Penaeus monodon) is one of the most commercially important aquaculture species, a systematic assessment of genetic divergence and structure of wild-caught and domesticated broodstock populations of the species is yet to be documented. Therefore, we used skim sequencing (SkimSeq) based genotyping approach to investigate the genetic structure of 50 broodstock individuals of P. monodon species, collected from five sampling sites (n = 10 in each site) across their distribution in Indo-Pacific regions. The wild-caught P. monodon broodstock population were collected from Malaysia (MS) and Japan (MJ), while domesticated broodstock populations were collected from Madagascar (MMD), Hawaii, HI, USA (MMO), and Thailand (MT). After various filtering process, a total of 194,259 single nucleotide polymorphism (SNP) loci were identified, in which 4983 SNP loci were identified as putatively adaptive by the pcadapt approach. In both datasets, pairwise FST estimates high genetic divergence between wild and domesticated broodstock populations. Consistently, different spatial clustering analyses in both datasets categorized divergent genetic structure into two clusters: (1) wild-caught populations (MS and MJ), and (2) domesticated populations . Among 4983 putatively adaptive SNP loci, only 50 loci were observed to be in the coding region. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that non-synonymous mutated genes might be associated with the energy production, metabolic functions, respiration regulation and developmental rates, which likely act to promote adaptation to the strong artificial selection during the domestication process. This study has demonstrated the applicability of SkimSeq in a highly duplicated genome of P. monodon specifically, across a range of genetic backgrounds and geographical distributions, and would be useful for future genetic improvement program of this species in aquaculture.The domestication of a wild-caught aquatic animal is an evolutionary process, which results in genetic discrimination at the genomic level in response to strong artificial selection. Although black tiger shrimp ( The world production of inland aquaculture reached 51.3 million tonnes in 2018, with their dominant production of 97.2% comprising of finfish, while mariculture produced 30.8 million tonnes, with 56.2% represented by mollusc ,5,6. To Population structure is the organization of genetic diversity, and it is influenced by multiple evolutionary process, such as genetic drift, mutation, gene flow, natural selection and demographic history ,9. It isPenaeus monodon, commonly known as black tiger shrimp is widely distributed across the Indo-Pacific region, and is one of the most commercially important aquaculture species [P. monodon have only been limited to several traditional markers such as mtDNA RFLP [ species ,30. To dDNA RFLP , allozymDNA RFLP , microsaDNA RFLP ,33 and SDNA RFLP ,35, mitoDNA RFLP  and elonDNA RFLP , while nDNA RFLP ,42,43,44Skim sequencing (SkimSeq) is one of the less complex NGS methods, which uses low coverage (1–10X) whole genome sequencing of multiple individuals for high resolution genotyping ,46,47,48P. monodon from different origins (wild vs. domesticated), we genetically assayed 50 individuals with the SkimSeq approach using the short read Illumina sequencing platform. This study aimed to: (i) investigate the genetic structure of wild and domesticated populations, (ii) quantify the genetic differentiation between populations and (iii) examine the presence of putative loci causing the genetic variation. The genomic analyses and genetic resource acquired from this study will be useful to support future genetic improvements in P. monodon culture and brood-stock selection activities.Given that the SkimSeq is known as low coverage genome sequencing approach , and so P. monodon individuals consisting of both domesticated and wild-caught broodstocks were collected from five sampling sites  across Indo-Pacific regions (P. monodon broodstock populations from MMD (Madagascar), MT (Thailand) and MMO  were obtained from domesticated shrimp farms, while MS  and MJ (Japan) were caught from the wild. The muscle tissues were extracted from broodstock individuals and preserved in 99.5% v/v ethanol and stored at −20 °C until DNA extraction. All samples were collected in accordance with the animal care and tissue collection protocol as approved by the Universiti Malaysia Terengganu’s Animal Care and Biosafety Committee.A total of 50  regions , and theP. monodon broodstocks were isolated from the