Aedes albopictus is a very important vector for pathogens of many infectious diseases including dengue fever. In this study, we explored the genetic polymorphism of Aedes albopictus strains in different geographical regions using DNA barcoding of mitochondrial COI (MT-COI) gene. We collected MT-COI sequence of 106 Aedes albopictus mosquitos from 6 provinces in China including Fujian, Guangdong, Hainan, Yunnan, and Taiwan. The length of the sequences is 709bp with the content of A+T (67.7%) greater than that of G+C (32.3%). We identified mutations in 90 (13.68%) loci, of which 57 (63.33%) are transitions, 28 (31.11%) are transversions, and 5 (5.56%) are hypervariable loci. In addition, we obtained 42 haplotypes, 4 (9.52%) of which are shared among different populations. The haplotype diversity of Aedes albopictus is 0.882 and nucleotide diversity is 0.01017. Moreover, the pedigree network diagram shows that most haplotypes are under parallel evolution, suggesting a local expansion of Aedes albopictus in history. Finally, the Neighbor-Joining tree of MT-COI haplotypes reveals a certain correlation between haplotype clusters and geographical distribution, and there are differences among Aedes albopictus in different geographical regions. In conclusion, DNA barcoding of MT-COI gene is an effective method to study the genetic structure of Aedes albopictus. Aedes albopictus, belonging to the Diptera Nematocera Culicidac Aedes genus, are widely distributed all over the world [ Aedes albopictus was observed from Hainan island in the south all over to Liaoning province in the north, it was most gathered mostly in the south of 30° north latitude such as the Fujian province [ Aedes albopictus is critical to its prevention and control.he world –4. It wahe world , 5. Intehe world . In Chinprovince . UnderstMT-COI) gene (about 500-600 bp), which was first introduced by Canadian biologist Paul Hebert in 2003 [ Aedes albopictus at species level based on DNA barcoding at present. The reason might be due to the high cost in collecting and sequencing Aedes albopictus strains in a wide region, which might even be infeasible in a small country.DNA barcoding is a biometrics based on the mitochondrial cytochrome C oxidase subunit I , Guangdong province (Guangzhou), Hainan province , Yunnan province (Mengla), Liaoning province (Xishan), and Taiwan  .μL, 10XEx Taq Buffer 5μL, dNTP Mixture 4μL, Template DNA 3μL (if the light belt can be properly increased), and upper and lower primer (10 μM) with each 1μL, making a total volume of 50μL by adding ddH2O. In addition, the amplification conditions are set as follows: predenaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 sec, a total of 30 cycles, and final elongation at 72°C for 5 min.Nucleic acid was extracted using the TIANamp Genomic DNA Kit after removing the head of mosquitos. We then used a pair of universal primers including LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAAT-CA-3′) to amplify them . The reaThe PCR products were cloned by Universal DNA Purification kit, equipped with LB liquid medium, LB solid medium, and 50 mg/ml IPTG. The PCR products were digested with PCR Identification Kit for Recombinant pGM-T Clone, which were sent out for sequencing.Sequence Verification and Alignment. By referring to the NCBI sequence database, homology search was performed against existing COI gene sequences of Aedes albopictus in GenBank, and sequences with identity greater than or equal to 98% were selected [selected .Sequence Analysis. The aligned sequences were input into the multiple sequence alignment tool Clustal X (v1.8) for multiple sequence alignment [ Chironomidae nepeanensis (GenBank: KC750313.1) as the outgroup, we constructed Neighbor-Joining (NJ) tree and Maximum Likelihood (ML) tree according to the genetic distance Kimura 2-parameter method and bootstrapped 1000 times to test the reliability of the branch trees [ Aedes albopictus. The median joining method of Network4.6 software was used to construct haplotype pedigree network diagram. The analysis of molecular variance (AMOVA v3.1) was used to calculate the genetic differentiation within and between populations, including Fst (F-statistics) and Nm (Nm = (L − Fst)/4Fst) [ Aedes albopictus population. Using the Tajima's D [lignment , 20. Thelignment . Taking ch trees –23. The ch trees  was usedt)/4Fst) . Arlequit)/4Fst) , 27, drejima's D  and Fu'sjima's D  of the nθ) is calculated as follows: θ = 2Nu, where N represents the effective number of female mosquitos in the population, u represents the mutation rate per generation, and u = μk . We used τ = 2ut to estimate the approximate generation number (t) of population expansion [ Aedes albopictus which is 1 × 10−8 [The ancestral population size (xpansion  and set 1 × 10−8 .Mantel test was performed by online software IBD Web Service (IBDWS)  to evalu Aedes albopictus in NCBI database, the sequence identity was 99%-100% showing very small difference among the 106 sequences.The mitochondrial COI gene was obtained with length 709bp. According to the sequence alignment ofAfter sequence alignment, we took the 658 bp fragment  for subsequent analysis. The overall base composition of the fragment is A (28.5%), T (39.2%), C (16.7%), and G (15.6%). The A+T content (67.7%) is greater than that of G+C (32.3%). There were 90 (13.68%) polymorphism sites, of which 57 (63.33%) are transitions, 28 (31.11%) are transversions, and 5 (5.56%) are hypervariable loci . Aedes albopictus MT-COI gene  and nucleotide diversity (Pi) were used to indicate the degree of haplotype differentiation and the degree of nucleotide sequence variation. The haplotype diversity of Aedes albopictus is 0.882 and nucleotide diversity is 0.01017. The haplotype diversity of geographical strains is ordered as Yunnan > Fujian > Hainan > Taiwan > Guangdong > Liaoning strains. The nucleotide diversity is ordered as Fujian > Yunnan > Hainan > Taiwan > Guangdong > Liaoning strains. Fujian strains had the largest number of polymorphism sites (s = 66) and haplotype number (n = 12) and thus also had the largest nucleotide diversity (Pi = 0.03316). Its haplotype diversity is 0.87895, only slightly smaller than that of Yunnan strains (Hd = 0.88421). There were no differences between the 5 individuals in Liaoning populations.We obtained 42 haplotypes on 6 geographical strains ofCOI gene , 4 , but shared haplotypes H2 , H18 , and H19  are highly distributed in the population, which may be the sources of expansion. It can be seen that Yunnan + Hainan strains and other geographical strains are roughly divided into two categories, but there are individual haplotypes intersecting with each other.The median joining method in software Network4.6 was used to construct haplotypes pedigree network of 42 haplotypes from 6 geographical strains ofCOI gene . In the  MT-COI gene haplotypes of different geographical strains and the NJ and ML methods were used to construct the phylogenetic trees respectively with 1,000 times bootstrapping to assess branch confidence. The trees constructed from the two methods are quite similar, so we took NJ tree for illustration  among geographical strains and the results were summarized in  Aedes albopictus population structure mainly comes from the population interior. The total of Fst value (0.21871) and Nm value (0.893) indicates that the overall gene flow of Aedes albopictus failed to prevent the population differentiation caused by genetic drift and there is a certain level of genetic differentiation in the population.The AMOVA analysis  shows th Aedes albopictus indicates that the p-values of SSD and HR are all greater than 0.05, suggesting an expansion of Aedes albopictus in history. From the perspective of geographical strains, the p-values of SSD and HR of Hainan, Yunnan, and Taiwan strains are all greater than 0.05, suggesting an expansion of Aedes albopictus in these areas in history. In Fujian and Guangdong, only p-value of HR parameter is greater than 0.05, suggesting that Aedes albopictus in these areas do not have significant expansion in history.We used the mismatch analysis of Arlequin  to asses Aedes albopictus MT-COI gene  and Fs value (−24.983) of overall population both reach a significant level, indicating a significant population expansion in history. As for specific geographical strains, except for Fujian strains with positive D value and Yunnan strains without significant negative D value, other geographical strains are with statistically significant negative D and Fs values. The general results are consistent with the mismatch analysis.Tajima's D value and Fu's Fs value  were calθ = 2Nu, the effective size of female mosquito population was estimated based on θ0 and θ1 before and after expansion. When θ0 = 0.443 and θ1 = 99999, the effective number of female mosquitos in the population is estimated to be 3.4 × 104~7.6 × 109. According to τ (95% CI) = 2ut = 3.943, the population expansion occurred in 3.0 × 105 years ago.According to the formula  Aedes albopictus MT-COI gene sequences. The correlation curve between genetic differentiation coefficients Fst and geographical distances was illustrated in The Mantel test was performed on the geographical distances between different collection points and Aedes albopictus by different methods. Preliminary studies have confirmed that there is a certain degree of genetic differentiation among Aedes albopictus populations. Therefore, further studies on genetic variation of Aedes albopictus population are needed to provide valuable information for the prevention and control of dengue fever. Also, new mosquito species can become the vector of new diseases, and it is critical to surveil the mosquito species in a geographical region.The genetic diversity of insect species can arise from two sources: internal and external causes. The internal causes are genetic mutation and adaptability of insect itself while the external factors are closely related to ecological environment of insects. The accumulation of genetic difference will lead to reproductive isolation and thus the formation of new species. A few scholars have studied the diversity of Aedes albopictus in this study  are higher than those in Aedes aegypti , indicating high diversity of Aedes albopictus population. Aedes albopictus exhibited high Hd and low Pi mode probably because of rapid population expansion after the bottleneck effect. The formation of a new haplotype is due to a single base variation, which has less effect on nucleotide diversity, so increasing nucleotide diversity requires more time to accumulate than increasing haplotype diversity. The bottleneck effect can eliminate the accumulation of nucleotide diversity in the past but can also cause mutations at the base site, resulting in a pattern of high Hd and low Pi. The possible reasons for the bottleneck of Aedes albopictus may be as follows: on the one hand, in terms of neutral test of Aedes albopictus  and the bifurcation distribution representing a single peak, Aedes albopictus has been or is in a state of population expansion. On the other hand, dengue fever has caused great harm to human health since it was recognized that controlling the number of media mosquitos is critical for controlling the spread of the disease. Therefore, a wide range of remediation media mosquitos have a greater impact on Aedes albopictus population.Haplotype diversity (Hd) and nucleotide diversity (Pi) are two important indicators to measure the diversity of a species population . The hap Aedes albopictus in different regions of Guangzhou about 0.000~0.007 in a previous study. Among them the genetic distances between Fujian and other geographical strains were greater than 0.02 , indicating that the level of gene exchange failed to prevent population differentiation caused by genetic drift and there is a certain level of genetic differentiation between populations. In terms of Nm value of different geographical strains  and Guangdong, Taiwan and Liaoning strains are less than 1, indicating that Aedes albopictus gene exchanges between (Yunnan + Hainan) and the four regions are hindered. This is consistent with NJ tree that Yunnan + Hainan was clustered into one category. The NJ tree and gene flow show that the genetic exchanges frequently happen among Taiwan, Fujian, and Guangdong mosquitos, suggesting that Taiwan strains have the same type of geographical populations as Fujian and Guangdong strains. No relevant reports have been found yet.Morton  believed strains , the Nm  Aedes albopictus population are larger than those between populations. Due to the different ecological and climatic conditions in different regions, there are various kinds of natural barriers. However, Aedes albopictus belongs to the semihabitat mosquito and is closely related to human activities, the geographical barrier failed to completely prevent the gene flow, so genetic differentiation mainly which comes from internal and genetic differentiation among the populations is not high. The result of the Mantel test  shows that the degree of CO I gene sequence variation is positively correlated with geographical distance. Further experiments will be conducted to confirm this conclusion. Giving more and more genetic sequences of mosquitos in different regions has been published; it would be interesting to study the genetic differences of mosquito populations throughout China or across a continent in the future.Finally, this study shows that the genetic differences within Aedes albopictus populations in different geographical regions; (2) there is a positive correlation between the genetic differences of Aedes albopictus population and their geographical distances; and (3) DNA barcoding of MT-COI gene is an effective method to study the genetic structure of Aedes albopictus.In conclusion, our results suggest that (1) there are genetic differences among


