Peronospora effusa is an obligate pathogen that causes downy mildew on spinach and is considered the most economically important disease of spinach. The objective of the current research was to assess genetic diversity of known historical races and isolates collected in 2014 from production fields in Yuma, Arizona and Salinas Valley, California. Candidate neutral single nucleotide polymorphisms (SNPs) were identified by comparing sequence data from reference isolates of known races of the pathogen collected in 2009 and 2010. Genotypes were assessed using targeted sequencing on genomic DNA extracted directly from infected plant tissue. Genotyping 26 historical and 167 contemporary samples at 46 SNP loci revealed 82 unique multi-locus genotypes. The unique genotypes clustered into five groups and the majority of isolates collected in 2014 were genetically closely related, regardless of source location. The historical samples, representing several races, showed greater genetic differentiation. Overall, the SNP data indicate much of the genotypic variation found within fields was produced during asexual development, whereas overall genetic diversity may be influenced by sexual recombination on broader geographical and temporal scales. Spinacia oleracea) is believed to be native to the Middle East and has been cultivated for more than 1,300 years [th century and was introduced to the new world and southern hemisphere in the 16th century [Spinach . As a result, the higher plant population densities have influenced disease pressure. Increased market demand has also affected the spinach seed production industry. Although some open pollinated lines of spinach are produced, hybrid seed represents over 90% of production. Hybrid spinach lines are produced by the use of separate male and female inbred lines with different resistance characteristics for downy mildew disease ,9.Peronospora effusa causes downy mildew disease in spinach, and is one of the most economically important diseases constraining spinach production [oduction . It is aoduction . Initialoduction . If condoduction . Sporangoduction .P. effusa can be classified into distinct races based on infection phenotypes on a standardized set of different spinach cultivars [P. effusa has exponentially increased in recent years with 15 races described [P. effusa was identified in 1824, and in 1950, resistance to race 1 was determined to be conveyed by a single dominant gene. Race 2 was described in 1958 and soon thereafter resistance to both race 1 and race 2 was discovered [P. effusa have been identified [Isolates of ultivars . The ratescribed . The firscovered . In 1976entified ,13. In 2entified . CurrentManagement of spinach downy mildew is problematic. Cultural controls include crop rotation, reduction of “green bridges”, and efforts to reduce free moisture on leaves. Although there are some fungicides available for management of spinach in conventionally grown production fields, approximately 40% of spinach grown in California and Arizona is produced organically where synthetic fungicides are prohibited. Thus, disease resistance is the most economical and environmentally sound means of managing downy mildew in both conventional and organically produced spinach.P. effusa. When used to assess seed lots, a high percentage of seeds tested positive for contamination via PCR, but infected seedlings were not observed in grow-out studies (6). This discrepancy may reflect the presence of inviable propagules of the pathogen, lack of seed transmission among certain isolates, or inadequate conditions for seed transmission in the experimental conditions.Oospores have been detected in washings of seeds and infected seedlings were produced from contaminated seed . OosporeP. effusa, assessments of population diversity on various spatial and temporal scales are critical. For example, new races could originate via spontaneous mutation among prevalent genotypes, or could originate from selective sweeps due to the introduction of new genotypes on infected plant material. Initial population genetics studies in P. effusa were based on assessments of rDNA sequences and suggested low levels of genetic diversity among isolates [P. effusa is an obligate pathogen, the disease is postulated to have spread with the crop and is thought to have originated in a small geographic area [P. effusa has hindered efforts to utilize more sophisticated molecular markers to assess genetic diversity in the context of evolutionary biology.To better understand the emergence of new races of isolates ,6. Howevhic area . HoweverP. effusa and to characterize diversity in a historical panel of race types and contemporary field populations in Arizona and California.Our goal was to develop novel single nucleotide polymorphism (SNP) markers for All samples were collected from commercial production fields of spinach and in each case, at every location; the grower (owner of the crop) gave permission to collect infected plant tissue. The specific geographic coordinates for each of the fields is not included as the growers requested this information remain private. Infected leaves were collected from spinach production sites in Yuma, Arizona in March of 2014 and from the Salinas Valley in May of 2014 and stored for 3 to 7 days on ice or in a refrigerator at 4°C. A disc (approximately 7mm diameter) of infected tissue was excised from each leaf and processed for genomic DNA extraction in a 96-well plate as previously described . BrieflyThe historical race-type panel of isolates is maintained at the University of Arkansas . The racP. effusa, UA2209 and UA4410 (race 12 and 14 respectively) and sequenced using 100bp paired-end Illumina sequencing on a HiSeq 2500 device . The sequencing is part of a larger effort to develop a reference genome for P. effusa (underway and to be reported separately). Here the sequence data was exclusively used to identify putative SNP sites. Raw sequence data (≈145M paired reads per isolate) was assembled de novo into contigs using CLC Genomics Workbench 7.5  at default settings. All further sequence manipulation, mapping and genotyping were accomplished with CLC. The resulting 300 largest contigs for isolate UA2209 were processed further to identify all open reading frames (ORFs) greater than 1000 amino acids (3000bp). Of these, the largest 100 contigs with ORFs showing high predicted protein similarity, as assessed using Blastx in CLC, to other oomycete organisms (e.g. P. infestans) were used as a reference. The raw data from isolates UA4410 and UA2209 was then mapped separately to the 100 contig reference using settings of 90% similarity and 90% sequence coverage to identify heterozygous positions. Variable positions were assigned when the alternate allele frequency was >15% and <85%. Polymorphic SNP sites that fell within the 1000 amino acid putative genes and are predicted to be synonymous (non-amino acid changing) were then catalogued and used to choose 72 SNPs residing on separate contigs for further analyses.Genomic DNA was extracted from two isolates of http://probes.pw.usda.gov/batchprimer3/) at default settings for generic primers with total amplicon size set as an optimum of 100bp with the amplified region containing the target SNP  clustering model using the program STRUCTURE v2.3.4 ,20. The arvester . There warvester –24.P. effusa isolates sampled. For each SNP locus, the number of genotype is 2 when no heterozygous individuals are observed or 3 when there are heterozygous individuals. Heterozygosity is the proportion of heterozygous individuals in the population; gene diversity is defined as the probability that two randomly chosen alleles from the population are different; and PIC is a closely related diversity measure [http://www.ualberta.ca/~fyeh/popgene.html) [For each SNP locus the sample size, number of observations, availability, genotype number, allele number, major allele frequency, heterozygosity, gene diversity, and polymorphism information content (PIC) were calculated using the PowerMarker V3.25 software [ne.html) .Genomic DNA from 375 samples, including 33 samples from a historical panel of race types, was submitted for targeted sequencing. Of these, 167 field samples and 26 of the historical race types amplified well enough to be included in further analysis; including 68 samples from Yuma, Arizona and 99 from the Salinas Valley, California. The 26 race types included races 3, 4, and 10–14. Forty seven markers had a minimum of 10X sequence coverage for all 193 samples. One marker was fixed for homozygosity and removed and the remaining 46 markers were used for subsequent analysis. The alternate allele coverage for most samples generally fell into a distribution around 50%, suggesting most samples are diploid (data not shown).The genotypes for the 12 previously sequenced SNP positions were replicated and showed identical genotypes using the Ion Torrent, Ion Proton and MiSeq sequencing devices. The 96 field samples, analyzed on all three sequencing platforms, produced identical multi-locus genotypes for all 46 SNPs included in the study.In total, there were 82 unique multi-locus genotypes. Of these, 21 genotypes were found more than once and assigned a genotype identifier of G1-G21 . The altWhile most identical genotypes were recovered from field samples, there were two cases, G13 and G14, where identical genotypes were recovered for two race 10 (G13) and two race 13 (G14) samples .Analysis using the program STRUCTURE indicated a K value of 5 based on the largest ln Pr(X|K) value . Fig 2 iP. effusa genotypes had no missing data , was the only isolate from the race panel that was found in STRUCTURE group five, the largest group of field samples. This isolate is a new race and is currently in a ring test being conducted by the International Working Group on Peronospora to be nominated