dispersion models, 5 showed positive and statistically significant risk estimates. Studies using traffic-related air pollutants concentrations at fixed-site monitoring stations, and studies using LUR modelling generally showed an increased asthma risk associated with TRAP. For example, out of 22 studies using LUR models, 17 showed positive and statistically significant risk estimates. The one study that measured NOd asthma ; so did d asthma . Some ofd asthma ,42,45,782 was the most commonly studied pollutant. Most studies estimated TRAP exposures at the residential address and only a few considered the mobility of the children and/or their residential address changes. Most studies estimated the TRAP exposures at the first year of life (birth year) and only a few studies assessed the effects of cumulative exposures and/or exposures at different time-windows. Validation was undertaken for LUR and dispersion models estimates only and no study has validated exposure estimates against personal monitored exposures. Although our previous meta-analysis found positive and statistically significant associations for various TRAP exposures  with the onset asthma [We found 42 studies that examined the association between TRAP and the subsequent onset of childhood asthma defined as incidence or lifetime prevalence. Exposures metrics differed in terms of their spatial and temporal resolution and their specificity to traffic. LUR modelling was the most commonly used exposure assessment method and NOt asthma , further2 in the literature is probably related to the wide availability of this pollutant measure, the ease and relatively low cost to measure it and its relative specificity to road traffic [2 in air quality guidelines, plans and mitigation strategies in the EU, and beyond, is perhaps reinforcing the study of this pollutant. Fewer studies measured or modelled PM2.5 or particulate components, even though it is more widely implicated in the health effects of air pollution [2.5, suggesting that this exposure metric may be closer to the underlying mechanisms [2 differ; to determine with more confidence which metric predicts respiratory health better.The prominent focus on NO traffic . The focollution . The cosollution ,48,75. Aollution . The worollution . PM compchanisms . These d2, PM10 and PM2.5 were 0.75 (0.19–0.89), 0.39 (0.23–0.66) and 0.29 (0.22–0.81) for 112,971 (13 study areas), 69,591 (7) and 28,519 (4) addresses respectively, suggesting a much better agreement for NO2 than for PM, probably because the main source for NO2 is traffic and PM has other sources. The median Pearson R correlation coefficients (range) between air dispersion model estimates and measurements were 0.74 (0.09–0.86) for NO2; 0.58 (0.36–0.88) for PM10 and 0.58 (0.39–0.66) for PM2.5. Wang et al. [2.5, NO2, and black carbon, but not for PM10. Health effect estimates did not depend on the type of model used in their population of Dutch children. Yet, with a very limited number of comparison studies, the extent to which estimates of air pollution effects are affected by the choice of exposure model remains unclear. A combination of the LUR and dispersion models may further improve the exposure assessment estimates, possibly accounting for some of the imperfections in the emission databases [Many studies have used LUR modelling to estimate TRAP exposures, partly because of its relatively low costs, ease of implementation and possibility to consider traffic determinants of exposure such as the road network and traffic density. LUR models also tend to provide a good spatial coverage and resolution for TRAP exposure. The LUR method is an empirical method and uses least squares regression to combine measured data with geographic information system (GIS)-based predictor data reflecting pollutant sources, to build a prediction model applicable to non-measured locations, e.g., residential addresses of cohort members. An advantage of LUR models is that they are stable over time ,92,93. Hg et al.  comparedCompared to estimates from routine monitoring stations LUR and air dispersion model have the advantage that they provide a better spatial resolution, but also require more effort and are costlier. The better spatial resolution may be quite important when the study area is small and clear exposure differences can be observed by detailed exposure assessment. At least, all three methods provide some level of pollutants which may be important for policy reasons, while surrogate measures like distance from roads do not. A relatively new method, remote sensing  has the 2 exposure, there were only 20 studies entering the meta-analysis, 12 of which used LUR models and 1 used dispersion modelling. The meta-analyses though suggested considerable heterogeneity, especially in the case of NO2 where most studies where