Cotesia plutellae, which is an important larval parasitoid of the worldwide pest Plutella xylostella. A subsequent test was performed for a wild C. plutellae population (N = 32) from Fuzhou, Fujian, southeastern China, to verify the effectiveness of the 14 microsatellite loci in future studies on C. plutellae genetic diversity. The observed number of alleles ranged from two to six. The expected and observed heterozygosity ranged from 0.123 to 0.316 and from 0.141 to 0.281, respectively. The polymorphism information content (PIC) value ranged from 0.272 to 0.622. Potentially due to the substructure of the sampled population, three of the 14 microsatellite loci deviated from Hardy—Weinberg equilibrium (HWE). Further, loci C6, C22, and C31 could be amplified in Cocobius fulvus and Encarsia japonica, suggesting the transferability of these three polymorphic loci to other species of Hymenoptera.Fourteen polymorphic microsatellite loci were isolated in this transcriptome-based data analysis for  Plutella xylostella) is one of the most devastating pests for the global Brassica industry, and the estimated annual losses and management costs are approximately US $4–5 billion [P. xylostella is attacked by natural enemies in all developmental stages; biological factors  thus play a significant role in the integrated management of this species. There are more than 60 reported parasitoid species of P. xylostella, of which 12 have been widely applied for pest management in the field [Cotesia plutellae is a larval parasitoid of P. xylostella, and its global distribution covers 38 countries on all continents except Antarctica [P. xylostella, successful introduction of C. plutellae has been documented more than 20 times [P. xylostella larvae by C. plutellae may increase their demand for food [C. plutellae moves out of the body of its host, eventually leading to its death.The diamondback moth ( billion . TraditiC. plutellae.Knowledge of the genetic variation and structure of the parasitoid populations favors parasitoid conservation and better pest control. Polymerase Chain Reaction (PCR) and molecular marker-based techniques are increasingly becoming recognized as valuable tools in ecological studies, and have been widely used in evaluating the genetic diversity of natural enemy populations . VariousA comprehensive understanding of the biological characteristics, ecological characteristics, genetic variation, and interrelationships in different geographical populations of pests are prerequisite for the development of an effective pest control strategy . Using mC. plutellae transcriptome dataset [http://www.ncbi.nlm.nih.gov/sra/DRX001445). Primer 3.0 [A total of 19,309 microsatellite loci—among which 17,070 had complete motifs—were detected by MISA (MicroSAtellite identification)  based on dataset , downloaimer 3.0  was usedC. plutellae colony , and the subsequent identification of polymorphic loci and population genetic analysis was performed using individuals from wild C. plutellae populations . The C. plutellae were collected from nine 20 × 30 m plots with an average distance of 1.47 km  between plots. Considering the arrhenotokous parthenogenesis of Hymenopteran species  [C. plutellae individuals were used for population genetic analysis (N = 32).Primer effectiveness was verified on a lab-reared ed eggs) , only feM-13 (TGT AAA ACG ACG GCC AGT) at their 5’ ends, and M-13 labeled with FAM, HEX, or TAMRA fluorescent dye was also used in the genotyping system.Genomic DNA was individually isolated with the DNeasy Blood and Tissue Kit  following the manufacturer's instructions. We initially performed PCR to validate the effectiveness of the primer pairs. Polymorphism identification was based on the primer pairs and the expected product sizes. The forward primers of the validated primer pairs were linked with the universal primer M-13. The temperature conditions were at 94 °C for 10 min, and then 30 cycles at 94 °C for 30 s, Tm , 0.2 µL forward primer, 0.8 µL reverse primer, and 0.8 µL After testing by agarose gel electrophoresis (AGE), sizes of the amplification products were detected using an ABI 3730 sequencer . GeneMapper 4.1  was used to assign alleles based on the sizes of PCR amplifications.OH), observed heterozygosity (EH), and polymorphism information content (PIC) were calculated using genepop V4  [HWE) and linkage disequilibrium (LD) were also tested using genepop V4.Fourteen polymorphic loci were isolated in this study. Number of alleles, expected heterozygosity ( France) . Hardy—WBlastophaga javana (Hymenoptera: Agaoninae) [Trypoxylon (Trypargilum) albitarse (Hymenoptera: Crabronidae) [N = 30) for Pachycrepoideus vindemmiae (Rondani)  [PIC value of <0.25; and to show high polymorphism with an associated PIC value of >0.5. The polymorphism level of the 14 identified microsatellite loci in the present study were medium-to-high, with PIC values ranging from 0.272 to 0.622. The EH and OH values of the 14 microsatellite loci ranged from 0.123 to 0.316 and 0.141 to 0.281, respectively. Compared with the values of EH, the relatively lower OH values suggest a lack of heterozygosity in the examined population  , the numronidae) ; and themalidae) . A locuspulation .HWE, and significant LD was observed between loci C6 and C32. Samples for this study were collected from different fields patchily distributed within the sampling site. Considering the weak migration capacity of C. plutellae, the structures of sub-populations from different fields might be responsible for such a deviation from LD and HWE.Loci C6, C32, and C53 deviated significantly from Cocobius fulvus and Encarsia japonica, and three of the fourteen microsatellite loci showed amplified products with the expected sizes  was also verified in ed sizes . Therefo


Lutjanus campechanus) young-of-the-year (YOY) sampled at six localities and in adults sampled at two localities in the northern Gulf of Mexico. Significant genetic heterogeneity was detected between the two adult samples, separated by ~600 km, and at spatial scales less than five kilometers among samples of  YOY. Genetic differences between YOY samples and between YOY samples and adult samples were not associated with geographic distance, and a genome scan revealed no evidence of loci under selection. Estimates of the effective number of breeders, allelic richness, and relatedness within YOY samples were not consistent with sweepstakes recruitment. Instead, the data demonstrate, at least within one recruitment season, that multiple pulses of recruits originate from distinct groups of spawning adults, even at small spatial scales. For exploited species with this type of recruitment pattern, protection of spawning adults over wide geographic areas may be critical for ensuring productivity and stability of the fishery by maintaining larval supply and connectivity.Conservation and management of exploited species depends on accurate knowledge of how genetic variation is partitioned across a fishery, especially as it relates to recruitment. Using double-digest restriction-site associated DNA sequencing, we surveyed variation in 7,382 single nucleotide polymorphisms (SNPs) in red snapper ( Genetic heterogeneity observed among recruits in marine organisms (genetic patchiness) is thought to be generated by a number of different processes, including: 1) variance in reproductive success or sweepstakes recruitment in extreme cases, 2) natural selection operating on early life stages, and/or 3) contribution from localized subdivided groups of breeders3Lutjanus campechanus) is the target of the largest combined recreational and commercial fisheries in the United Statesth century when abundance decreased by 90%7891011Red snapper , raw sequence data has been deposited in the Short Read Archive of NCBI (BioProject PRJNA329407), and Raw SNPs, Final SNPs, and haplotype calls can be found on Figshare (Please see Data Accessibility Section).Samples of YOY red snapper were collected during the late fall of 2012 and early spring of 2013 from six, near-shore reefs and represent a single recruiting cohort. Reefs were a combination of natural patch reef (NR) and small experimental reefs (AR). Adults sample were collected between 2011 and 2013 from two localities bracketing the YOY samples . MetadatBayescanq-value which directly corresponded to a false discovery rate of 0.05. Loci with a q-value less than 0.1 were removed from other analyses. Homogeneity of haplotype distributions was tested using single-level analysis of molecular variance (Amova), and pairwise FST values and Nei’s genetic distanceGenoDiveMegaNb) was estimated for each YOY sample, using the linkage disequilibrium methodNeEstimatorML-RelateHeirfstatFST = 0.001; P = 0.001); no outlier loci were detected , whereas sites PB1 and AR2 are ~443 km apart and genetically homogeneous . No coherent geographic clustering of YOY localities was identified  and 3,060 , respectively. Genetic diversity, measured by rarefied allelic richness, did not differ significantly among localities .Average, within-site pairwise relatedness differed significantly among localities but was low Bioinformatic code (Raw sequence data (SRA: BioProject PRJNA329407)https://figshare.com/articles/TotalRawSNPs_vcf_gz/3490226)Raw SNP calls Filtered SNPs Final haplotype calls .