as a new race. Closely related genotypes to this isolate dominated the field populations in 2014. The appearance of this new race (and the many genotypes similar to it) is likely due to selection pressure imposed by growers who have increasingly relied on spinach cultivars resistant to races 1–15 in Yuma and the Salinas Valley.Although some historical races cluster together (e.g. race 14) there is not a strict correlation between genotype and race type. Therefore, it is not possible with this panel of neutral SNP markers to strictly assign a race type based on multi-locus genotype. However, as more markers become available, and more information is generated on the genome of this pathogen, progress can be made toward this goal.Phytophthora capsici, and was found to be associated with changes in pathogenicity, virulence and mating type [It is becoming increasingly clear that oomycete plant pathogens have the potential to produce significant amounts of genotypic variation during asexual population expansion . The majing type . How thiAlthough the 2014 epidemics appear to be dominated by a few clonal lineages with sub-clonal variation, there appears to be sufficient genotypic diversity in the historical race panel to suggest sexual as well as asexual reproduction contributes to diversity over time. Sexual reproduction of the spinach downy mildew pathogen has been documented based on the observation of oospores in California  and in sThe current study documents a relatively high level of genotypic diversity within a regional sample of the spinach downy mildew pathogen and indicates genomic variation within a clonal context is common. This initial study is a useful baseline to address how asexual plasticity may impact evolving virulence of the pathogen.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Candida albicans is a ubiquitous opportunistic fungal pathogen that afflicts immunocompromised human hosts. With rare and transient exceptions the yeast is diploid, yet despite its clinical relevance the respective sequences of its two homologous chromosomes have not been completely resolved.C. albicans as a function of the indel rate, and provides a general framework to interpret repeat abundance in species ranging from bacteria to humans.We construct a phased diploid genome assembly by deep sequencing a standard laboratory wild-type strain and a panel of strains homozygous for particular chromosomes. The assembly has 700-fold coverage on average, allowing extensive revision and expansion of the number of known SNPs and indels. This phased genome significantly enhances the sensitivity and specificity of allele-specific expression measurements by enabling pooling and cross-validation of signal across multiple polymorphic sites. Additionally, the diploid assembly reveals pervasive and unexpected patterns in allelic differences between homologous chromosomes. Firstly, we see striking clustering of indels, concentrated primarily in the repeat sequences in promoters. Secondly, both indels and their repeat-sequence substrate are enriched near replication origins. Finally, we reveal an intimate link between repeat sequences and indels, which argues that repeat length is under selective pressure for most eukaryotes. This connection is described by a concise one-parameter model that explains repeat-sequence abundance in The phased genome assembly and insights into repeat plasticity will be valuable for better understanding allele-specific phenomena and genome evolution. The advent of short-read DNA sequencing has resulted in super-exponential growth in the quantity of available sequencing data. Along with a dramatic increase in the number of assembled reference genomes for different species, much recent effort has been focused on defining the sequence variants - such as SNPs and insertions/deletions (‘indels’) - between individuals of the same species. The focus of many such studies is the resolution of haplotypes , which sCandida albicans is a model fungal pathogen that almost exclusively exists in a diploid state and does not achieve genome diversity via a typical meiotic cycle with frequent recombination. Instead, it employs one of two strategies, both involving mating and whole chromosome loss, where the order of these events is inverted. First, in the so-called ‘parasexual cycle’ [C. albicans, and both leave the homologs largely intact. Thus, the phasing of polymorphisms in C. albicans has fewer entropic, degenerating forces than in most other organisms, making the assembly of its phased genome particularly desirable.Most of the effort in haplotype discovery has focused on humans; thus, there are few phased genomes available in other multiploid model organisms. Knowing the phasing information in model organisms, however - for example, those that are single-celled, have compact genomes, double rapidly, and are easily manipulated genetically - is useful for a variety of reasons, including ease of measurement of allele-specific phenomena in different genetic backgrounds and observation of homolog-specific evolution on laboratory timescales. l cycle’ , two dipl cycle’ ,13. Altel cycle’ . ImportaC. albicans and many closely related Candida species has yielded important insight into the pathogenicity of C. albicans[C. albicans genome, Assembly 19, was partially diploid and identified thousands of polymorphisms using low-coverage Sanger sequencing, but had long haploid spans and did not assemble the genome into full chromosomes [Extensive sequencing of  albicans, as wellomosomes . The nexomosomes , assemblomosomes . Here weC. albicans, we performed deep sequencing on genomic DNA prepared from a panel of nine strains, including wild-type SC5314 and eight related strains, each known to be homozygous for one of the eight C. albicans chromosomes  at each position across the genome Figure D, top. NRegions of unexpected homozygosity enhanced our SNP-identification procedure. Our initial strategy Figure B assumedThe length-scale of our phasing results is on the order of whole chromosomes. Interestingly, for the entirety of chromosome 5 and nearly a megabase of chromosome 3, at least one strain was homozygous for the opposite homolog as the other(s) . Positions with distance <0.195 - where the marginal true- and false-positive rates are equal . C. albicans SC5314 strain. We succeeded in mapping 98.25%  of the previously identified SNP positions from the contig-based coordinates of Assembly 19 into the chromosomes of Assembly 21. Of these, 75%  were corroborated and phased in this study. The average heterozygous max-to-sum ratio of the remaining 25% that were not confirmed as SNPs was 0.92, strongly suggesting that these positions are not polymorphic but were perhaps misidentified as such due to the lower coverage of previous assemblies. The 69,688 total SNPs we identified here thus represent an increase of nearly 69%  in the number of known SNPs.Our study appreciably revises and expands the number of SNPs in the laboratory standard ALS2, ALS4, and ALS9), which are largely identical but divergent enough to complicate read-mapping and SNP resolution [An independent test of phasing fidelity confirms our results. Since paired-end fragments necessarily originate from a contiguous DNA molecule, they are properly phased by definition. Thus, we assessed the validity of our phasing method - which treats each nucleotide position independently of all others - by determining the consistency in phasing across all SNP positions included in the 72 nucleotides (2 ends × 36 nucleotides/end = 72 nucleotides) sequenced in each set of paired-end reads. We found that 94% of SNPs were part of a paired-end molecule in which both ends had SNPs, suggesting that this assay to compare phasing at opposing paired ends was nearly exhaustive. Of these, 99.8% of SNPs were consistently phased between the two paired ends. Further, the molecules with phasing disparity between the two ends were highly localized in a few positions in the genome, nearly all corresponding to adhesion genes of the ALS family .SNP phasing increases the precision with which allele-specific phenomena, such as allele-specific messenger RNA expression, can be measured. Using an RNA-seq dataset generated from the wild-type SC5314 strain grown in rich media , we mappBy pooling reads across many SNPs, small but significant allele-specific biases are detectable. There are 11 SNP windows across orf19.3556, and summing reads across SNP windows indicates a 30% bias in expression of the B allele over the A allele  relative to their partner alleles . We tabulated the gap positions into a genome-wide histogram, identifying thousands of putative indel positions in the genome, many with hundreds of reads supporting an indel  are expected to yield minor expression effects (<20%) based on fluorescence reporter systems that isolated their relative contribution [Since indels in the repeats of promoters could affect gene expression via their role in nucleosome positioning, we tested whether indel density in the promoter correlated with allelic bias from the RNA-seq data , but we ribution , and (2)The indels identified were not artifacts of systematic errors in the sequencing of repeats. We plotted the average indel length reported by all reads at each validated indel position and summarized the results in a histogram Figure G. If ranIndels are not uniformly distributed throughout the genome. This clustering of indels is conspicuous in a genome-wide string indicating indel positions with an ‘X’ and each likely indel substrate  with a dash  are enriched near replication origins. We measured indel density in 7.5 kb windows surrounding the 142 high-confidence replication origins mapped across the s genome   is the probability of a single repeat unit , IR(n) is the observed indel rate for repeats of length n, and α is a scalar