muscle tissues using Wizard Genomic DNA Purification Kit following the manufacturer’s protocols . The concentration and purity of the extracted genomic DNA were quantified based on A260/280 nm ratio using BioDrop . DNA quantifications were conducted using real-time PCR fluorescence measurements of double stranded DNA [® DNA PCR-Free Library Prep Kit  and Covaris M220 , following the kits’ protocol at Monash University Malaysia Genomics Facility . Accurate quantification and quality checking of the DNA libraries were conducted using the KAPA Library Quantification Kit , while qualitative estimation of the libraries was performed by Agilent Technologies 2100 Bioanalyzer . Detailed QC parameters, including library cluster density, library complexity, percent duplication, GC bias, and index representation were generated on the MiSeq system  to ensure a uniform concentration of all samples prior to SkimSeq. The pooled library was denatured based on the Illumina NextSeq denaturation guideline and paired-end sequencing was carried out using NextSeq 500/550 High Output v2 300 cycles kit on a NextSeq 500 .Genomic DNA of nded DNA  and the http://bowtie.cbcb.umd.edu) [http://jhhung.github.io/PEAT) [http://www.usadellab.org/cms/index.php?page=trimmomatic) [P. monodon had about 30% low-quality reads, which were discarded before de novo genome assembly (ftp://public.genomics.org.cn/BGI/SOAPdenovo2) [ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST) [http://maq.sourceforge.net) [https://github.com/brentp/vcfanno) [From the Illumina platform, 151 base-pair paired-end (2 × 151) sequencing reads were obtained in FASTQ format. Sequencing reads matching to PhiX DNA sequences were first removed by aligning reads against the PhiX DNA sequence using Bowtie 2 software version 2.2.3 (umd.edu) ,56. The io/PEAT) , to ensumomatic) . The quaassembly . In the denovo2)  into a s/LATEST)  was used/LATEST)  to the arge.net) . The rearge.net)  to find vcfanno) . If a vaP. monodon, 5 individual sequences  were removed from the dataset due to the inconsistent SNP profiles and/or greater than 20% missing genotypes, while the remaining 45 samples were used for all downstream analyses. Furthermore, variants filtering was conducted using VCFtools software version 0.1.16 (https://vcftools.github.io/) [http://samtools.github.io/bcftools/bcftools.html). At first, the variants filtering steps included the removal of complex indels, SNPs with more than two alleles, and composite insertion and substitution events. Further filtering steps included the removal of sites with less than 5% overall minor allele frequency, missing genotypes in >90% of the samples in any population, and SNP sites with genotypes not in Hardy-Weinberg equilibrium in any population (PHWE < 0.001). After all filtering steps, a total of 194,259 individual SNP loci remained in the dataset. To detect putatively adaptive SNP loci among different wild and domesticated broodstock populations of P. monodon, we identified outlier SNPs from the 194,259 filtered individual SNP loci using pcadapt version 3.0.2 [p-values of each locus to detect adaptive loci. Default parameters were used for pcadapt analysis, and the “number_of_samples” parameter was set to 5 . The false discovery rate (FDR) threshold value set to 0.05, to control the false positive. Finally, an overall SNP loci dataset and putatively adaptive SNP loci dataset were created and used for all down-stream spatial clustering analyses.SNP profiles were analyzed and visualized using SNPRelate version 1.16.0 , to exclhub.io/)  and BCFton 3.0.2 ,68. The ST values (10 and 20 generations), as described previously [ST in POWSIM assumes the independence of the subpopulations, power was expressed as the proportion of significant outcomes for 1000 alterations per batch and a statistically significant test (p < 0.05).A power analysis using POWSIM v. 4.1  was carreviously . As FST http://cran.r-project.org/mirrors.html) [ST to determine the genetic differences between collection sites for the all SNP loci and outlier datasets using the default settings, with the samples grouped by collection sites. Clustering analysis, discriminant analysis of the principal component (DAPC), was conducted using the adegenet R package  on all SNP loci dataset and outlier datasets. Neighbor-joining trees were generated using all the SNP loci dataset and outlier datasets using Nei’s genetic distance method. The Bayesian clustering method, implemented in the STRUCTURE software v. 2.3.4 , was used to genetically assign individuals to clusters [The