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Pyrus) is one of the most ancient and widely cultivated tree fruit crops in temperate climates. The Mount Etna area claims a large number of pear varieties differentiated due to a long history of cultivation and environmental variability, making this area particularly suitable for genetic studies. Ninety-five pear individuals were genotyped using the simple sequence repeat (SSR) methodology interrogating both the nuclear (nDNA) and chloroplast DNA (cpDNA) to combine an investigation of maternal inheritance of chloroplast SSRs (cpSSRs) with the high informativity of nuclear SSRs (nSSRs). The germplasm was selected ad hoc to include wild genotypes, local varieties, and national and international cultivated varieties. The objectives of this study were as follows: (i) estimate the level of differentiation within local varieties; (ii) elucidate the phylogenetic relationships between the cultivated genotypes and wild accessions; and (iii) estimate the potential genetic flow and the relationship among the germplasms in our analysis. Eight nSSRs detected a total of 136 alleles with an average minor allelic frequency and observed heterozygosity of 0.29 and 0.65, respectively, whereas cpSSRs allowed identification of eight haplotypes (P. amygdaliformis, few P. pyraster genotypes exhibited higher genetic similarity to local pear varieties. Our analysis revealed the presence of genetic stratification with a ‘wild’ subpopulation characterizing the genetic makeup of wild species and the international cultivated varieties exhibiting the predominance of the ‘cultivated’ subpopulation.The pear (genus plotypes . These r Pyrus spp.) is one of the most cultivated fruit crops in temperate zones. Pyrus species are traditionally divided into two groups based on domestication area and geographic distribution. European pears (P. communis) are cultivated mainly in Europe and the U.S., and Asian pears  grow in East Asian countries. The genus Pyrus belongs to the family Rosaceae, subtribe Pyrinae and contains at least 22 widely recognized primary species, all indigenous to Asia, Europe, and the mountainous area of North America. The worldwide production of European pear relies on a few main cultivars released from the late 18th century onward (or derived from those cultivars).The pear  as rootstocks, an agronomical practise that increases the hardiness and longevity of the trees [P. pyraster = P. communis ssp. pyraster L.) is a woody plant closely related to the European pear. This species comes from the western Black Sea region. Its distribution extends from the British Isles to Latvia [Although Sicily is not important in pear production and does not include large areas of production, it exhibits significant germplasm diversity. In the Mount Etna area, the large amount of pear biodiversity could be related to ancient practises of cultivation and seed propagation combined with the variety of favourable microclimates, soils and orographic conditions. In addition, pear cultivation in Sicily was historically characterized by a wide use of wild pears  present a high level of transferability and polymorphisms in the subtribe Pyrinae , allowing their use to assess genetic diversity and cultivar identification in pear [Pyrus [Pyrus for both parentage and taxonomy studies [P. pyraster, P. communis, P. pyrifolia and P. ussuriensis using nuclear and chloroplast SSR markers for improving preservation measures [Pyrus resulting from the self-incompatibility of the genus. The use of molecular markers could have a direct positive implication for the genetic characterization of the germplasm collection, laying a foundation for use of genetic polymorphisms to make predictions of phenotype changes through marker-trait association analysis.DNA markers have become powerful tools for cultivar identification, evaluation of genetic diversity and parentage analysis. Studies on the genetic relationships of pear genotypes were performed using nuclear  and chlo species –22. In a in pear –26. Markr [Pyrus –30. The r [Pyrus –32. Thusr [Pyrus . These mr [Pyrus . Nuclear studies , 35–36. measures –40. The In the present study, nuclear (nSSR) and chloroplast (cpSSR) microsatellites were used (i) to estimate the level of differentiation within the cultivated genotypes and the wild accessions; (ii) to elucidate phylogenetic relationships between the cultivated genotypes and the wild accessions; and (iii) to estimate the potential genetic flow between and the relationship among local Sicilian pear genotypes, native wild species and international varieties.Ninety-five pear genotypes were used in this study , includiGenomic DNA was extracted from fresh leaves using ISOLATE II Plant DNA Kits . The quantities and qualities of the extracted DNA samples were determined using a Nanodrop 2000  spectrophotometer and agarose gel electrophoresis. DNA samples were stored at -20°C.CAC GAC GTT GTA AAA CGA C) tagged with 6-FAM, NED, VIC or PET and 1U of MyTaq DNA polymerase (Bioline). Amplifications were conducted using a programme with an initial denaturation step at 95°C for 15 min followed by 35 cycles at 95°C for 30 sec, 52–55°C for 30 sec and 72°C for 45 min with a final cycle of 72°C for 15 min. A 0.4- to 0.6-μl aliquot of PCR product (depending on the performance of amplification of each primer pair) was mixed with 13 μl of formamide and 0.3 μl of LIZ-500 size standard and denatured at 95°C for 5 min. Up to four PCR products labelled with 6-FAM, PET, VIC or NED were pooled before separation in the ABI 3130 Genetic Analyser  and subjected to subsequent analysis using GeneMapper 4.0 software.PCR amplification was performed using four chloroplast SSR primer pairs derived from the pear genome and eight nuclear SSR primer pairs derived from the pear and apple genomes . PCR reaGenetic distance was estimated by analysing dissimilarity indices calculated using allelic data by simple allele matching to obtain the genetic dissimilarity matrix. Dendrogram trees were obtained using Dissimilarity Analysis and Representation for Windows software version 5.0 (DARwin5) by the neighbour-joining method . The robST) was calculated using GenePop software [The numbers of genotypes, the numbers of alleles, the major allele frequency (MAF), the expected heterozygosity (exp-het), the observed heterozygosity (obs-het) and the polymorphism information content  for each SSR marker were calculated using PowerMarker . Pairwissoftware .qI) was greater than or equal to 0.8 [http://www.fluxus-engineering.com/sharenet.htm) with default settings.The level of genetic stratification within the germplasms in the analysis was assessed using STRUCTURE v.2.3.1 . This anl to 0.8 –49. Prinl to 0.8  http://wP. pyraster n. 6 and P. amygdaliformis n. 10) genotypes exhibited no PCR amplification and were excluded from further analysis.Capillary electrophoresis analysis produced clear profiles for all four cpSSR and eight nSSR loci for 91 pear genotypes. In contrast, two local (‘Savino’ and ‘Angelico’) and two wild .The nSSRs detected a total of 136 alleles with sizes ranging from 115 to 256 bp with average values of 17 and 0.29 for the number of alleles and the MAF, respectively . The meaThe cpSSR analysis detected a total of 11 alleles with an average value of 2.75 alleles and sizes ranging from 182 to 216 bp . The chlOverall, cpSSRs and nSSRs discriminated 81 of the 91 analysed genotypes, detecting a total of 147 alleles with an average value of 12.25 and an average MAF of 0.47. The mean values of exp-het, obs-het and PIC were 0.63, 0.43 and 0.61, respectively.P. pyraster n. 2, 3, 9 and 10). Among these, ‘Max Red Bartlett’, ‘William’s’ and ‘Virgolese’-’Pergolesi’ were undistinguished.The genetic relationship among analysed genotypes is presented in the neighbour-joining dendrogram constructed using both nSSR and cpSSR data . The cluP. pyraster n. 1 and 11). Among these, the two ‘Bianchetto’ accessions (1 and 2) presented the same SSR profile. The second subgroup includes nine local genotypes, among which ‘Faccibedda’-’Pauluzzo’ and ‘Moscatello maiolino’-’Franconello’ exhibited the same SSR profile. The third subgroup includes all nine wild genotypes of P. amygdaliformis, four wild genotypes of P. pyraster  and the genotypes ‘Spineddu’ (NCV) and ‘Sciaduna’ (LV). The local genotypes ‘Moscatello’ (NCV) and ‘Chiuzzu’ (LV) represented cluster C.Cluster B includes three subgroups (B1-B2-B3). The first subgroup B1 includes three international varieties, ‘Butirra Hardy’, ‘Abate Fetel’ and ‘Kaiser’; nine NCV; 20 LV; and 2 RS . This analysis was performed following a plateau criterion , a non-pQI > 0.8) ‘wild’ genetic configuration, whereas twenty-two exhibited a predominance of the ‘cultivated’ sub-population. The remaining nine samples exhibited QI values less than 0.8 for both subpopulations and were therefore considered ‘admixed’ , a summary statistic quantifying the variation in allelic frequencies between groups. The FST between these two subpopulations was 0.096, whereas the pairwise FST estimates between ‘admixed’ and ‘wild’ or ‘admixed’ and ‘cultivated’ exhibited considerably reduced values .The distinction between ‘wild’ and ‘cultivated’ subpopulations was further confirmed by the analysis of the fixation index , and an additional round of structure analysis was separately performed on each of the two subsets following the approach presented by Urrestarazu and colleagues (2012) .P. pyraster and P. amygdaliformis accessions were assigned to the same subpopulation. In contrast, at K = 5, the two species were assigned to different sub-populations . Local cultivars (LV) and national cultivars (NCV) exhibited a similar pattern characterized by a unimodal distribution of the samples centred in the range between -2 and +2 PC1 values. PC2 allowed a distinction within the RS, NCV and LV groups. The RS group is composed of individuals belonging to P. amygdaliformis and P. pyraster species. Extreme negative values indicated P. amygdaliformis individuals, whereas values of PC2 greater than -3.5 indicated the presence of P. pyraster. Similarly, NCV and LV can be further differentiated according to PC2, whereas PC3 can efficiently discriminate within the ICV group (data not shown).Both nSSR and cpSSR were employed for PCA. The first two principal components (PC1 and PC2) accounted for 14.5% of total genotypic variability, allowing a distinction of cultivars according to their genetic makeup. The four groups of cultivars defined in Pyrus genus includes important cultivated species that have been widely studied by means of different molecular markers, mostly nuclear markers. Polyploidy was occasionally observed in several Pyrus species [Pyrus were inferred by coupling cytoplasmic and nuclear markers. Two different types of SSR markers were adopted to combine the investigation of the maternal inheritance property of cpSSRs with the high informativity of nSSRs [The Mount Etna area claims a large number of differentiated pear varieties due to ancient cultivation practises and the variability of soil and climatic conditions. Local varieties display significant variability in many agronomic traits, including fruit size, flowering and ripening periods and harvesting time . Local g species . In thisof nSSRs .Pyrus and Malus is confirmed by the high heterozygosity of CH02h11a and CH05d04 with the latter exhibiting the highest allelic diversity within the nSSR marker set. On the other hand, CH04e03 exhibited the lowest heterozygosity but was still efficiently used in the germplasm characterization analysis [P. communis for nSSRs [P. ussuriensis for cpSSRs [P. communis using different sets of SSRs (ranging from 5.9 to 12.8) [The study presented here employed eight nSSRs . Among tanalysis . Althouganalysis , 56–58. analysis , 59–60. or nSSRs –17, 55 ar cpSSRs . Overallto 12.8) , 61–66.Molecular characterization using SSR markers highlighted high genetic variability among the analysed accessions. It was possible to characterize most of the local varieties and to highlight the genetic relatedness of Italian varieties (NCV + LV) with wild genotypes .As expected, the international varieties ‘Williams’ and ‘Max Red Bartlett’ exhibited the same SSR profile, confirming the origin of ‘Max Red Bartlett’ as a bud mutation of ‘Williams’. Fingerprint analysis confirmed other previously known pedigree records, such ‘Harrow Sweet’ (‘William’ x ‘Purdue 80–50’), for which the observed SSR profiles were consistent with the known origin. Some local varieties exhibited the same SSR profile , indicating possible cases of synonymy or, at the very least, a close genetic relationship. This question could be further investigated through phenotypization and/or deeper molecular analysis.Interestingly, most of the local varieties clustered together in B1 and B2 subclusters of the dendrogram. In structure analysis, most of the local varieties exhibited an increased contribution of the ‘wild’ subpopulation to their genetic makeup.P. pyraster genotypes were reported in every cluster, exhibiting increased genetic similarity to the local pear varieties compared with P. amygdaliformis. In contrast, P. amygdaliformis appeared to be more conserved.Among the wild species, few P. amygdaliformis and P. pyraster and a second group of cultivated pears. The relative contribution of one