multiplier of the indel rate. For short repeats, indels are rare (IR(n) ~ 0), so the model predicts that the number of repeats is unbiased, dominated simply by the random probability of incorporating a single repeat unit, p(R). However, once the indel rate rises, it modulates the probability of adding more repeat units: for positive values of α, longer repeats are more favored than random, as we observed in C. albicans.where IR(n) was the indel rate for single units  was the indel rate for AT or TA dyads . Strikingly, positive α values are common across a broad range of species, but only in eukaryotes, not in prokaryotes . Such determinants can be elucidated because measuring differences between thousands of allelic pairs provides a broad scope of sequence variants while simultaneously facilitating the attribution of expression differences to particular sequence features. In other words, since each allelic pair has only a few polymorphisms, phenomenological deviations between alleles can be more easily attributed to specific sequence features than is possible when comparing totally different genes. Due to the technical difficulties associated with mapping haplotypes, allele-specific measurements have predominantly relied on inbred strains or unnatural hybrid diploid organisms, where viable haploids could be sequenced prior to hybrid construction such that the respective haplotypes were known [C. albicans is a natural organism that is almost exclusively diploid and frequently heterozygous, its alleles may have evolved complex and physiologically relevant interactions that would not have developed in a multi-species hybrid or highly inbred population.A fully phased diploid genome for a unicellular model organism like re known ,31-33. SC. albicans; however, such events typically involve whole chromosomes , and it thus remains unclear whether there is an alternative LOH mechanism that occurs on a shorter length scale that can effectively erase indels through homozygosis.We found that indels are spatially clustered throughout the genome, with nearly 93% of indels in multikilobase spans that collectively comprise only 45% of the genome. In that both indel-dense and indel-sparse regions contain a similar level of coding sequence, it seems unlikely that the disparity in indels arises from gross differences in sequence context . We speculate that these spans could result from regions undergoing loss-of-heterozygosity (LOH) events, which are frequently observed in C. albicans erase indels but preserve repeats.The subtle increase in indel and repeat-sequence density that we observe near replication origins poses an interesting conundrum. In particular, it is not actually clear whether indels are favored near replication origins over an evolutionary timescale. At first, it seems that there are simply more indels near origins because there are also more repeats, arguing that indels have no higher propensity to occur near origins than elsewhere. However, since indels may be the driving force behind the creation of repeats in the first place - a possibility reinforced by our one-parameter model - indels may indeed be more likely near origins. For instance, one could imagine that a DNA polymerase prone to introducing indels is not as processive as ordinary polymerases, thus leading to an indel bias  near origins. Ultimately, it is nontrivial to resolve this problem since the LOH events that occur in C. albicans genome. Though we cannot exclude the possibility that this correspondence is purely coincidental, it seems unlikely that one of the few mechanisms that locally alters the length of DNA  is not involved in the generation of DNA sequences whose length is the chief determinant of their function . A causative relationship is further supported by the correlation between sharp increases in the indel rate and marked increases in repeat abundance for both homopolymers and dyad repeats.Due to their role in positioning nucleosomes and relevance to human disease, long repeats in eukaryotic genomes have been the topic of many experimental and quantitative modeling studies ,29,34-36C. albicans and Saccharomyces cerevisiae) suggests that the net effect of indels is to lengthen repeats   Figure F. The sidirectly ,29,37, dC. albicans genome reveals phenomena that are both expected, such as allele-specific expression, and unexpected, such as indel clustering. We anticipate that the higher degree of genomic resolution provided here will empower not only future researchers of this important model organism, but also those who study allele-specific traits and expression characteristics more generally.We have shown that the fully phased In addition to the SC5314 wild-type strain, the following homozygosed strains - all from  and geneGenomic DNA was prepared from saturated overnight cultures from single colonies grown in YPD. Cell pellets were ruptured by vortexing with glass beads; DNA was extracted using phenol/chloroform/isoamylalcohol (25:24:1) and then precipitated in isopropanol and sodium acetate. After ethanol washing, DNA was resuspended in TE + RNase. Library construction methods were previously described . LibrariFor SNP identification, reads were mapped to the Assembly 21 reference genome using Bowtie (v1.0) , allowinReads from  - accessThe two-state HMM Figure  was fit HMM: Hidden Markov model; indel: Insertion/deletion; LOH: Loss-of-heterozygosity; NMD: Nonsense-mediated decay; ORF: Open reading frame; PTC: Premature termination codon; SNP: Single-nucleotide polymorphism.The authors declare that they have no competing interests.DM, JSW, and GS designed the study. DM prepared samples, and KS finished library preparation and performed DNA sequencing. DM performed analysis of the data and drafted the manuscript. JSW and GS offered insights into the analysis and edited the manuscript. All authors read and approved the final manuscript.Figures S1 to S5, Table S1.Click here for fileTable listing characteristics of C. albicans ORFs, including coordinates in phased assembly, allele-specific expression levels, number of SNPs and indels in coding region, and number of indels in promoter.Click here for filePhased FASTA file (SNPs only).Click here for filePhased FASTA file (SNPs plus indels).Click here for file


Bemisia tabaci is one of the most damaging agricultural pests world-wide. Although its control is based on insecticides, B. tabaci has developed resistance against almost all classes of insecticides, including neonicotinoids.B. tabaci (Q1 biotype). Twelve libraries from insecticide resistant and susceptible whitefly populations were sequenced on an Illumina Next-generation sequencing platform, and genomic sequence information of approximately 73 Gbp was generated.We employed an RNA-seq approach to generate genome wide expression data and identify genes associated with neonicotinoid resistance in Mediterranean (MED) de novo assembly and functionally annotated. A total of 146 P450s, 18 GSTs and 23 CCEs enzymes (unigenes) potentially involved in the detoxification of xenobiotics were identified, along with 78 contigs encoding putative target proteins of six different insecticide classes. Ten unigenes encoding nicotinic Acetylcholine Receptors (nAChR), the target of neoinicotinoids, were identified and phylogenetically classified. No nAChR polymorphism potentially related with the resistant phenotypes, was observed among the studied strains.A reference transcriptome was built by DE analysis revealed that among the 550 differentially (logFC > 1) over-transcribed unigenes, 52 detoxification enzymes were over expressed including unigenes with orthologues in P450s, GSTs, CCE and UDP-glucuronosyltransferases.B. tabaci. Using quantitative qPCRs, a larger screening of field MED B. tabaci from Crete with known neonicotinoid phenotype was performed to associate expression levels of P450s with resistance levels. Expression levels of five P450s, including CYP6CM1, were found associated with neonicotinoid resistance. However, a significant correlation was found only in CYP303 and CYP6CX3, with imidacloprid and acetamiprid respectively.Eight P450 unigenes belonging to clades CYP2, CYP3 and CYP4 were highly up-regulated (logFC > 2) including CYP6CM1, a gene already known to confer imidacloprid resistance in B. tabaci. No evidence of target site neonicotinoid resistance has been found. Eight P450 unigenes, including CYP6CM1, were found significantly over-expressed in resistant B. tabaci. This study suggests at least two novel P450s (CYP303 and CYP6CX3) as candidates for their functional characterization as detoxification mechanisms of neonicotinoid resistance in B. tabaci.Our work has generated new toxicological data and genomic resources which will significantly enrich the available dataset and substantially facilitate the molecular studies in MED The online version of this article (doi:10.1186/s12864-015-2161-5) contains supplementary material, which is available to authorized users. Bemisia tabaci (Hemiptera: Aleyrodidae) (sweet potato or tobacco whitefly) is a broad phloem feeding herbivore. B. tabaci is a complex of 31 putative morphocryptic species [B. tabaci as a pest refers mainly to two species of the complex, MEAM1  and MED  which are both highly invasive and polyphagous (over 1000 host plants reported) and particularly detrimental by transmitting viruses which cause serious crop diseases.The whitefly  species . The curB. tabaci relies on insecticides. However, their extensive and chronic use has imposed strong selection pressures for resistance to many insecticide classes in B. tabaci, including neonicotinoids, which are the world market leaders of insecticides [Currently, the control of cticides –7. Neonicticides .B. tabaci, it was shown that the cytochome P450 CYP6CM1 is the major resistance mechanism, as the gene expression is highly associated with the