available, and part of this heterogeneity could be caused by different exposure assessment methods. Given the rapid increase in the number of studies in this field, it may become possible to conduct such analysis in the near future.We attempted to evaluate the effects of the exposure assessment method on the health risk estimates observed in the included studies, for example with meta-regression, but the number of studies available are still too small to conduct such analyses. Even for NO2.5 in Beijing found that there is a strong correlation between indoor and outdoor PM2.5 mass concentrations, and that the ambient data explained ≥ 84% variance of the indoor data [2.5 levels in an Australian primary school were mainly affected by the outdoor PM2.5  [2.5 and black smoke variation could be explained by daily outdoor variation for those pollutants [Only a small number of studies considered children’s mobility at ages when exposure at the residential address becomes less relevant and assigned time-weighted TRAP exposures at day care centres and schools and other locations where the child spends significant time alongside residence. Children may spend only around 50%–60% of their time at home, and the rest elsewhere e.g., at school . TRAP exoor data . Another < 0.01) . Anotherllutants .Further, investigating different exposure time windows may highlight other relevant exposure windows beyond the birth year and early-life that are commonly studied. The differences between effects of early exposure versus later exposures or exposures with greater duration is yet unclear and is difficult to detangle due to the limited number of studies investigating different time windows. Some authors have suggested that exposures of longer duration at elevated TRAP levels may be necessary to generate pathophysiological changes leading to asthma development and therefore may be behind the observed effects .Novel approaches to exposure assessment are underway including the use of OMICS technologies that measure biological molecules and/or activity in the body  to identify fingerprints of air pollution ,102. AltAlthough there appear to be statistical significant associations between TRAP and the development of childhood asthma, there is a further need to improve the exposure estimates, and therefore improve the exposure–response functions and the consistency of the study findings. This is important for example when these exposure–response functions are used for burden of disease and health impact assessment studies, and for better understanding the underlying mechanisms of TRAP and childhood asthma and the potential differential pollutant effects and drivers of heterogeneity. Over the past few years, there has been an epidemic increase in the number of studies in the field, and there are likely to be more studies over the next few years given the importance of the topic. Improvements in exposure assessments, as we discuss in this paper, may well increase the scientific value of these new studies. More refined exposure models are needed, and will arguably produce the most robust associations when investigating the potential health effects of TRAP. Furthermore, we also emphasize the need to incorporate mobility patterns in the exposure estimates and to undertake personal exposure monitoring to cross validate modelling estimates.Although our previous meta-analysis found statistically significant associations for various TRAP exposures and childhood asthma, further refinement of the exposure assessment may improve the risk estimates and shed light on critical exposure time windows, putative agents, underlying mechanisms and drivers of heterogeneity.


Histomonas meleagridis, were included. For tissue samples from SPF chickens RPL13 and TBP were found to be the most stable reference genes. Further testing of RPL13 and TBP in the same organs of uninfected and infected SPF broiler chickens with the intracellular pathogen fowl aviadenovirus confirmed this finding. In tissue samples from turkeys, a stable expression of RPL13 and TFRC genes was noticed. Overall, the determined reference genes should be considered whenever gene expression studies in spleen, liver, caecum and caecal tonsil of chickens and turkeys are performed.Evaluation of reference genes for expression studies in chickens and turkeys is very much limited and unavailable for various infectious models. In this study, eight candidate reference genes HMBS, HPRT1, TBP, VIM, TFRC, RPLP0, RPL13 and RPS7 were evaluated by five different algorithms  to assess their stability. In order to analyze a broad variation of tissues, spleen, liver, caecum and caecal tonsil of different aged specific pathogen free (SPF) layer chickens and commercial turkeys, uninfected or infected with the extracellular pathogen The online version of this article (doi:10.1186/s13567-016-0388-z) contains supplementary material, which is available to authorized users. Gallus gallus), a number of studies validated reference genes under different physiological conditions [Meleagris gallopavo) [Histomonas meleagridis at different ages. In SPF broiler chickens, preselected reference genes were further evaluated using samples from birds infected with the intracellular pathogen fowl aviadenovirus (FAdV).Gene expression analysis provides insights into complex biological regulatory processes and has become an essential part in various molecular biology studies. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is, in many studies, the method of choice for the detection and quantification of mRNA . Howevernditions –20. Howellopavo) . This prth day of life, to cover age related changes in gene expression. In addition, the effect of infectious pathogens on the expression of reference genes in host birds was investigated by collecting samples during the course of disease. For that, the same organs were sampled on three different time points from SPF layer-type chickens and on two time points from turkeys following infection with the extracellular pathogen H. meleagridis between the 35th and 49th day of life. Infected birds showed inflammation of the caeca and the livers with the exception of chickens sampled on the day 49, when two out of three birds had no lesions. Healthy SPF broiler chickens were sacrificed at the age of 4, 7 and 21 days of life to collect spleen, liver, caecum, and caecal tonsils of three birds each time point. The same organs of three additional SPF broiler chickens that were infected with the intracellular pathogen fowl aviadenovirus were sampled at day 7 of life. Swollen marble-like livers with a colour ranging from yellow to brown were observed in infected birds [A total of 252 different tissue samples from 27 SPF layer-type chickens , 12 SPF broiler chickens  and 24 commercial turkeys  were included in the analysis  at −80 °C.All samples were collected separately during post mortem investigations directly after euthanization of birds and stored in RNAH. meleagridis were processed. Further analyses of RPL13 and TBP were performed using the same organs from healthy and fowl aviadenovirus infected SPF broiler chickens.Based on findings from previous gene expression studies on different bird tissues , 17, 18,later® (Qiagen) stabilized liver, spleen, caecum and caecal tonsil tissues. Tissue samples were homogenised separately using QIAshredders (Qiagen) and total RNA was extracted by RNeasy® mini kit (Qiagen) according to manufacturer’s instructions and RNA was stored at −80 °C before further use. Every sample was assessed according to following criteria: the nucleic acid purity was analyzed with A260/280 and additionally A260/230 ratio by NanoDrop 2000  to ascertain that the RNA was free from contaminates like guanidine, glycogen and EDTA. The RNA quality and quantity of every sample was further surveyed by chip-based capillary electrophoresis Bioanalyzer 2100 . In this process, the RNA concentration and the integrity of total RNA together with the presence or absence of degradation products were estimated by measuring the entire electrophoretic trace of each sample, given by the RNA integrity number (RIN) [Total RNA was prepared from RNAer (RIN) .An identical set of primer and probe sequences were designed for both poultry species, chickens and turkeys, to target highly conserved regions of reference genes. Primers and probes were selected by using the respective sequence information from the NCBI database Table  and GenSOrgan samples of healthy and infected SPF layer chickens and turkeys were analyzed separately. The stability of gene expression was determined by calculations using different software algorithms: GeNorm, NormFinder, BestKeeper© and delta CT. In addition, RefFinder was used to rank the genes on the basis of stability from the most to the least stable reference genes.The GeNorm algorithm was used to calculate the average of pairwise variation of one gene with all the other potential reference genes and to identify their average expression stability (M). The gene with lowest M value was assumed to be the highest stable gene . Genes wNormFinder software generates a stability measure (Sv) which indicates an increased stability in gene expression by a low value. NormFinder software allows direct estimation of expression variation between different organ groups and ranked genes according to the similarity of their expression profiles by using a model-based approach. The chance to introduce systemic errors in genes with a low Sv value were found to be marginal, as previously described .p), as explained earlier [BestKeeper© calculated the standard deviation (SD) based on raw crossing point (CP) including the inter-gene relationship with the help of Pearson correlation coefficient matrix. Highly correlated genes were combined into an index which is called BestKeeper© index. The comparison of the correlation of each gene with BestKeeper© index gave a correlation coefficient value (r) with the probability value . The integrity of each RNA sample considered for RT-qPCR analysis was ensured by reaching a RIN value of 6.5 to 10 , TFRC (1.302) and TBP 1.364) were within the threshold range of M value ≤ 1.5 indicating a reliable stability and in case of infected chickens RPL13 (0.753), TBP (0.753), TFRC (0.848) showed even more stable threshold with M values ≤1.0 , TBP (0.978) and TFRC (1.192), whereas it was slightly perturbed in tissues from infected chickens, with TBP (0.377) as the most stable followed by RPL13 (0.509) and TFRC (0.719)  and infected layer chickens (0.7) Table .The delta CT results supported GeNorm, NormFinder, BestKeeper© findings. RPL13 , TFRC , TBP  genes showed the most constant CT values in chicken samples , TBP  and TFRC  were most stable in contrast to RPLP0, HPRT1, HMBS which were found to be the least stable genes for healthy and infected layer chickens Table .Additionally, the variation in gene expression of the two most stable genes in layer-type chickens, RPL13 and TBP, was investigated in SPF broiler chickens. The same organs sampled from SPF layer chickens were also investigated from healthy and fowl aviadenovirus-infected SPF broiler chickens (Additional file Recent studies focused on the validation of reference genes applied on different avian tissues , 17, 18.In a first step candidate reference genes, namely TBP, HPRT1 and HMBS, were selected in the present work according to their previously described stability in gene expression studies of various avian tissues or cells, such as muscular tissues, liver and leukocytes isolated from spleen, thymus and Bursa of Fabricius of chickens , 14, 17.th day of life, and SPF broiler chickens from day-old to 21st day of life. Finally, the impact of different kind of pathogens on the gene expression in organs of host birds was investigated. For that, chickens and turkeys were infected with the extracellular pathogen H. meleagridis following a well-defined infection model [According to the MIQE guidelines reference genes can vary between different species and have to be validated even between closely related species . Consequon model . Hence, H. meleagridis. TBP and TFRC were both shown to be stable as well; however, there were slightly higher variations in the expression levels of TFRC in tissues of infected SPF layer chickens. Depending on the experimental setup TFRC also can be used as reference gene along with RPL13 and TBP for chickens. All other reference gene candidates calculated with RefFinder were not considered as stable for SPF layer chickens due to severe variations in their expression profiles. Hence, RPL13 and TBP were further investigated in tissues of non-infected and infected SPF broiler chickens with fowl aviadenovirus. These additional investigations confirmed the stable expression of both genes in SPF broiler chicken spleen, liver, caecum and caecal tonsil samples. Our findings demonstrated that neither the genetic background of chickens nor the nature of an infectious agent caused remarkable variations of RPL13 and TBP expression in spleen, liver, caecum and caecal tonsil, suggesting them as optimal candidates for normalization of RT-qPCR experiments. Similar to the results found in chicken tissues, RPL13 was also determined to be the most stable gene, followed by TFRC in spleen, liver, caecum and caecal tonsil samples from healthy and infected turkeys with the extracellular pathogen. In contrast to data from chickens, presented here and elsewhere [The expression of each reference gene for every sample was analyzed with different algorithms: GeNorm, NormFinder, BestKeeper© and delta CT. The rankings of assayed genes were not always identical due to variations in statistical calculations by different algorithms, a phenomenon recently reported . Therefolsewhere , TBP of lsewhere .According to the MIQE guidelines, a reference gene needs to be validated and established for every species and for different physiological conditions. The guidelines furthermore specify verification of a used reference gene for every experimental settings, otherwise the variation of reference genes expression may adversely affect the results and with it the biological conclusion. The RPL13 and the TBP genes of chickens and the RPL13 and the TFRC genes of turkeys were shown to be highly stable in the present experimental setting and are therefore recommended to be first priority candidates for gene expression studies in case spleen, liver, caecum and caecal tonsil tissues are targeted.