of the two sub-populations clearly demonstrates the different genetic structure of the ICV and RS groups characterized by a predominant contribution of the ‘cultivated’ and ‘wild’ sub-populations, respectively. In contrast, increased levels of admixture are registered for NCV and LV groups. These results suggest an increased contribution of the ‘wild’ subpopulation to the genetic makeup of many Italian varieties, especially local varieties, compared with internationally cultivated varieties. The FST between the two subpopulations was 0.096, revealing increased population differentiation compared with other studies on pear germplasm collected in Spain [The use of nSSRs in structure analysis  allowed in Spain  or Bosniin Spain .P. ussuriensis population [Pyrus. Additionally, the lack of a haplotype unequivocally representing one of the two sub-populations  or considering the second PC.Within related species, a different level of genetic admixture was observed. For example, analysis , in whicFor pear, the identification of traits of agronomic importance, including adaptability to different pedoclimatic conditions and resistance to biotic stresses, is crucial for breeding new varieties. Most of these traits can be found in local germplasm that to date has been less exploited for genetic improvement programmes. The Mount Etna area represents an important biodiversity repository for many woody species due to the presence of many climatic and pedological conditions and a wide range of altitudinal levels. In the case of cultivated species, such as pear, this richness has also been increased by the long history of diffusion and cultivation. In the present work, a set of ninety-five individual trees representing local varieties, wild genotypes and nationally and internationally cultivated varieties have been genotyped to obtain novel insights into pear genetic structure and to elucidate the influence of wild species on the genetic makeup of local germplasm. The molecular insight presented in this work could also be useful for future association studies at the genomic level, such as Genome Wide Association Studies (GWAS), in which a prior knowledge of the genetic diversity of the germplasm collection is a prerequisite of paramount importance.Overall, our results allow a better understanding of the variability of the cultivars from the Etna area. We have provided evidence of allelic interchange between wild and cultivated sub-populations and provided useful information for better management and conservation of germplasm and the direction of pear breeding programmes.S1 FigA: Plot of the complete pear collection, B: Plot for the substructure analysis of the ‘wild’ accessions, C: Plot for the substructure analysis of the ‘cultivated’ accessions.(TIF)Click here for additional data file.S2 FigNested structure analysis for the ‘wild’  and ‘cultivated’  groups. For each analysis, the results according to the two most likely K value are reported: ‘wild’ K = 2 (A), ‘wild’ K = 5 (B), ‘cultivated’ K = 3 (C), ‘cultivated’ K = 4 (D).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Tablend order rate of change of the likelihood distribution |Ln''(K)| and the ΔK (mean (|Ln''(K)|) / sd(L(K)).Structure descriptive statistics. For increasing values of K, the number of replications are reported together with the mean and standard deviation of the estimated Ln probability of data, the mean rate of change of the likelihood distribution Ln'(K), the mean absolute value of the 2(XLSX)Click here for additional data file.S3 TableQI values less than 0.8 were considered ‘admixed’. All other individuals are grouped as ‘wild’ or ‘cultivated’ according to the predominant subpopulation. The status of the individual is also reported .Individuals exhibiting both (XLSX)Click here for additional data file.S4 TableThe four cpDNA markers allowed the definition of eight haplotypes named with progressive numbers from 1 to 8. Genotypes of the single cpDNA markers are reported in the last four columns.(DOCX)Click here for additional data file.


Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific this should be ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of the multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e.,  Multiple sclerosis (MS) is the most common debilitating neurological disease of young adults in the developed world. It is a complex heterogeneous, inflammatory disorder characterized by a loss of the myelin sheath surrounding the nerve axons in the central nervous system (CNS). MS is considered an autoimmune disease in which the possible role of infections as triggering events is actively debated. In fact, it is unclear how adaptive immune responses are involved in CNS damage, and whether such responses are the pathogenic root cause or just an epiphenomenon in MS. The defined cause of MS is still unknown, but it is established that complex interactions between environmental factors and multiple gene products are involved12578d-glucopyranosyl moiety20et al. first reported that protein asparagine-glucosylation is a modification found on HMW1 adhesin in non-typeable Haemophilus influenzae23Actinobacillus pleuropneumoniae was reported in 2011Ap HM1WC revealed a relaxed substrate specificity. Nevertheless, UDP-Glc is unequivocally the preferred substrate over UDP-Gal A. pleuropneumoniae and H. influenzae are some of the first examples of soluble bacterial protein glycosyltransferases that are capable of performing N-glycosylation with simple hexoses  on asparagine residues in conserved Asn-Xaa-Ser/Thr motifsH. influenzae (and homologues of A. pleuropneumoniae), we produced and purified glycoproteins with defined N-glucosylation patterns. These antigens were preferentially recognized by antibodies from MS patients. Moreover, the purified antibodies were shown to cross-react with the myelin in the spinal cord tissue in an experimental animal model of MS.Myelin Oligodendrocyte Glycoprotein (MOG), a glycoprotein in the CNS, has emerged as a potential autoantigen involved in demyelination processes fundamental to MS. While the importance of anti-MOG autoantibodies has been established in pediatric demyelination111315H. influenzae and autoantibodies from MS patient sera, we established methods to generate a well-defined set of N-glucosylated protein antigens using biochemical techniques. We selected the C-terminal fragment of the HMW1A adhesin , which has been reported to be well expressed, soluble, and stably folded29H. influenzae, but other reports suggest different glycosylation patterns both in vitro and in vivo31In order to identify the cross-reactivity between N-glucosylated adhesin fragments from A. pleuropneumoniae was employed for N-glucosylation because of its high expression and stability and the ability to produce glucosylated HMW1ct fragmentsI(Glc), was prepared by simultaneous co-expression of adhesin HMW1ct and N-glucosyltransferase HMW1C in E. coliI(Glc) was obtained as a mixture of three N-glucosylated HMW1ct variants containing 7, 8, and 9 glucose moieties on Asn in NX(S/T) sequons in a 1:1:1 ratio (II(Glc), 4 spatially proximal N-Glc moieties (antigen III(Glc)), a set of four unrelated N-Glc positions (antigen IV(Glc)), and an antigen containing just two proximal N-Glc moieties (antigen V(Glc)) (I-V).Similarly, the N-glucosyltransferase (HMW1C) homolog from :1 ratio . Tryptic:1 ratio . Inspire V(Glc)) . These aI(Glc) and the non-glucosylated protein I to detect IgM and IgG antibodies in sera of MS patients (We investigated the ability of patients  and normpatients  using SoI(Glc) detected IgM and IgG in MS patient sera and discriminated between the recruited population of 126 MS patients and 112 NBDs , favorably affects antibody recognition in MS. At variance, the non-glucosylated protein I did not discriminate between MS and NBD sera, and NBD sera presented a similar level of antibodies interacting with both the glucosylated I(Glc) and non-glucosylated I antigens. The relatively high titers observed for both antigens I and I(Glc) with NBD sera is not surprising as H. influenzae is a rather ubiquitous human pathogen to which most adults have been exposed. This results in the development of antibodies against epitopes that do not include the Glc moiety, but are shared by both I and I(Glc) (I(Glc), which suggests the recognition of an epitope specifically displayed on I(Glc).The new hyperglucosylated protein d I(Glc) . SignifiI(Glc) and I may interfere with the detection of significant and distinct epitopes introduced by protein N-glucosylation. To quantify the antibody binding to I(Glc) and non-glucosylated analog I in both MS patient and NBD sera, an inhibition experiment was performed. A significant difference in antibody binding between glucosylated antigen I(Glc) (pIC50 = 7.67 ± 0.28–8.65 ± 0.10) and non-glucosylated antigen I (pIC50 = <5.0) was observed for MS patient sera (I(Glc) (pIC50 = 6.37 ± 0.52–7.20 ± 0.29) and I (pIC50 = 5.92 ± 0.52–7.84 ± 0.42) in NBD sera  and I, an immunoaffinity-based fractionation was performed for representative MS (MS 1) and NBD (NBD 1) sera, as outlined schematically in Next, we explored the hypothesis that the N-Glc modification is a key determinant only for MS serum antibodies and that antibodies in NBD sera recognize protein epitopes shared by both the modified and unmodified proteins. Using the purified antigens I and the flow-through (FT1) and elution (Elu2) were collected. In the case of MS1, the un-retained FT1 fraction was loaded on a second column, bearing the immobilized glucosylated protein antigen I(Glc). Retained IgGs were eluted from both columns separately (Elu2 and Elu4), and their activities were determined by SP-ELISA (The NBD1 and the MS1 sera were applied separately to a Sepharose column bearing immobilized non-glucosylated adhesin SP-ELISA .I(Glc) and the non-glucosylated I, suggesting that all antibody reactivity had been retained on the I-immobilized column, and that these antibodies have a similar titer for both antigens -immobilized Sepharose (second column), a high antibody reactivity with I(Glc) resulted .The FT1 from NBD1 displayed a remarkable decrease in IgG antibody recognition both to antigens . This coantigens . In contt column . Indeed,ed Elu4, , which wed Elu4, . These rHMW1ct-Glc and anti-CSF114(N-Glc) IgG antibodies by competitive ELISAI(Glc)-V(Glc)) was tested in a selected population of representative MS patient and NBD sera (I(Glc) was able to inhibit the binding of anti-CSF114(N-Glc) IgG antibodies in sera from a selected population of representative MS patients (mean pIC50 = 9.07 ± 0.42). In contrast, non-glucosylated protein I did not inhibit, or only inhibited MS serum binding at significantly higher concentrations (mean pIC50 = <6.0), confirming the fundamental role of the N-glucosyl moieties on the adhesin protein for the interaction with anti-N-glucosyl antibodies in MS sera (II(Glc)-V(Glc) and II-V) were also tested for their binding to anti-CSF114(N-Glc) IgG antibodies. Interestingly, antigens II(Glc)-IV(Glc), containing 5/6 and 4 differentially-spaced N-Glc moieties, displayed a similar binding affinity to the hyper-N-glucosylated antigen I(Glc) (II-IV (V(Glc) however, displaying only two N-Glc moieties, showed a significant reduction in binding affinity to anti-CSF114(N-Glc) IgG antibodies (mean pIC50 = 7.19 ± 0.40), to an extent comparable to the binding affinity of CSF114(N-Glc) itself (I(Glc) and V(Glc) is almost 2 log units , this is a very significant decrease, even taking into account the different number of N-Glc epitopes in the various samples . Convincingly, a similar trend in binding of antigens I-V and I(Glc)-V(Glc) with anti-I(Glc) antibodies was obtained in a similar inhibition experiment (I(Glc) and CSF114(N-Glc) for purified anti-I(Glc) IgG antibodies, confirming their cross-reactivity (We then set out to investigate the cross-reactivity between the N-glucosylated adhesin antigen NBD sera . Resultsperiment . Moreoveactivity . These rI(Glc), immunohistochemistry experiments on normal and experimental autoimmune encephalomyelitis (EAE) mouse spinal cords were performed. In this experiment, the cross-species ability of