resistance phenotype in a number of populations worldwide [Insecticide resistance is typically characterized by a variety of molecular aberrations, such as transcriptional changes, gene amplification and point mutations in coding regions, which in turn result in phenotypic novelties like increased rates of insecticide detoxification or reduced sensitivity of the target protein(s). The most significant mechanisms of resistance to neonicotinoid insecticides are decreased target-site sensitivity and increased metabolic detoxification by cytochrome P450 monooxygenages , 10. In orldwide , and it B. tabaci [, a major vegetable crop production area in Greece, especially towards the neonicotinoid insecticide imidacloprid [The phenomenon of insecticide resistance is particularly striking in Mediterranean countries, where suitable climatic conditions allow year-round crop production and the survival of . tabaci , 12. Higacloprid . Pymetroacloprid .B. tabaci cytochome P450 CYP6CM1 was found to be over-expressed in Cretan populations; however, in some cases resistance ratios were not strongly correlated with CYP6CM1 expression level, suggesting that additional neonicotinoid resistance mechanisms may operate in Crete [The in Crete .de novo transcriptome sequencing and has already been applied in transcriptome analysis of insects, such as Aedes aegypti, Anopheles funestus, Nilaparvata lugens, Locusta migratoria, B. tabaci, Trialeurodes vaporariorum [A. aegypti [Cimex lectularius [B. tabaci, there is a number of transcriptome resources generated by Roche 454 pyrosequencing or Illumina deep sequencing which have substantially improved our understanding on topics such as; symbiosis, virus-vector interactions, Bauveria bassiana infection, gut role or gene expression in different developmental stages and genetic groups within B. tabaci [B. tabaci transcriptome data available explores the transcriptome of the MED lineage of this complex species [RNA sequencing (RNA-Seq) was the decisive tool to perform rariorum –19 and mrariorum , providirariorum . Some re aegypti  and Cimetularius . In B. t. tabaci , 24–28. . tabaci . Moreove species , 28.B. tabaci from Crete. Twelve libraries from three resistant and one susceptible B. tabaci strain including biological replicates were sequenced with the Illumina platform, and generated about 73 Gbp of sequence data. This high throughput sequencing technique has allowed for global expression profiling of neonicotinoid resistant and susceptible populations. A reference transcriptome was built by de novo assembly and functionally annotated. It was then used to map the reads from insecticide-susceptible and resistant strains, study the levels of gene expression and gain genetic information for resistance mechanisms.In the present study bioassays and transcriptome data from the MED species were analyzed to further characterize the underlying molecular mechanism responsible for neonicotinoid resistance in B. tabaci MED, we chose the whitefly populations used in the present study from those displaying the highest resistance phenotypes and heaviest neonicotinoid treatment history. Three field derived strains originally collected from eggplants maintained in the laboratory on cotton plants under Imidacloprid  or Acetamiprid Selection (GR4-AS) for few (six) generations were used in this study, along with their Parental ‘relaxed’ strains , namely the respective original strains maintained in the absence of any insecticide contact. All comparisons were performed with a local susceptible reference strain (S-GR6), collected in Crete in 2006, in order to access differences at the transcription level linked with differences at the resistance phenotypes.Based on recent resistance monitoring in Cretan 50 was reduced from 423 to 185 mg/L for GR4-AS, from 301 to 207 mg/L for GR9-IS and from 177 to 52 mg/L for GR0-IS) after pre-exposure to PBO  to the three neonicotinoid insecticides imidacloprid, acetamiprid and thiachloprid were determined by classical leaf dip bioassays on adult females Table . CompareCompared to their relaxed parental strains, the respective selected strains exhibited higher resistance levels towards acetamiprid and imidacloprid but not towards thiacloprid, indicating that the mechanism underlying resitance to thiacloprid is not selected by imidacloprid or acetamiprid treatments.B. tabaci transcriptom changes linked to the resistance, we compared also strains with the same genetic background which differ only in their resistant level. The pair GR4-AP and GR4-AS displayed the higher differences (6× with imidacloprid and 3.6× with acetamiprid) among the three selected-parental pairs of strains . These two strains were therefore chosen for styding their transcriptomic profile in the RNA-seq experiment in comparison to the susceptible S-GR6, which was used as a reference strain. In order to better understand the part of ns Table . TherefoB. tabaci strains  yielded more than 396 million paired-end reads. The pre-processing technique  resulted to 369.04 million reads (72.75Gbp).Illumina Hiseq 2000 sequencing of 12 cDNA libraries  from four The short read sequences were deposited into the Sequence Read Archive (SRA), (BioProject ID: PRJNA293094). The full list of transcript sequences is available upon request from the corresponding author.The number of reads was evenly distributed among the 12 libraries, with an average of 30.75 +/− 2.4 million reads. The summary of reads per library used in the subsequent analysis is shown in Additional file The intra- and inter-sample fraction for the contaminant ribosomal RNA as well as the mitochondrial cDNA was also similar among them  indicating moderate variation between libraries.De novo assembly performed using Trinity produced 170,377 contigs , all the usable sequencing reads were aligned onto the transcripts using Bowtie2. A high percentage of reads (97.14 %) were successfully back-mapped on this assembly and at least 71.6 % of the mapped reads were proper pairs.nr NCBI protein database was examined using Blastx and disclosed 31,505 contigs with coverage higher than 80 %. In addition, similarity to a dataset of 129 complete mRNA of B. tabaci downloaded from NCBI, using blastn, disclosed 134 contigs and 106 B. tabaci mRNA sequences that met the criteria initially set  returned 37,393 contigs with at least one blast hit. Additionally, 1,988 contigs were found with blastn performed on the remaining (with no blastx hit) contigs  (Table The blastx search (0) Table . InterPrB. tabaci [T. vaporariorum [B. oleae [To identify the possible functions, Gene Ontology (GO) assignments were used to classify the sequences. A total of 25,392 contigs with at least one GO term  followed by oxydoreductases , hydrolases , lyases  ligases  and isomerases .The potential enzymes were characterized based on the chemical reaction they catalyze using the prediction of Enzyme Commission (EC) numbers for each sequence. Among the 37,393 annotated contigs, 6,994 contigs were classified to 795 different EC numbers. Enzyme classification revealed that transferases are the largest group of B. tabaci MED, we mapped all contigs with EC number  to the reference canonical pathways in Kyoto Encyclopedia of Gene and Genome (KEGG). In total, we assigned 6,731  contigs to 137 KEGG pathways. The pathways mostly represented by the assembled sequences were purine metabolism , arginine and proline metabolism  and pyrimidine metabolism . Contigs for pathways that could be involved in detoxification like metabolism of xenobiotics by cytochrome P450 , drug metabolism - cytochrome P450 , drug metabolism - other enzymes , oxidative phosphorylation , glutathione metabolism  and nicotinate and nicotinamide metabolism  were also represented  are ligand gated ion channels (LGIC) and serve an important role in nerve signaling, at the post-synaptic membrane, by mediating the fast actions of acetylcholine (ACh) at cholinergic synapses. Neonicotinoid insecticides and spinosad have high and selective affinity for many arthropod nAChRs .B. tabaci MED encoding putative receptor subunits  and T. castaneum α10 (44 %). However, in this subunit one of the two vicinal cysteines of the motif YxCC at Loop C, defining α subunits, was replaced by a histidine and is therefore named β2  or GR4-AS  and for subunit α7 between S-GR6 and GR9-IS . Whether this differential gene expression between resistant and susceptible strains for subunits β2 and α7 contributes to the observed resistance phenotypes needs further examination. However, it is interesting to point to recent studies that established a link between nAChR expression and response to neonicotinoids. Taillebois et al. [Musca domestica was correlated with reduced expression of the nAChR subunit α2.We found substantial variation in expression levels between the different subunits of nACh receptors. In all strains, subunit β2 was the most highly expressed (average FPKM across libraries = 52 to 100) followed by the α2 (FPKM = 6 to 8) and the similarly expressed α3, α7 and α8 (FPKM = 3 to 6). The lower expressed subunit was α 6 with only ten mapped reads . Most of them (52 %) were found in the coding regions, 19 % in the 5′UTR and 29 % in the 3′UTR.We investigated the occurrence of SNPs in unigenes encoding nAChRs and possible differences between resistant and susceptible The number of SNPs differed between the subunits of nAChR with the subunits α8 and β1 showing higher variation  and subunits α5 and α7 lower variation . In subunit α6, no variable site was detected; however, the shorter transcript was recovered for this subunit.Comparative polymorphism analysis identified several alleles differentially represented between insecticide resistance strains and the reference susceptible strain . Twenty-seven out of the 347 SNPs were differential SNPs (10 in the coding region and 17 in the 5′UTR or in