purified anti-hyperglucosylated adhesin IgG antibodies from sera of a representative MS patient to recognize CNS tissue was compared to the binding of the total IgG fraction from NBD sera. The CNS myelin of mice is an accepted system for screening pathogenically relevant and immunoreactive antigens because these antigens are well conserved relative to the human counterparts. Moreover, the murine tissues are available for rapid processing thereby avoiding problems with protein degradation as is often encountered in human brain autopsy samples. Additionally, EAE is a relevant and broadly accepted model for MS and considerable insight into MS pathophysiology has been achieved using this modelI(Glc) antibodies from the representative MS1 patient serum were used in immunohistochemistry experiments (I(Glc) antibodies, purified by immunoaffinity chromatography, is too low in NBD serum. The anti-I(Glc) antibodies positively stained the mouse spinal cord (I(Glc) antibodies, staining by anti-I(Glc) antibodies from MS1 was restricted to the white matter region, overlapping the myelin . Intense anti-I(Glc) staining was particularly manifested in the spinal cord white matter of naïve mice, whereas in EAE-mice, at sites of inflammation (depicted by Hoechst nuclear staining of the infiltrating cells), staining by anti-I(Glc) antibodies was lost, in parallel to the demyelination. Anti-I(Glc) antibodies did not bind to the spinal cord neurons located in the grey matter, or to the myelin in the peripheral nervous system. These findings indicate that anti-glucosylated adhesin antibodies from MS patients can bind to the myelin in the mouse CNS. The observation that after demyelination in the EAE model little myelin remains for recognition by anti-I(Glc) antibodies further supports the hypothesis that these antibodies recognize myelin.To demonstrate the biological significance of the binding of antibodies from MS patient sera to hyperglucosylated antigen eriments  and 5a, nal cord  and 5a, nal cord  and 5b. Campylobacter jejuni, which is the leading cause of gastroenteritis in humansC. jejuni infection symptoms clear in a few days, epidemiological studies show a significant correlation between C. jejuni infections and Guillian-Barré syndrome (GBS), which manifests in acute neuromuscular paralysis and approximately 25% of GBS patients have developed symptoms after infection with C. jejuniC. jejuni lipooligosaccharide ultimately results in the autoimmune neuropathy conditionet al. recently reported 4 cases of encephalomyelitis correlated to an unknown pathogen. On the basis of the genomic studies and the ultrastructural analysis of the pathogen, they proposed a new disease entity having archaeal featuresGenome sequencing, in combination with biochemical, bioanalytical, and bioinformatic approaches373839414243The current study starts from the critical observation that antibody titers against a glycopeptide designated as CSF114(N-Glc) are increased in the sera of MS patientsWith these considerations in mind, we turned our attention to the bacterial glycoconjugates of human pathogens. N-Glc is a prokaryote-specific modification that is found in selected Gram-negative bacteria, where it is most commonly found on cell-surface proteins such as (autotransporter) adhesins – biosynthesized as part of the three-protein HMW cluster including the N-glucosyl tranfserase HMW1C. These adhesins are abundantly displayed on the bacterial cell surface, and because they are often extensively glycosylated, the glycan structures are likely to be presented in a multivalent manner, which would potentially favor the emergence of a robust immunologic response.Haemophilus influenzae, which is a common cause of upper respiratory infection in humans. The biochemical construction of N-glucosylated protein antigens using the biosynthetic machinery from H. influenzae and A. pleuropneumoniae was critical to isolate purified antibodies from MS patient sera. Evidently, introduction of at least four N-glucosyl moieties on the H. influenzae C-terminal adhesin fragment HMW1ct(1205–1526) is essential for the identification of the highest affinity antibodies in multiple sclerosis. The purified MS antibodies were used in SP-ELISA titrations, inhibition, and SPR experiments, to demonstrate specific recognition of the N-Glc epitope. Finally, in immunohistochemistry experiments, the purified MS antibodies showed intense and specific staining to the myelin in the spinal cord white matter, in contrast to the absence of staining by total IgG antibodies from normal blood donor.We present evidence of a strong connection between MS and the pathogen H. influenzae hyperglucosylated adhesin I(Glc) is the first example of an N-glucosylated antigen that can be considered a relevant candidate for triggering pathogenic antibodies in multiple sclerosis. Our data are in agreement with a recent report on microbial infections and degenerative neurodiseasesWe conclude that the With these new findings, the foundation is established for determining the nature of the molecular mimicry mechanism, and for elucidating the human protein target(s), which are cryptic mimics recognized by anti-hyperglycosylated adhesin antibodies in Multiple Sclerosis.Multiple Sclerosis (MS) patient sera samples were collected in two Multiple Sclerosis Centers: The Multiple Sclerosis Clinical Care and Research Centre, Department of Neurosciences, Reproductive Sciences and Odontostomatology, Federico II University  and the Azienda Ospedaliera Universitaria Careggi, Clinica Neurologica, University of Florence . Normal Blood Donor (NBD) patient sera samples were collected by F. Lolli at the Azienda Ospedaliera Universitaria Careggi, Immunotransfusion Centre, University of Florence .I(Glc). MS1, displaying high IgG titer also to I (>1), was selected for immunoaffinity purification of anti-I(Glc) antibodies used for immunohistochemistry and Surface Plasmon Resonance experiments. The age of disease onset ranged from 10 to 40 years. The clinical findings, the EDSS and the clinical course of the MS patients were evaluated at the time of blood sampling and follow-up. MRI examinations were made for diagnostic purposes with typical MS findings. In seropositive patients, disease duration was between 6 and 9 years, the EDSS score was between 2.5 and 4. Four of these patients (MS2-MS5) were in treatment with interferon-beta and MS1 was an untreated patient.MS patient sera samples were collected from 126 patients  aged from 19 to 65 years (mean age 39 years). The patients fulfilled established international diagnostic criteriaI(Glc) and I; two out of these four were IgG negative to CSF114(N-Glc) (NBD1 and NBD3), while two were positive (NBD2 and NBD4). NBD1 was selected as control in immunoaffinity purification experiments.As controls, we collected 112 Normal Blood Donor (NBD) sera samples matched for age and sex with MS patient sera samples. Total IgM and IgG titers were measured by SP-ELISA using all the NBD sera samples as controls. Four NBD sera samples (NBD1-NBD4) were subsequently selected for detailed analysis among those displaying IgG > 1 to The present study was conducted in accordance with the Declaration of Helsinki. All experimental protocols performed were approved by the Ethics Committee of the University Hospitals above mentioned involved in this study. Written informed consent forms were obtained from all MS patients and NBD subjects for sampling and research. All the methods were carried out in accordance with relevant approved guidelines and regulations.E. coli, and obtained in the pUC57 vector (Genscript). Using KpnI and SacI restriction sites, the gene was amplified by PCR and ligated into the pET-45b (+) vector encoding carbenicillin resistance (Novagen). A tryptophan residue was introduced in the N-terminal overhang by transforming Gly to Trp using QuickChange mutagenesis. HMW1ct mutants were designed by either substituting the Asn residue for Glu (N → Q) or substituting the Ser/Thr residue for Ala (S/T → A). The resulting mutants were constructed using the Quik Change II site-directed mutagenesis kit and the mutagenic primers sets (Actinobacillus pleuropneumoniae genomic DNA (ATCC: 27088D-5) and inserted into the pET-24a (+) vector (Novagen), as described previously336-tag was removed by introducing a stop codon after the hm1C gene using the primers in The gene encoding the HMW1ct corresponding to amino acids 1205–1536 was synthesized commercially after gene optimization for ers sets  accordinHis6-hmw1ct and pET24::hmw1C-stop was initiated by mixing both plasmids (0.5 μL) into BL21 cells (50 μL). After heat shock, the cells were diluted with SOC media (200 μL) and incubated at 37 °C for 90 min. A 100 μL aliquot was plated out on carbenicillin/kanamycin/chloramphenicol plates and incubated at 37 °C overnight. Colonies were selected for growing in a 5-mL overnight culture. Subsequently, the bacteria were grown to an OD600 of 0.6–0.8 at 37 °C, and expression was induced with 1 mM IPTG, followed by incubation at 16 °C overnight. Subsequently, bacterial cells were harvested, resuspended in lysis buffer  and disrupted using sonication (3 × 1 min at 40% amplitude) in the presence of protease inhibitor (10 μL per gram of cells). After centrifugation at 35,000 × g for 65 min at 4 °C, the clear supernatant was applied to a suspension of Ni2+-NTA beads at 4 °C for 1 h. The beads were transferred to a frit-containing column, the flow-through was collected, and the beads were subsequently washed with washing buffer  and both glucosylated HMW1ct and HMW1C were eluted with elution buffer . Protein fractions were pooled and dialyzed into 20 mM Tris pH 8.0 and 20 mM NaCl. The protein mixtures were loaded onto a 5-mL anion exchange column (HiTrap Q FF) and eluted with a linear gradient of 20 mM Nacl-1M NaCl using an AKTA FPLC system, to facilitate separation of glucosylated HMW1ct and HMW1C. The fractions containing I(Glc) were pooled, concentrated, and dialyzed  to give a total yield of ~16 mg of I(Glc) from a 1 L culture.Transformation of the two plasmids pET45::V was observed. Instead of glycosylation of the two remaining sites, a mixture of proteins with 2, 3, and 4 glucose units was observed in a ratio of 2:2:1 respectively. Glucosylation of non-canonical sites has been reported previouslyin vitro glucosylation protocol, the glucosylation level of antigen V was greatly reduced, resulting in pure twice glycosylated antigen V(Glc).Using the co-expression protocol, over-glucosylation of antigen V and equivalents of HMW1C were varied, optimal conditions were obtained to produce V(Glc) with only two Glc moieties: 50 μM V, 500 nM HMW1C, 500 μM UDP-Glc in a final volume of 10 mL. The reaction was incubated at RT overnight, and subsequently purified using Ni2+-NTA.After optimization studies, in which the concentration of I(Glc) was diluted with 100 μL 8 M urea (in 50 mM ammonium bicarbonate pH 8.5). The sample was heated at 55 °C for 1.5 h, after which time 12 μL 300 mM iodoacetamide in ammonium bicarbonate was added. The mixture was incubated at RT in the dark for 60 min, and subsequently transferred to a 10,000 MWCO spin filter. A 300 μL aliquot of ammonium carbonate was added, and the mixture was centrifuged at 14,000 × g to reduce the volume in the filter to below 50 μL. This was repeated with 300 μL and 400 μL ammonium carbonate, followed by transfer of the residue to a clean eppendorf tube. Then 6 μL of a freshly prepared trypsin solution (1 μg per 30 μL 50 mM ammonium carbonate) was added to the sample, and the mixture was incubated at RT overnight. Subsequently, 5.6 μL 10% formic acid in water was added to acidify the mixture, and it was analyzed using LC-MS/MS (A sample containing 4.6 μg of the purified LC-MS/MS .I(Glc), followed by incubation with a serum in the presence of a concentration range of the competing species  or I). The resultant antibody binding to the immobilized antigen is quantified using a fluorescent secondary antibody, and the absorbance units across the concentration range can be converted to a pIC50 value  (3 mg) and the non-glucosylated adhesin I (3 mg) were dissolved in NaHCO3  containing 0.5 M NaCl, pH 8.3, and were coupled to CNBr-Sepharose (200 mg) matrix (Sigma). The NBD1 and the MS1 sera (3 mL) positive to both CSF114(N-Glc) and I(Glc) (diluted 1:10 in PBS) were passed three times through the non-glucosylated adhesin I column pre-equilibrated with PBS, pH 7.2. In the case of MS1, the un-retained flow-through fraction was directly loaded onto the column and recirculated three times. Adsorbed antibodies were eluted using glycine  and recovered in D-PBS buffer pH 7.2. Protein content of specific anti-I(Glc) purified IgG antibodies was determined to be 46.5 μg/ml by UV spectroscopy measuring the absorbance of the solution at 280 nm (Shimadzu UV-1601PC) considering that the extinction coefficient is 1.4 for a solution of 1 mg/mL. The efficiency of this sequential immunoadsorbtion was confirmed by SP-ELISA.The total IgG fraction from NBD1 serum was obtained by Protein G affinity chromatography using a prepacked column  according to manufacturer’s instructions. Protein content of the total IgG fraction from NBD1 serum was determined to be 1.7 mg/mL using the method above described.I(Glc) IgG and anti-I antibodies were evaluated in SP-ELISA following a previously validated protocolSpecific anti-I(Glc) IgG antibody fraction  was diluted in running buffer to final concentrations of 16.6, 33.3, 66.6, and 133.3 nM. Diluted samples were injected over each immobilized antigen according to the method published elsewhere. Kinetic and affinity parameters were obtained using Biacore Evaluation Software 2.0 according to the 1:1 binding model.All experiments were carried out on a Biacore T100 . All solutions and buffers were prepared with milliQ water obtained by a Sartorius system (arium 611 VF). Sensor chips CM5 (carboxymethylated dextran), amine coupling kit (EDC/NHS (50:50), and Running Buffer HBS-EP+ 10×  were purchased by GE Healthcare. Running Buffer was diluted ten times with milliQ water at pH 7.4. Sodium acetate and sodium hydroxide . Each antigen was immobilized on a different channel of the chip CM5-type according to the amine coupling strategy using a concentration of 10 μg/mL in immobilization buffer at 10 μL/min for 420 s and 60 s. Unreacted groups on the sensor chip surface were blocked by injecting 60 s-pulses of ethanolamine-HCl (1 M pH 8.5) until complete deactivation. The reference channel was activated and subsequently blocked with ethanolamine. The selected buffers and the immobilized protein levels are reported in I(Glc) IgG antibodies from the representative MS patient serum or with total IgG fraction from the normal blood donor, as well as with rat anti-myelin basic protein antibodies . The second step was performed by labelling with species-specific antibodies , namely, biotinylated anti-human antibody followed by Cy3-conjugated streptavidin, and with a Cy2-conjugated anti-rat antibody. We used 535/40 nm band pass filter for detection of Cy2 and 605/50 nm band pass filter for detection of Cy3. Control slides were incubated with the non-relevant fluorescent secondary anti-IgG antibody. Sections were counter-stained with Hoechst 33258  for nuclear staining. Stained sections were examined and photographed by fluorescence microscope , equipped with Plan Fluor objectives connected to CCD camera . Images were assembled using Adobe Photoshop (Adobe Systems).Lumbar segments of spinal cords from naïve mice, as well as from mice inflicted with EAE induced by the 35–55 peptide of myelin oligodendrocyte glycoprotein (MOG) as describedHow to cite this article: Walvoort, M. T. C. et al. Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae. Sci. Rep.6, 39430; doi: 10.1038/srep39430 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Brassica oleracea var. botrytis) is an important vegetable crop for human nutrition. We characterized 192 cauliflower accessions from the USDA and IPK genebanks with genotyping by sequencing (GBS). They originated from 26 different countries and represent about 44% of all cauliflower accessions in both genebanks. The analysis of genetic diversity revealed that accessions formed two major groups that represented the two genebanks and were not related to the country of origin. This differentiation was robust with respect to the analysis methods that included principal component analysis, ADMIXTURE and neighbor-joining trees. Genetic diversity was higher in the USDA collection and significant phenotypic differences between the two genebanks were found in three out of six traits investigated. GBS data have a high proportion of missing data, but we observed that the exclusion of single nucleotide polymorphisms (SNPs) with missing data or the imputation of missing SNP alleles produced very similar results. The results indicate that the composition and type of accessions have a strong effect on the structure of genetic diversity of ex situ collections, although regeneration procedures and local adaptation to regeneration conditions may also contribute to a divergence. Fst-based outlier tests of genetic differentiation identified only a small proportion (<1%) of SNPs that are highly differentiated between the two genebanks, which indicates that selection during seed regeneration is not a major cause of differentiation between genebanks. Seed regeneration procedures of both genebanks do not result in different levels of genetic drift and loss of genetic variation. We therefore conclude that the composition and type of accessions mainly influence the level of genetic diversity and explain the strong genetic differentiation between the two ex situ collections. In summary, GBS is a useful method for characterizing genetic diversity in cauliflower genebank material and our results suggest that it may be useful to incorporate routine genotyping into accession management and seed regeneration to monitor the diversity present in ex situ collections and to reduce the loss of genetic diversity during seed regeneration.Cauliflower ( The extent and type of genetic variation present in the germplasm of a crop is an important component of efficient breeding programs, because it provides useful information for the broadening of breeding pools, the utilization of heterosis and the selection of parental lines. Also, this information helps breeders to narrow the search for new alleles at loci of interest and assists in the identification of markers linked to desirable traits for introgression into new varieties . An asseEx situ conserved plant genetic resources (PGR) are plant genotypes that are stored in central storage facilities. PGR are utilized to improve modern cultivars by the introgression of new and exotic genetic variation into breeding pools . Howeven cycles , 5. In aBrassica oleracea var. botrytis) is an important vegetable crop worldwide and a valuable component of a healthy diet because of a high content of glucosinolates with anticancer properties [Cauliflower  or simpoperties , 11). Thoperties . More reoperties . Whole goperties –17. In toperties –20. In Boperties .ex situ genebanks, which harbor large collections of cauliflower accessions. We also investigated whether imputation of missing genotypes improves diversity estimates and whether genetic and phenotypic diversity among cauliflower accessions are correlated. Cauliflower is a predominately outcrossing species whose pollination depends on insects. A self-incompatibility (SI) system prevents self-fertilization, but high variation in the extent of SI was reported in different landraces and varieties [The density of polymorphisms identified by reduced representation sequencing methods like GBS are sufficient to conduct genome-wide analyses of diversity and to construct core collections for further phenotyping or breeding. Our objective was to use GBS for assessing the genetic diversity and genetic relationship of randomly selected accessions of the USDA and IPK arieties . This vaBrassica oleracea was obtained from Heidelberg Botanic Garden and Herbarium (HEID), Germany. All accessions of this study including accession type and country of origin are listed in Table A in A total of 191 cauliflower accessions were randomly selected and ordered from the genebanks of the United States Department of Agriculture (USDA), USA and IPK Gatersleben, Germany. They represent 47% (100 of 212) of the USDA and 40% (91 of 227) of the IPK cauliflower accessions, respectively. We selected accessions from these two large genebanks because they harbor large collections of cauliflower that are expected to reflect the worldwide diversity of this vegetable crop. According to the passport information, the sample consists of traditional cultivars, breeding material, hybrids, unverified genotypes, collector material, commercial vegetable seeds and landraces . AccessiAll accessions were evaluated for six morphological traits at six environments consisting of two cultivation methods  and three growing seasons (June 2011 and April/August 2012) in a randomized complete block design (RCBD) with two replicates in each environment . Five rahttp://www.deenabio.com/services/gbs-adapters and 32 barcodes were taken from [Genomic DNA was extracted from leaf tissue three weeks after sowing in the green house from a single individual of each accession according to a standard CTAB protocol . The quaken from . All barken from . The twoBrassica oleracea sp. capitata cabbage line 02-12 [Sequence reads were filtered for sequencing artifacts and low quality reads with custom Python scripts, bwa  and Fastne 02-12  with bwane 02-12 , bcfutilp = 0.05/6 = 0.0083 to test results. The correlation between genetic and phenotypic distances based on the PCA was determined with a Mantel test [Fst values between genotypes calculated with R adegenet R package [K = 1–10 and cross-validation was used to estimate the value of K which best fits the data [Fst) between the two genebanks was estimated with the pairwise.fst function in the R package adegenet [Ho and Hexp, as well as the inbreeding coefficient (F) with adegenet. We also calculated percent polymorphic loci, %P, and nucleotide diversity, π, with the R package pegas [The genetic structure of the sample was investigated by various methods for comparison. They included principal component analysis (PCA), as implemented in the R package adegenet . Also, atel test  in the atel test . Princip package , was use package . In addi package . Populat package . The numthe data . An Analthe data . The extadegenet . For eacge pegas . To compge pegas .Fst values under an island model. LOSITAN was run in two steps: in the first step, an initial run with 50,000 simulations was performed with all SNPs and using the mean Fst. After excluding a candidate subset of selected SNPs determined in the initial run, the distribution of neutral Fst values were calculated. We also used Arlequin 3.5 [Ω, which is a variance-covariance matrix of allele frequencies, for the two groups of accessions from each genebank was calculated from the final run of the MCMC after 100,000 iterations. We used 360 (25%) randomly selected SNP markers from the SNP dataset without missing values. To evaluate the robustness of this matrix, we repeated the calculation with five randomly drawn SNP sets and calculated the average correlation coefficient of estimated variance and covariance parameters from all 10 pairwise comparisons of matrices. The resulting average correlation coefficient was 0.99, which indicates a high convergence of Ω matrices based on different random SNP sets. The Ω matrix was then used to control for the genome-wide genetic relationship among groups in the calculation of the XTX statistics for each SNP by using 100,000 iterations of the MCMC. The XTX statistic is equivalent to the Fst value as a measure of population differentiation.Outlier tests for highly differentiated SNPs between the sets from the two genebanks (USDA and IPK) were based on a coalescent-based simulation method  implemenquin 3.5  to detecquin 3.5  which isquin 3.5 . We firsB. oleracea reference genome . The mean percentage of missing data across all genotypes was 42% and values ranged from 19% to 77% per genotype. The number of SNPs and percentage of missing data per genotype are shown in Table