the 3′UTR). Seventeen polymorphisms concerned SNPs with high frequency differences between the S-GR6 and any of the resistant strains, which were equally affected . Only five differential SNPs between S and R strains were in the coding region and all of them concerned synonymous substitutions.Nilaparvata lugens conferring imidacloprid resistance [B. tabaci, could be explained , α3 and α4, resulted in an amino acid change  by the persicae . In addiotinoids , where aotinoids , 50).We compared gene expressions of the susceptible and the three neonicotinoid resistant strains and performed differential expression analysis (DE). Data of three biological replicates of each condition were used in this analysis . The biological coefficients of variation (BCV) observed in this study ranged from 0.11 to 0.19  and are close to those observed in genetically identical model organisms, indicating small variability between replicates . The lowThe DE analysis revealed in total 3,745 differentially expressed unigenes  in the three R strains compared to the S strain based on their TMM-FPKM  Fig. .Fig. 3GrThe total number of DE unigenes against S-GR6 was unbalanced Fig.  suggestiFor a better understanding of the molecular functions and biological processes underlying the nature of resistance to insecticides, Gene Ontology term enrichment analysis was used to detect differentially expressed unigenes over or under represented specifically in each strain. Here, the term enrichment results for the comparison between the DE unigenes of each strain against the reference transcriptome. For the up-regulated unigenes, the GO terms were generally negatively enriched in the resistant strains including terms such as response to stimulus (GO:0050896), signalling (GO:0023052) and cellular process (GO:0009987) in all three resistant strains and terms such as binding GO:0005488) only in GR4_AS. Among the higher significant positively enriched GO terms corresponding to up-regulated unigenes figured those related to catalytic activity such as transferase (GO:0016740) and oxidoreductase activity (GO:0016491) in all three resistant strains together with terms related to heterocyclic compound binding (GO:1901363) and organic cyclic compound binding (GO:0097159) in GR4_AP and GR9_IS  and peptidase activity (GO:0008233) are among the terms related with catalytic activity (GO:0003824), for which the frequency increased significantly   and five fatty acid synthases  should represent genes selected by neonicotinoid or other insecticides applied in the field and maintained their expression levels in GR4-AP strain even after 6 generations without insecticide exposure. The four significantly up regulated unigenes shared only by the GR9-IS and GR4-AS, which may have been commonly selected by either neonicotinoids, include three P450s and one CCE with moderate logFC ranging from 1 to 2  were specifically up regulated in GR9-IS strain, with one CCE and one P450 having the higher logFC (2.7 and 3.7 respectively). Similarly, 17 unigenes were specifically up regulated in GR4-AS including 10 P450, 4 UGTs, 2 GSTs and 1 CCE. These specifically GR9-IS or GR4-AS up regulated unigenes reflect a genetic background related specificity, a selection agent (imidacloprid or acetamiprid) specificity, or a combination of both. Our experimental design does not allow distinguishing between the two cases. However, taking into account that these strains should belong to the same genetic cluster (south Crete) identified by microsatellites , it is pGSTs metabolism as a mechanism of resistance to organochlorines, carbamates, organophosphates and pyrethroids has been reported in many arthropod species –56. SimiM. domestica [D. melanogaster [N. lugens [H. armigera [T. urticae [B. tabaci [B. tabaci [M. domestica [M. persicae [D. melanogaster [Among detoxification enzyme systems, P450s are the most common genes found up-regulated in a wide number of resistant insects with a subset of these P450s confirmed to be able to metabolize pesticides by functional in vitro studies in mosquitoes species reviewed by , 61, M. omestica , D. melanogaster , N. luge. lugens , H. armiarmigera , 66, T.  urticae , 68 and . tabaci , 69, 70.. tabaci , 71, 72,omestica , M. perspersicae  and D. mnogaster . P450s eD. melanogaster (88), A. gambiae (105), A. aegypti (160), T. castaneum (134), A. pisum (64) , B. oleae (55) and T. vaporariorum (57) [P450s are one of the largest super-families, playing a dominant role in plant-insect interactions and insecticide/xenobiotic metabolism. They are divided in 4 major clades, named CYP2, CYP3, CYP4 and mitochondrial . A variaiewed in ) and in rum (57) , 31, 33.B. tabaci transcriptome  were included together with other known insect P450s (345 protein sequences from 14 species including B. tabaci).A total of 367 contigs (146 unigenes) were identified as P450 encoding genes in the B. tabaci P450s (76 out of 171) belonged to the CYP3 clade, 73 to the CYP4, 18 to the CYP2, and 4 to the mitochondrial clade . However, this separation is not as clear in CYP3 B. tabaci P450s.In this dataset, representatives of all 4 major insect P450 clades were found. The majority of Finally, we performed a Bidirectional Best Hit (BBH) with 1:1 relationship between the 171 P450s contigs of the current study and the 345 P450 proteins from different species that were also included in the phylogenetic analysis. The BBH returned 35 P450 orthologs. For the remaining P450s, a corresponding name was assigned according to the first Blast hit. . They were both significantly up-regulated in the resistant strains compared to the S-GR6 however, comp61334_c0 displayed higher up regulation (logFC = 5.57–6.58) compared to comp33028_c0 (logFC = 1.4–3.2). These two specific P450s belong to clade CYP2 and are presumed 1:1 orthologs with CYP303a1 family members. Interestingly, CYP303A1 is one of few highly conserved P450s found in all insect species sequenced to date, but not in Daphnia pulex [D. Melanogaster, CYP303A1 has a putative external sensory development function and it has been hypothesized that it probably metabolizes a small signal molecule essential in the development and structure of external sensory organs [Unigenes comp61334_c0 and comp33028_c0 shared the higher similarity (higher best hit) with a putative P450 CYP303a1-like of y organs . WhetherL. stratiella (orthologous to CYP417). They belong to the CYP4 clade, with which they formed a separate cluster with other P450 unigenes of B. tabaci issued from this study. This cluster was a sister group with members of CYP417, CYP425, CYP426 and CYP439 of L. stratiella. Comp62212_c0 was highly up-regulated in the three resistant strains (logFC = 4.78–6.07) while comp43434_c1 was up-regulated only in the GR9-IS strain (logFC = 3.73).Components 62212_c0 and 43434_c1 shared the higher similarity with CYP417B1 of A. pisum, with which formed a distinct group separated from other CYP4C1 A. pisum sequences. This unigene was highly over-expressed in the GR4-AS strain (logFC = 5.36) and was also significantly increased level of expression after selection with acetamiprid (logFC = 2.45 in GR4-AS compared to GR4-AP). Over-expression of members of CYP4 family has been found associated with a number of insecticide resistant insect strains  [A third unigene of clade CYP4, comp43065_c0, shared the higher similarity and was 1:1 ortholog with CYP4C1 of ble 7 of ) includimethoxam , 32, or  aegypti , or in DCYP4G70) .B. tabaci (100 % similarity) and was differentially unregulated in the three resistant strains with similar, very high expression levels, compared to the susceptible strain (logFC = 5.55–5.84).Unigene comp50040_c0 had a significant Blast hit with CYP6CM1vQ of CYP6 family P450s are widely known to be involved in metabolic resistance to pyrethroids and organophosphates in a number of insect species –83 incluB. tabaci. These two unigenes showed comparable over-expression levels in the three resistant strains (logFC = 1.23–2.06 for comp57969_c124 and logFC = 1.61–2.45 for comp57969_c113). Although there is no significant difference, their expression levels slightly increase with acetamiprid selection in GR4_AS compared to GR4_AP. In the phylogenetic analysis, they were grouped together with the third member of clade CYP3 (CYP6CM1) that has also been found highly over-expressed in this study. All three  displayed the higher similarity with the respective published sequences of B. tabaci as well as with CYP6CX1 and CYP6CX4. CYP6CX1 has been associated with B. tabaci (biotype B) resistance to fenvalerate, chlopyrifos, and avermectin in the field [B. tabaci MEAM1 (B-biotype) strain [For components 57969_c113 and 57969_c124 the bidirectional best hits (1:1) were respectively CYP6CX5 and CYP6CX3 of he field . CYP6CX1) strain .D. melanogaster where only a single P450, cyp6g1, is associated with neonicotinoid resistance [B. tabaci, is a heavily treated agricultural pest and different insecticides, of the same or different chemical groups, may select several P450s conferring unpredictable cross resistance. For example, CYP6CM1 the only functionally characterized P450 in B. tabaci, confers multiple resistant phenotypes and metabolizes some but not all neonicotinoids, as well as other insecticides from different chemical groups. Our differential expression study provides a good base to select additional candidate genes for further functional characterization and validate their role in resistant phenotypes.All of the eight above mentioned P450s may be potentially associated with insecticide resistance and this could constitute a discrepancy compared to other insects, ie sistance . HoweverR2 = 0.85; P < 0.0001)  with logFC > 2 . Using this approach, we investigated their expression levels in several selected strains (some of them used in the RNA-seq experiment), as well as in additional whiteflies