D in To analyse genetic diversity in the sample we included one individual plant per genebank accession. The sequencing data of the 192 accessions consisted of 455 Mio. reads with a length of 100 bp. Quality filtering removed 5.2% of reads because they mapped to the PhiX genome or were of low quality, and read number per accession ranged from only 80 to 7.1 Mio with an average of 2.4 Mio reads. Eighteen accessions with less than 200,000 reads were excluded from further analysis because the proportion of missing data in these accessions was too high. A total of 133 Mio reads mapped to the e genome . The pern = 174) was analyzed with PCA, PCoA and ADMIXTURE. Here, we present the results for the set of 1,444 SNPs without missing data, but the same results were obtained with the other two data sets with missing or imputed data , which are provided as Supplementary Information.The genetic structure of the whole collection . The mean pairwise Fst of accessions within each genebank was 0.301 for the USDA and 0.160 for the IPK accessions , showing that the USDA accessions are more differentiated from each other than the IPK accessions.Despite the clear genetic differentiation between the two genebanks, a very large proportion of genetic variation (90%) segregates within rather than between genebanks , which did not differ between both genebanks.For a further comparison between accessions from the two genebanks, we calculated various genetic diversity parameters . Values FST value suggests that a small number of SNPs are responsible for the differentiation. To identify polymorphisms whose allele frequencies differ between the two genebank collections, we performed outlier tests using the GBS data without missing values . LOSITAN identified 182 (12.6%) SNPs as outliers . Arlequin identified only 79 (5.5%) SNPs as outliers . The phenotypic variance was different between accessions from both genebank, and the MANOVA with genebank as grouping factor strongly supported the phenotypic differentiation . A post-hoc analysis of single-factor ANOVA with Bonferroni correction revealed that only the traits curd width  and cluster width  differed between genebanks.To test which traits are significantly differentiated between genebanks , we usedr = 0.291, p < 0.001).Finally, a Mantel test showed a positive correlation of phenotypic and genetic similiarities between pairs of accessions  from which the reference genome was produced and cauliflower, B. oleracea var. botrytis. This interpretation is confirmed by the pan-genome analysis of B. oleracea cultivars [Previously, the genetic diversity of cauliflower was assessed with different types of markers and based on small data sets. This limitation can be overcome by sequencing-based methods like GBS . Althougin maize . Such a ultivars , 44. Anoultivars .Ho, are influenced by missing data, whereas estimates of the inbreeding coefficient and nucleotide diversity, π are very similar . A geographic population structure was found in previous survey of cauliflower cultivars [Despite the high proportion of missing data, GBS allowed to analyze genetic diversity and population structure in ultivars  despite ultivars –20. The B. oleracea germplasm. Unfortunately, limited passport information did not allow us to account for the breeding history and relationships among varieties as co-variate in the population structure analysis and to disentangle the effects collection date and geographic origin on diversity estimates. In addition, the absence of a strong geographic structure may result from a combination of low genetic diversity in cauliflower [First, the accession types (landraces vs. cultivars) and collection dates may result from different collection strategies between genebanks that may be responsible for the observed clustering of accessions. For example, the USDA sample includes a much higher proportion of unverified material than the IPK sample which mostly consist of modern varieties. Passport data suggest that many accessions in the ‘unverified’ group of USDA and the ‘cultivar’ group of IPK reflect common cultivars (or possibly landraces) that were commercially available during the time of collection. Five accessions in the IPK set differed markedly from the other IPK accessions in all analyses and they clustered together with the set of USDA accessions. They consist of four landraces and one hybrid  with mesh covers to prevent cross-pollination by insects and a population size of at least 100 plants per accession. At IPK, cauliflower accessions are cultivated in small glass houses containing other species as well. The total area is about 6 m2 and population size is 20-25 plants. At IPK, seeds are regenerated after 20 years and at USDA after 15 years, which on average corresponds to 1.5 and 4 regeneration cycles for the material included in this study. Using the formula for calculating the expected decay in heterozygosity, Ht = H0 × (1 − 1/Ne)t [H = (1 − 1/100)4 = 0.96 under the assumption that on average each accession has undergone 4 regeneration cycles, and of IPK accessions ΔH = (1 − 1/20)1.5 = 0.93. Although a more rapid decay in heterozygosity is expected in the IPK collection, the difference is rather small.A second explanation for differentiation between genebanks are seed regeneration procedures which may affect genetic diversity –50. In sulations –53 or hiulations . A reducom drift , 54, 55.− 1/Ne)t , the expex situ genebanks. The cauliflower accessions were collected in different regions of the world, and selection of genes controlling photoperiod sensitivity, flowering time, and other traits caused by local adaptation to the propagation environment may have occured. The effect of selection on fitness and genetic diversity can be quite strong. A significant correlation between genetic and phenotypic distance shown by the Mantel test and a high heritability of traits like flowering time [et al. [ex situ genebank material, and facilitates the close monitoring of allele frequency changes and a better management of ex situ germplasm collections.Natural and/or artificial selection during seed propagation of genebank accessions may also impact genetic diversity and differentiation in ing time  indicate [et al.  suggeste [et al. . Hence, A major advantage of GBS and related methods is their applicability to any species. These methods do not entail setup costs like SNP arrays and do not cost much per individual genotype, but provide sufficient power for genome-wide analyses of population structure and genetic relationships. On the other hand, GBS has a high proportion of missing data that may reduce the power for correct estimation of population parameters. Data imputation was suggested as a solution because it can be accurate and then increase the quality of genomic selection or association mapping , 60. In ex situ conserved plant genetic resources, in particular for outbreeding crops such as cauliflower.Our study outlined the usefulness of GBS to characterize the genetic diversity of genebank accessions of a minor crop like cauliflower. A key result was the strong differentiation of genetic diversity between the two genebanks which most likely reflects the different collection histories of the two genebanks. Due to a lack of detail in the passport information, factors influencing genetic diversity like sampling strategy, regeneration procedures and selection during regeneration could not be well reconstructed, although the type of accessions included (landraces vs. cultivars) likely has a strong influence. The low cost of GBS and low-coverage genome sequencing suggest that a lack of passport information can be substituted by high-resolution genotyping and suitable analysis methods to characterize the diversity of germplasm from different sources. This facilitates the exchange of material between genebanks and the construction of core collections that harbor a high proportion of species-wide genetic and phenotypic diversity for a more efficient utilization of plant genetic resources . GBS-derS1 File(PDF)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


The city has proved to be the most successful form of human agglomeration and provides wide employment opportunities for its dwellers. As advances in robotics and artificial intelligence revive concerns about the impact of automation on jobs, a question looms: how will automation affect employment in cities? Here, we provide a comparative picture of the impact of automation across US urban areas. Small cities will undertake greater adjustments, such as worker displacement and job content substitutions. We demonstrate that large cities exhibit increased occupational and skill specialization due to increased abundance of managerial and technical professions. These occupations are not easily automatable, and, thus, reduce the potential impact of automation in large cities. Our results pass several robustness checks including potential errors in the estimation of occupational automation and subsampling of occupations. Our study provides the first empirical law connecting two societal forces: urban agglomeration and automation's impact on employment. In a world struggling between localism and globalism, a question emerges: how will different cities cope with automation? Answering this question has implications on everything from urban migration to investment, and from social welfare policy to educational initiatives.Cities, which accommodate over half of the world's population , are modTo construct a comparative picture of automation in cities, our first challenge is to get reliable estimates of how automation impacts workers. Existing estimates are wide ranging. Frey & Osborne  estimateabsolute risk of computerization of jobs with skepticism, these estimates can provide useful guidance about relative risk to different cities that is robust to errors in the estimates provided by Frey & Osborne [et al. [Nevertheless, even if we take current estimates of the  Osborne  and Arnt [et al. . We can A priori, it is not obvious whether large cities will experience more or less impact from automation. On one hand, an influx of occupational diversity explains the wealth creation, innovation and success of cities [are the forces of diversity, specialization and the division of labour shaping a city's ability to accommodate automation?f cities –22. On tf cities . This enf cities –26 throuimpact includes unemployment, but may also manifest itself through the changing skill demands of occupations as automation diminishes the need for individual types of skills [Here, we undertake a comparative examination of cities while measuring the relative impact of automation on employment. We also contextualize these measurements through a detailed analysis of the skill composition of different cities. Note that f skills ,18. In t2.2.1.m having job j according tom denotes the set of job types in city m according to BLS data, and fm(j) denotes the number of workers in city m with job j.The US Bureau of Labor Statistics (BLS) data identify the employment distribution of about 700 different occupations across each of 380 US metropolitan statistical areas (MSAs) and combined statistical areas (CSAs) in 2014. (We refer to both CSAs and MSAs as ‘cities’.) We consider MSAs in isolation only when they are not part of a CSA. CSAs have arisen as the best approximation for determining cities ,6,27–31.raw skill values.For each occupation, the BLS O*NET dataset details the importance of 230 different workplace skills, such as Manual Dexterity, Finger Dexterity, Complex Problem Solving, Time Management and Negotiation. BLS obtains this information through several separate surveys which group the raw O*NET skills into the following categories: Abilities, Education/Training/Experience, Interests Knowledge, Skills, Work Activities and Work Context. We normalize the raw survey responses to obtain a value between 0 (irrelevant to the occupation) and 1  indicating the absolute importance of that skill to that occupation. We refer to these values of skill importance as 2.2.m by calculating the normalized Shannon entropy. Shannon entropy [m|) normalizes the entropy score so that