sampled in the field. The levels of gene expression (folds regulation) in resistant strains compared to the susceptible S_GR6 found in qPCRs were highly correlated with those observed in RNAseq experiment  Fig. . In geneB. tabaci and were collected from eggplants grown in greenhouses in Crete, setting the frequent application of neonicotinoids, pyrethroids, spiromesifen and pymetrozine as the common point in their treatment history. Compared to the laboratory susceptible strain S_GR6, their RR ranged from 6 to 65-fold for imidacloprid and 2 to 26.5-fold for acetamiprid  , with logFC close to 1. For this reason, its expression levels were not examined in more populations by qPCR and we were are not able to assess the presence of any possible correlation with resistance levels in the Greek populations. Components 61334_c0 and 57969_c124 are the number one candidates for further studies, i.e. functional analysis, given their stronger positive correlation with the neonicotinoid resistant phenotype. However, the absence of significant correlation between RR and relative fold regulation cannot exclude the possibility of another P450 being involved in imidacloprid or acetamiprid resistance. Rather, it indicates that this P450 is probably not the major, in terms of frequency, resistance mechanism for the given populations. This is illustrated by the case of CYP6CM1 whose implication in neonicotinoid resistance has been functionally demonstrated; although in the present study, no significant correlation was found. It is worth noting the total absence of a correlation between CYP6CM1 and RR to acetamiprid is in agreement with the fact that acetamiptid is not metabolized by this P450. The unpredictable diverse range of chemistries detoxified by CYP6CM1, which includes some - but not all - neonicotinoids [In field MED (Q biotype)  CYP4C64 . In our otinoids , 69 as wotinoids  and pyriotinoids , exempliB. tabaci. Twelve libraries from three neonicotinoid resistant B. tabaci strains from Greece were sequenced by Illumina platform, and generated about 73 Gbp. A reference transcriptome was built by de novo assembly and functionally annotated. The large number of sequences, obtained by the investigation of expression profile of different neonicotinoid resistant strains, provided useful insights into the underlying molecular mechanisms associated with resistance. We further focused on the differential expression analysis and phylogenetic classification of nAChR subunits and cytochrome P450s, the main genes related to neonicotinoid resistance, enabling useful genetic information of resistance mechanisms to be gained. No nAChR polymorphism or differential expression of the different nAChR subunits were observed among the studied strains that could be related to the resistant phenotypes. Differential expression analyses disclosed a number of significantly up regulated detoxification genes (mainly cytochrome P450s), some of which specifically over-expressed in each selected strain, providing a list of putative candidate resistance genes. CYP6CM1 already known as a major mechanism of imidacloprid resistance in B. tabaci from Crete was also deferentially expressed in the resistant strains. Using quantitative qPCRs, a larger screening of field MED B. tabaci from Crete with known neonicotinoid phenotype was performed to associate expression levels of P450s with resistance levels. Expression levels of five P450s including CYP6CM1 were found associated with neonicotinoid resistance, however, a significant correlation was found only in two of them, CYP303 and CYP6CX3, with imidacloprid and acetamiprid respectively. Further functional expression analysis of the genes and the encoded proteins are currently being investigated to confirm their role in resistance.Our work has generated new toxicological data and genomic resources which will significantly enrich the available dataset and substantially facilitate the molecular studies in MED B. tabaci populations , collected in 2012 in Crete on eggplant greenhouses were maintained in laboratory conditions under short term selections and were chosen based on their LC50s  to neonicotinoids and their insecticide application history. Insecticide selection experiments were carried out in parallel for the three strains during six generations with the neonicotinoids acetamiprid (GR4_AS) and imidacloprid (GR9-IS and GR0-IS). During the experiment, the initial parental strains  were also maintained without insecticide selection. Under these relaxed conditions, it is expected that the Resistance Ratio (RR) in the parental strains would be lower than the respective selected ones, given that a fitness cost is often associated to the insecticide resistance.Three B. tabaci populations collected in Crete in 2013 from eggplants grown in greenhouses, were also used in our study without any prior rearing in the laboratory .Five additional All populations were identified as the Mediterranean species MED (Q1 mitotype) by sequencing part of their COI mtDNA gene and applying a diagnostic PCR to 50 females of each strain .In all experiments, the laboratory MED strain S-GR6 susceptible to neonicotinoids was used as reference strain. It was collected in 2006 in Crete and has been since maintained in the lab without any insecticide treatment.The selected strains were established by > 500 field collected individuals and were reared on cotton plants in standard insectary conditions . Selections were performed by exposing adult females and males for 72 h to a lethal concentration of commercial formulations of imidacloprid or acetamiprid. Survivors were transferred onto untreated cotton plants and allowed to lay eggs to start a new generation. The concentration of insecticide was adjusted at each generation  in order to reach 60–80 % mortality after 72 h exposure. Surviving adults were transferred onto untreated cotton plants to obtain the next generation. In order to limit bottleneck effects, each generation was set off with > 300 individuals.−1 SL , acetamiprid 20 SG  and thiacloprid 240 g litre−1 SC . Final mortality was assessed after 72 h. Lethal concentrations corresponding to 50 % mortality (LC50) and their 95 % confident intervals (CI95%) were then calculated with a probit approach for each strain using PoloPC . Resistance ratios  were calculated by comparison to the susceptible strain.Full dose bioassays on cotton leaf discs were performed with female whiteflies as previously described , 89. TheBioassays after exposure to piperonyl butoxide , an inhibitor of the P450 dependent monooxygenases, were used to determine P450 metabolism contribution to resistant phenotypes. Piperonyl butoxide was applied at 300 ppm via a tarsal contact method 4 h prior to the insecticide application as previously described .In total, four different strains -all of MED species originated from Crete- were used in the RNA-Seq experiments: 1) the neonicotinoid susceptible strain S_GR6 was used as a reference strain 2) the resistant strain R_GR9_IS maintained under Imidacloprid Selection 3) the resistant strain R_GR4_AS maintained under Acetamiprid Selection, and 4) its corresponding Parental strain R_GR4_AP issued from the same field collected population (R_GR_4) which has been maintained in the laboratory without any selection pressure. Strains R_GR9_IS and R_GR4_AS were used as they exhibited the higher resistance levels against neonicotinoids.Three biological replicates were included per strain in order to statistically validate the pair wise comparisons of transcript expression between the resistant and susceptible strains. For each biological replicate, total RNA was prepared by grinding approximately 300 frozen adults 1–2 days old, including males and females, using the Qiagen RNeasy mini kit according to the manufacturer’s protocol. Additionally, column digestion with DNAseI (Qiagen) was applied for the removal of genomic DNA contaminations. RNA concentration as well as the 260/280 and the 260/230 ratios were estimated using a Nanodrop spectrophotometer after which RNA samples were subjected to quality check with an Agilent Biolanalyzer, taking into account that in insects (and other taxa) the electrophoresis profile of rRNA significantly differs from the standard benchmark .RNA samples were sent to Macrogen (Korea) for mRNA paired-end library construction with the Illunina TruSeqTM RNA Sample Preparation Kits v2 following the manufacturer protocol  and sequencing on Illumina HiSeq 2000 platform. Barcoded libraries were prepared in a way that six samples could be run per lane. Each library was sequenced with the paired-end method for a read length of 100 base pairs.Bemisia tabaci mitochondrial genome were used. The reads from the fastq file were firstly collapsed  from all the 12 samples. A single reference transcriptome was generated using Trinity package .Based on the results of the preliminary assemblies, we selected to conduct our final de novo assembly using Trinity package. To increase transcriptome coverage and to facilitate downstream analyses , B. tabaci obtained from NCBI with blastn search, and d) with CEGMA (Core Eukaryotic Genes Mapping Approach) [B. tabaci transcriptome. In addition, based on BLAST NCBI taxonomy database we identified and removed 3, 467 host plant and fungus contaminant contigs (representing 2552 unigenes), as well as their respective mapped reads, from further analysis. No filtering against low-abundance contigs was performed in order to maximize the detection of low expressed transcripts. All the BLAST (http://www.ncbi.nlm.nih.gov/pubmed/2231712) searches were performed using NOBLAST program [http://sourceforge.net/projects/noblast/).To assess the assembled transcripts quality, we followed four procedures: a) we pooled all the reads and mapped them to the assembly using Bowtie2  and Samtpproach)  we deter program   was performed. Sequences that did not receive any hit were then searched via blastn against NCBI nucleotide database nt  using the parallel version of NOBLAST. For gene ontology (GO) mapping, Blast2GO [For sequence annotation of the assembled transcripts, first, a blastx similarity search against the NCBI protein database Blast2GO  was usednr.The sequences were further annotated using InterPro . A localGO terms were modulated using the annotation augmentation tool ANNEX  followedFor genes of particular interest expected to be involved in neonicotinoid resistance such as P450s and nAChR, similarity search and phylogenetic reconstructions were performed using known orthologs from other insect species.B. tabaci contigs (>200 bp) encoding P450s and nAChRs were identified using BLAST, Blast2Go and interpro as described above. In addition, P450s and nAChRs were further identified using tblastn search  in protein sequences of other insect species from [B. tabaci assembly transcriptome. Contigs were translated and searched by Blastp against themselves. Results showing more than 99 % similarity were considered as allelic variants. Specifically for nAChR subunits, which displayed high expression variation  was performed for the nAChR genes. Among all detected polymorphism variations, only SNP substitutions were considered for differential polymorphism analyses. Alignment files generated by Bowtie2 were used for SNP detection using SAMtools and Varscan  with theSNP allele frequencies were then computed between each insecticide resistant strain and the susceptible strain. Allele frequencies were considered as differential between an insecticide-selected strain and the susceptible strain  if the total read coverage at SNP position between both strains was ≥ 50 and difference in allele frequency between strains  > 50 % .SNPs were categorized to synonymous, non-synonymous and those that belong to the transcripts UTRs using a custom perl script. Non-synonymous SNPs were manually checked.run_RSEM_align_n_estimate.pl. The estimated expected counts for each sample (the sum of counts in each row >10) were extracted and used for the analysis of differential expression conducted in EdgeR-3.8.0 bioconductor package [Differential expression (DE) analysis was performed at the “gene” (unigene) level by pair wise comparisons between strains. For the quantification, the paired reads of each sample were mapped to the transcriptome assembly with Bowtie2 and abundance was estimated with RSEM v. 1.2.4, as implemented in the trinity script  package  using Ed package  with log20, according to the manufacturer’s instructions. The resulting cDNAs were diluted 20 times in ultra-high quality water for qRT-PCR reactions using a Mini Opticon System . PCR reactions of 25 μl contained Fast Start SYBR Green Master Mix (Kapa-Biosystems), 0.12 μM of each primer . Primers were designed using the Oligo7 Primer Analysis Software , based on cDNA sequences retrieved from RNA-seq and are listed in Additional file ficiency .All the computations were performed at the HCMR-Crete, high-performance computing bioinformatics platform. All the custom Perl, R and bash scripts are available from the authors upon request.


Serum concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) are highly heritable traits that are used clinically to evaluate risk for cardiovascular disease in humans. In this study, we applied a genome-wide association study (GWAS) in 1,075 pigs from two populations and gene expression studies on 497 liver samples to dissect the genetic basis of serum lipids in a pig model.2 population, the most prominent SNP was identified at the SSC3: 124,769,847 bp where APOB is the well-known candidate gene. However, in the Sutai population, the most number of significant SNPs was identified at SSC2: 64.97-82.22 Mb where LDLR was identified as the candidate gene. Furthermore, we firstly reported 4 novel genomic loci in pigs harboring the LDL-C-associated SNPs. We also observed obvious population heterogeneity in the two tested populations. Through whole-genome gene expression analysis, we detected 718 trait-correlated expressions. Many of these transcripts correspond to candidate genes for blood lipids in humans. The GWAS mapped 120 cis-eQTLs and 523 trans-eQTLs for these transcripts. One gene encoding the transcript gnl|UG|Ssc#S35330332 stands out to be an important candidate gene for LDL-C by an integrative analysis of GWAS, eQTL and trait-associated expression.We totally identified 8, 5, 2 and 3 genomic loci harboring 109 SNPs that were significantly associated with LDL-C, TC, TG and the ratio of HDL-C/LDL-C in two experimental populations, respectively. In the FWe identified the genomic regions or candidate genes associated with blood lipids by an integrative analysis of GWAS, QTT and eQTL mapping in pigs. The findings would benefit the further identification of the causative genes for blood lipid traits in both pigs and humans.The online version of this article (doi:10.1186/1471-2164-14-848) contains supplementary material, which is available to authorized users. GALNT2 [TRIB1 [SORT1 [Blood lipids are transported in the bloodstream of human and animal. They are important indicators for whole body lipid metabolism. The screening tests of blood lipid concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) are used clinically to evaluate the risk for cardiovascular diseases and to give the guidance for prescription of medicine. Serum concentrations of blood lipids are highly heritable phenotypes . To dateGALNT2 , TRIB1 [2 [TRIB1 , and SOR1 [SORT1  as causaLDL receptor (LDLR) mutations contribute to an autosomal recessive hypercholesterolemia in the specific pig strains [apolipoprotein B (APOB) mutations are associated with elevated plasma cholesterol and atherosclerosis in pigs in relation to atherosclerosis [The pig is an important biomedical model . Compare strains , 12, andclerosis , 14. Butclerosis .SORT1 and CELSR2 as candidate susceptibility genes for LDL-C using the integrative approach [AHNAK, SLC3A2 and MAP4K4 as candidate genes for meat drop loss by integrating data of gene expression, eQTL and phenotypic QTL [The GWAS is a powerful tool to identify genomic regions affecting phenotypic traits but not efficient for identifying causative mutations . The appapproach . Wimmer ypic QTL .2 intercross and a Chinese synthesized line (Sutai pigs). Genome-wide gene expression and quantitative trait transcript (QTT) analyses as well as eQTL mapping were also performed to facilitate the identification of candidate genes for these traits. This study provides useful information for the genetic architecture of blood lipids and for human cardiovascular diseases.In this study, a GWAS for porcine blood lipid traits was conducted in two populations including a White Duroc × Erhualian F2 resource population and Sutai pigs. The White Duroc × Erhualian F2 resource population was constructed as described previously [1 animals, and then 9 F1 sires and 59 F1 dams were randomly intercrossed, avoiding full-sib mating, to generate 1,912 F2 individuals. Sutai pigs were synthetized from Duroc × Erhualian crossing through selection of 18 generations. A total of 435 Sutai pigs from 5 boar families were used in this study. The animals were raised in a standard indoor condition with natural lighting and were fed three times a day using the feed containing 16% of crude protein, 3100 kJ of digestible energy, and 0.78% of lysine. Water was available ad libitum from nipple drinkers. These animals were slaughtered at 240 ± 3 days after fasting but water-free overnight (about 12 hours). All samples were collected according to the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China. Animal Care and Use Committee (IACUC) in Jiangxi Agricultural University specifically approved this study.All samples in this study were from the White Duroc × Erhualian Feviously . In brie2 animals  and 435 Sutai pigs  were measured by direct assay with diagnostic kits of Determiner-L LDL-C, Determiner-L HDL-C, Determiner-L TCII and Determiner-C TG , respectively, following the manufacturer’s instructions. All measurements were performed in an AU5421 Automatic Biochemistry Analyzer  at the First Affiliated Hospital of Nanchang University.Blood samples were collected from the major artery serum vessels near the heart when the animals were exsanguinated. After coagulation at room temperature, the clots were centrifuged at 3000 rpm at 4°C for 20 min to separate serums. All serum samples were then stored at -80°C until utilized. LDL-C, HDL-C, TG and TC levels for 760 F2 animals for RNA isolation within 30 min after slaughter. The tissues were put into the sterile and frozen cryopreservation tubes and dipped into liquid nitrogen, and then conserved in -80°C ultra freezer until RNA extraction. Total RNA was isolated with TRIzol  following the manufacture’s instruction. The residual DNA was cleared away from total RNA with RNase-free DNase I  for 30 min at 37°C. The quality of total RNA was assessed by a 2100 Bioanalyzer  and agarose gel electrophoresis.Liver samples were harvested from 497 FInfinium HD Assay Ultra protocol . The positions of all 62,163 SNPs from the Porcine SNP60 BeadChip on the current pig genome assembly (Sscrofa 10.2) were retrieved from the NRSP-8 Community Data Repository [P-value < 5 × 10-6, or the X-linked SNPs that were likely to be autosomal (odds > 1000) were excluded from further analysis. Finally, total 39,454 and 45,322 SNPs passed the quality control in the White Duroc × Erhualian F2 resource population and the Sutai pigs, respectively.All animals were genotyped using Porcine SNP60 BeadChips according to the pository . The quaY = Xb + Sα + Zu + e, where Y is the vector of phenotypes, b is the vector of fixed effects including sex and batch, α is the vector of the SNP substitution effect and u