we can compare the distributions of jobs in cities with different sets of job categories . The values for normalized Shannon entropy lie between 0  and 1 (diversity). Normalized Shannon entropy has been used in a variety of fields, including virology [We assess the specialization or diversity of the employment distribution in city  entropy , an infovirology , climatovirology  and cityvirology .pj(s)). Similarly to above, we measure the normalized Shannon entropy of the relative skill distribution of job j according toj denotes the set of O*NET skills with non-zero importance to job j. We employ normalized Shannon entropy here to facilitate a fair comparison of relative skill distributions between jobs which may have received the same raw O*NET value for a given skill, but have different numbers of non-zero raw O*NET skills.For a given occupation, we normalize each raw skill value by the sum of the values to obtain the relative importance of each skill to that occupation (denoted pm(s) is the relative importance of skill s in city m. Again, we use normalized Shannon entropy to assess the skill specialization in a city according tom represents the set of O*NET skills with non-zero importance in city m.We obtain a distribution of relative skill importance for a city according toT(m) = 1 indicates that each job specializes in exactly one skill, and T(m) = 0 indicates that the specialization of skills among jobs is equal to the specialization of skills on the city-level aggregation. We do not observe any jobs relying on exactly one skill, and so we expect the Theil entropy of any given city to be well below 1. We present 1 − Tm throughout the study for easy comparison to Shannon entropy. Note that both normalized Shannon entropy and Theil entropy are unit-less measures due to the normalizations employed; we therefore do not focus on their range of values across cities, but instead we focus on the relationship between labour specialization/diversity and other urban indicators.These aggregate skill distributions for a city may obfuscate the specialization of skills through the relative abundance of jobs in that city. For example, the city-level aggregation of skills may appear diverse, while the jobs within the city are actually specialized. The Theil entropy  of a cit3.3.1.m(j) denotes the employment share (as a percentage) in city m with occupation j according to the US BLS and pauto(j) denotes the probability of computerization for occupation j as estimated by [Em as the expected percentage of total employment in city m subject to computerization. Each city should expect between one-half and three-quarters of their current employment to be affected in the foreseeable future due to improvements in automation , b demonstrates that expected job impact decreases according to Em∝ − 3.2 × log10(city size), which suggests that larger cities are more resilient to the negative effects of automation. This relationship is significant with a Pearson correlation ρ = − 0.53 , and shows that labourers in smaller cities are susceptible to the impact of automated methods (R2 = 0.28). We confirm our finding using separate conservative skill-based estimates of the automatability of jobs [ρ = − 0.26  and Em∝ − 1.24 × log10(city size).  Despite the conservative nature of these alternative probabilities, we again observe increased resilience with city size. Furthermore, we demonstrate in electronic supplementary material, S3.1 that the observed negative trend relating city size to expected job impact from automation is robust to errors in the probabilities of computerization (i.e. pauto) produced by Frey & Osborne [What differentiates cities' resilience to automation? Figure 1 of jobs  (Pearson Osborne  and robu3.2.a) and, separately, in the aggregate distributions of skills (figure 2b). Additionally, we use Theil entropy to measure the proportion of specialized jobs (in terms of skills) in comparison to the skill specialization of the city on the whole. Figure 2c demonstrates an increasing proportion of specialized jobs in large cities (i.e. 1 − Tm decreases). See Material and methods for calculations of entropy measures.We explore the mechanisms underpinning resilience to automation by examining the most distinctive characteristics of urban economies: diversification and specialization. In particular, how does labour diversity, or specialization, mediate the relationship between city size and the expected job impact from automation? As automation typically targets workplace skills , we consfigure 2m), median household income (incomem), the per cent of population with a bachelor's degree (bachelorm), per capita GDP (GDPm) and the number of unique job titles (jobsm). This generic model captures 53% of the variance in expected impact from automation across US cities. Models 2, 3 and 4 use the information-theoretic measures in three separate linear regression models to reveal that skill specialization (i.e. Hskill(m)) is the most predictive of expected job impact in cities (R2 = 0.20) in the absence of other urban variables.In Hskill(m)) yields a model accounting for 60% of the variance in job impact (see model 6). Finally, we include all variables in a single model (see model 8) which produces the most predictive model accounting for 66% of the variance across cities . We confirm the stability of our regression results by alternatively training the regression model on half of the cities and measuring the performance of the regression on the remaining cities as validation .In models 5, 6 and 7, we demonstrate that the inclusion of each of the specialization measures produces models accounting for additional variance in expected impact over the use of generic variables alone. In particular, the inclusion of skill specialization  changes sign across the models in our analysis because of the strong relationship between city size and labour specialization, which we demonstrate in Each model that we tested yielded statistically significant coefficient estimates (note that variables were standardized before regression) and the inclusion of our labour specialization metrics yielded models with improved predictive power. Furthermore, we performed a formal mediation analysis that is presented in electronic supplementary material, §3.5. However, these observations should not be taken as conclusive evidence that labour specialization, or diversity, is causally related to the expected impact from automation in a city. This is due, in part, to the colinearity between variables used in model 8. For example, the city size coefficient (sizec). Examining these cities more closely may allow urban policy experts with a nuanced understanding of the policies in these cities to more easily identify causal mechanisms. The predictive power of this model and its reliance on workplace skills justifies our inclusion of skills data in addition to occupation data, and motivates us to characterize urban resilience to automation from the skills in cities.The residuals between the actual and modelled values according to model 8 highlight notably resilient cities (given the model), such as Boulder, CO, and Warner Robins, GA, and notably susceptible cities, such as Napa, CA, and Carson City, NV c. Examin3.3.m and n according toj according toHow do different types of occupations change with city size , and howj can increase or decrease the overall difference in expected job impact depending on the sign of the corresponding term in equation . In turn, this sign depends on the relative automatability of the occupation and the relative employment share. More details for this calculation and an example analysis comparing individual cities are provided in electronic supplementary material, S3.4.Occupation equation , or, equδ(j) for each occupation. Each occupation is assigned a quadrant and colour based on the sign of δ(j) and the relative automatability of occupation j. This visualization identifies both occupations that increase the differential impact (i.e. occupations on the right) and occupations that decrease the differential impact (i.e. occupations on the left). For example, increased employment for Cashiers, which is relatively susceptible to automation, in small cities contributes the most to the overall difference in expected job impact. Likewise, differences in employment for Software Developers, a relatively resilient occupation, also increases the overall difference. On the other hand, increased employment for Elementary School Teachers, which is another relatively resilient occupation, in small cities decreases the difference. On aggregate, differences in employment for occupations that are relatively resilient to automation contribute the most to the differential impact of automation in large and small cities  and examine the scaling relationship between job clusters and city size according to (number of workers)∝(city size)β in figure 5b. Note that the exponent, β, entirely describes the growth rate of these job clusters relative to city size. The job cluster comprising highly specialized jobs, such as Mathematician and Chemist, exhibits a notably superlinear scaling relationship with city size (β = 1.39). This scaling exponent is similar to the scaling relationship observed for Private R&D employment (β = 1.34) found in [β = 1.08). The job cluster exhibiting the slowest growth (β = 0.94) comprises entertainment and service jobs. We check the robustness of these scaling relationships using methods from [To explore the role of resilient occupations further, we focus on how employment for different occupation types changes with city size. We use the K-means clustering algorithm (i.e. occupations are instances and raw O*NET skill importance are features) to identify five clusters of jobs according to skill similarity . We confirm our findings using alternative definitions for aggregate workplace tasks and skills .We confirm that the fastest growing job cluster is indeed comprised of ‘technical’ jobs based on their constituent workplace skills. We employ K-means clustering (i.e. O*NET skills are instances and the correlation of raw O*NET importance of skills across occupations are features) to simplify the complete space of O*NET skills to 10 skill types based on the co-occurrence of skills across jobs . These simplified skill types allow us to intuitively explore which skills indicate specialization or indicate resilience in cities. Computational/Analytical skills and Management skills are more likely in faster growing (i.e. superlinear) jobs, while physical skills, such as Physical Coordination and Control/Perceptual skills, indicate notably slower job growth with city size d. We cona,c), while Physical Coordination and Control/Perceptual skills indicate both decreased specialization and decreased resilience in cities . We confirm our results using alternative groups of workplace tasks [Figure 6e reflects the same conclusion by comparing the relationship of each skill type to city size (right column) and expected job impact . Effectively, large cities employ workers whose skills better prepare them to interface with automation technology, while small cities rely more prominently on physical workers, who are more susceptible to automation.The skills that are relied on by fast-growing technical jobs suggest mechanisms for resilience and growth in cities. Existing work  identififigure 6ce tasks  providedce tasks   and by economic and educational policy . Nevertheless, we observe a strong trend relating city size to automation impact that is robust to errors in the automatability of individual occupations and occupational subsampling. For example, the estimates of occupational automation, which we employ in our analysis, would need to be severely flawed (errors over 50%) for the negative dependency on city size to disappear. Recognizing that small cities will experience larger adjustments to automation calls on policymakers to pay special attention to the pronounced risks we have identified.Despite being seemingly unrelated societal forces, we uncover a positive interplay between urbanization and automation. Larger cities not only tend to be more innovative ,6, but a