is the random additive genetic effects with u ~ N , where G is the genomic relationship matrix that was constructed based on SNP markers, and σα2 is the polygenetic additive variance. e is a vector of residual errors with a distribution of N , where I is the identity matrix and σe2 is the residual variance. X, S and Z are the incidence matrices for b, α and u. The analysis was conducted by mmscore function with GenABEL in R package. Bonferroni correction was used to adjust the multiple tests. A conservative threshold of P < 0.05/SNP number was applied for genome-wide significance, and 1/SNP number was set as suggestive significance. Quantile-quantile (Q-Q) plots and Manhattan plots were drawn using the corresponding P values by R package. We calculated the phenotypic variations explained by the top significant SNPs using (V reduce – V full)/V reduce, where V full and V reduce are residual variances of the models for association analysis with and without SNP term, respectively. We also searched candidate genes with functional relevance to serum lipids or lipid metabolism in an interval of 5.0 Mb centered at the top SNP at each significant locus.The associations of the filtered SNPs with serum lipid levels were evaluated using a mixed model based on the score test approach . The mod2 liver samples as described previously [Nla III and Mme I . The digested-cDNA was ligated to Illumina specific adapters 1 and 2. Polymerase chain reaction (PCR) was performed to enrich the cDNA library with two primers that annealed to the ends of the adapters. After purification and denaturation, the single chain molecules of each cDNA library were loaded onto the flowcell and sequenced on a GA II sequencer .Digital gene expression (DGE) analyses of genome-wide transcripts were performed on 497 Feviously . In brieNla III restriction site were created using in-house Perl scripts. The clean tag sequences were then mapped using SOAP2 that allowed up to one mismatches in 21-bp tag sequences [Tag data sets were analyzed according to the BGI bioinformatics protocols for digital gene expression. Briefly, the raw tags were firstly filtered to produce the clean tag data. To map the clean tags to reference transcript sets or to the pig reference genome, the reference transcript sets were downloaded from the database of PEDE  and pig equences . The expP < 0.0005 was set as threshold. eQTL mapping was performed for those trait-correlated transcripts using mixed linear model implemented by mmscore function of GenABEL in R package. Sex and batch were considered as fixed effects, the genetic co-variances among samples were also taken into account by fitting kinship matrix derived from genotypes of whole-genome SNP markers. A bonferroni correction was applied to adjust the multiple tests. All the above mentioned analyses were carried out with R package.The gene expression profiles and phenotypic data were further adjusted for gender, batch and kinship using a robust linear regression model. Associations of gene expression level with serum blood lipid concentrations were evaluated with spearman coefficient by R software. To adjust the multiple tests, a conservative h2) for each of the 5 serum lipid traits in the two pig populations. A medium to high h2 (0.31-0.71) was observed for these traits, suggesting considerable genetic contribution to serum lipid contents in pigs , 22 SNPs corresponding to 5 chromosomal regions were significantly associated with one or more phenotypes   was located at SSC1: 63,541,683 bp. For HDL-C/LDL-C, only one SNP (ASGA0016328) at SSC3: 124,769,847 bp showed significant association (P = 1.10 × 10-5). A total of 21 SNPs in 4 chromosomal regions showed signals of associations with LDL-C. The most significant association was found at SSC3: 124,769,847 bp (P = 1.90 × 10-10). Another 3 LDL-C associated SNPs located near this region . Two significant SNPs for this trait can’t be placed to the current genome assembly of Sscrofa 10.2. For TG, only one SNP at SSC4: 119,869,765 bp (ALGA0028032) showed association in this F2 population  for the SNPs near the gene cluster of APOA5-APOA4-APOA3-APOA1 (< 1.00 Mb) .We found 4 SNPs associated with TC in this resource population, of which 3 were mapped to the region around 125.00 Mb on SSC3 and achieved genome-wide significance level  as the suggestive significance threshold, we totally identified 91 SNPs corresponding to 8 chromosomal regions that were associated with one or more blood lipid traits , Except for SNP ALGA0109254 on SSC5, all genome-wide significant associations were detected on SSC2 . For the chromosomal region of SSC5: 3.55 Mb, only one SNP of ALGA0109254 was associated with LDL-C at genome-wide significance level  and the SNP ALGA0109254 at SSC5: 3,550,340 bp . Significant association with HDL-C/LDL-C was also observed for SNP ALGA0005129 at SSC1: 107,283,424 bp .A total of 28 SNPs in 3 chromosomal regions were associated with serum TC levels, including 23 SNPs at SSC2: 54.47-74.62 Mb  were discarded from further analysis. We used regression models to identify transcripts that were associated with each trait of blood lipids. At a significance threshold of P < 5 × 10-4, we found 58, 437, 115 and 108 transcripts whose expression levels were associated with TC, HDL-C, TG and LDL-C, respectively , and 523 trans-eQTL, those that mapped elsewhere in the genome . Only additive effect was considered in the model of GWAS. However, both additive and dominant effects were included in the QTL mapping model; (3). In the QTL mapping, the detected QTL was fixed as the genetic background for next round QTL identification but no conditional analysis was performed in the present GWAS. (4). The marker density (194 microsatellite markers across the genome) is much lower in the QTL mapping compared to the GWAS.The SNP effects at SSC1: 107.28 Mb for HDL-C/LDL-C, SSC1: 63.54 Mb for TC and LDL-C, SSC3: 124.77-125.64 Mb for TC, LDL-C and HDL-C/LDL-C, SSC2: 66.75-74.62 Mb for TC, LDL-C and HDL-C/LDL-C, and SSC4: 119.87 Mb for TG overlapped with the previously reported QTL for blood lipids in a White Duroc × Erhualian Fpulation , 28, 29.pulation . For inspulation . However2 and Sutai populations, it was unexpected to observe distinct associations for TC, TG and HDL-C/LDL-C in Sutai and F2 pigs. For example, the significant association of the SNP at SSC3: 124,769,847 bp with LDL-C and TC in the White Duroc × Erhualian F2 resource population was not repeated in the Sutai pigs. Although the F2 and Sutai populations were originated from the same founder breeds of Duroc and Erhualian, the principle component analysis showed a clear divergence of the two populations  of genome-wide significant SNPs associated with LDL-C in the Sutai pigs  The environment factors could be well controlled as all experimental pigs were managed in the uniform living conditions with the same diets. (3) As for the Sutai population, the moderate sample size likely caused the inflated phenotypic variance accounted for by the SNPs [The phenotypic variance explained by the top SNPs was larger than that by the most cases of identified SNPs in human studies , 4. The the SNPs .APOB, a candidate gene for LDL-C and TC in humans [SMARCA4 and LDLR are candidates for LDL-C and TC in humans [HMGCR on chromosome 5 (HSA5) for TC and LDL-C, and LIPG on HSA18 for LDL-C and HDL-C [SLC27A1 gene. SLC27A1 knock-out mice show abnormal lipid and triglyceride levels [GNA11, ABCA7, CSF1, CERS1, SIRT3 and SCP2, reside in the genomic regions related to TC, LDL-C, HDL-C/LDL-C and TG in this study  [ABCA7, CSF1) [CERS1) [SIRT3, SCP2) [We searched candidate genes with functional relevance to serum lipids or lipid metabolism in an interval of 5.0 Mb centered at the top SNP at each significant locus. The large interval was adopted as high LD extents were expected in the current experimental populations. A number of candidate genes for human blood lipids were also evidenced in this study Table . APOB, an humans , 4, 31 ie levels . Other pe GNA11) , 36, cir7, CSF1) , 38, abn [CERS1) , and cir3, SCP2) , 41 in kCYP8B1, SCD and TLR2 in liver were significantly correlated with LDL-C in this study  was higher than that of cis-eQTL (18.3%) [APOB and LDLR cause phenotypic variation in human blood lipids [eQTL were identified for about 31.8% of trait-correlated transcripts. Consistent with the finding in Ponsuksili et al., the proportion of  (18.3%) . In the  (18.3%) . It has  (18.3%) . HoweverTo our knowledge, this is the first study on identifying the genomic regions associated with blood lipids by an integrative analysis of GWAS, QTT and eQTL mapping in pigs. We firstly reported 4 novel genomic loci for porcine LDL-C level. Several chromosomal regions such as SSC2: 64.97-82.22 Mb and SSC3: 124.77-126.93 Mb are worthwhile for further fine-mapping and identifying the causative mutations for LDL-C and TC. Moreover, many genomic regions previously identified in humans and mice have been replicated here. A list of human or mice candidate genes were also evidenced in this study. This study would benefit the identification of causative genes for blood lipid traits and also gives useful information for studies of human cardiovascular diseases.P-values versus the expected P-values of association in GWAS for blood lipid. (DOC 588 KB)Additional file 1: Figure S1: Quantile-quantile (Q-Q) plots of the observed Additional file 2: Figure S2: Manhattan plots of genome-wide association analyses for serum TG, HDL-C and HDL-C/LDL-C. (DOC 1 MB)Additional file 3: Table S1: SNPs significantly associated with blood lipids in White Duroc X Erhualian F2 population and Sutai pigs. (XLS 38 KB)Additional file 4: Table S2: Transcripts significantly associated with blood lipids and its eQTL. (XLS 163 KB)Additional file 5: Figure S3: The PCA analysis showed a clear divergence of the two populations. (DOC 100 KB)