filtered data was imported as a genind object into R and down-stream spatial clustering analyses were largely conducted using the adegenet v2.0.1 R package (rs.html) . The Genrs.html) . The Genclusters . Simulatclusters . The calclusters . The cluclusters .L. vannamei in the NCBI database. For each detected 4983 SNP loci, flanking region of upstream and downstream 100 bp was extracted from the reference nucleotide sequence. Homology search between this extracted sequence set and CDS amino acid sequences of L. vannamei was performed under the homology threshold of e-value = 1 × 10−5. Homology search program of the SNP flanking sequences showed that the total of 50 coding regions containing nonsynonymous mutations were encoded by the putatively adaptive SNP loci. To know the functional distribution of the genes encoded within the adaptive SNP loci, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using KOBAS 3.0 software (http://kobas.cbi.pku.edu.cn/kobas3) and visualized with R [p < 0.05).To identify the genes encoded within the adaptive SNP loci, a homology search program was applied for each putatively adaptive SNP locus with the genome sequences available for the d with R . The hyphttps://ddbj.nig.ac.jp), with the DRA accession number: DRA010601. The data will be publicly available from 10 September 2020.Our raw data for 50 individuals were submitted to the DDBJ  broodstock populations of P. monodon for all SNP loci  and putatively adaptive SNP loci  datasets. Similar pattern of genetic differentiation was also observed between the three domesticated  broodstock populations of P. monodon, with pairwise FST values ranging from 0.003 (MMD vs. MMO) to 0.010 (MMO vs. MT) in all the SNP loci dataset, and 0.003 (MMD vs. MMO) to 0.042 (MMD vs. MT) in the putatively adaptive SNP loci dataset. In contrast, a high genetic divergence was observed between domesticated and wild populations, with pairwise FST values ranging from 0.145 (MMD vs. MS) to 0.153 (MJ vs. MT) in all the SNP loci dataset, and 0.836 (MJ vs. MMO) to 0.870 (MMD vs. MS) in the putatively adaptive SNP loci dataset. All of the pairwise FST comparison were significant (p ≤ 0.001), except for those between MMD and MMO for putatively adaptive SNP loci (p = 0.105)  .p = 0.000). A substantially low (9.8%) but significant divergence (p = 0.000) was observed among five broodstock populations of P. monodon . Interestingly, hierarchical AMOVA based on the putatively adaptive loci revealed a high genetic divergence among five broodstock populations of P. monodon , but no differentiation was observed among individuals , while the remaining variation within individuals was 19.2% , indicating a high level of genetic divergence of P. monodon broodstock populations. When all SNPs were used, genetic variation inferred by a hierarchical AMOVA reveals the largest component of genetic variability (74.1%) within the individual level . The por monodon . Overallas 19.2% . The hieP. monodon were observed. A discriminant analysis of principal components (DAPC) of all SNP loci datasets revealed that MT and MS populations were pronouncedly overlapped among each other, while others three populations were more or less discriminated from each other’s (P. monodon into two clusters: (1) wild-caught populations (MS and MJ), and (2) domesticated populations . However, NJ tree topology for all SNP loci dataset showed lower Nei’s genetic distance compared to the putatively adaptive SNP loci dataset, and only one wild-caught individual from Japan (MJ6) incongruently clustered with domesticated broodstock populations of P. monodon , and 50 of them yielded significant matches with CDS amino acid sequences  [AMOVA of putatively adaptive SNP loci showed a high total variance of 80.8% attributed to differences between the wild and domesticated groups. The low pairwise Fnbergii) ,87. The ST estimates, AMOVA, DAPC plot and STRUCTURE analysis agree with the genetic homogeneity of domesticated populations and their significant differentiation from the wild progenitor populations [Salmo salar) [Ctenopharyngodon idella) [Lates calcarifer) [Wild populations  co-locate on another branch in NJ tree, indicating they have a separate origin from the cultured populations . Besidesulations ,89, partulations , but in o salar) , grass c idella) ,92 and Acarifer) .In the present study, only one individual from MJ population was genetically identical to the cultured populations. Wild broodstocks, obtained as founders for shrimp domestication program, have been subjected to mass selection process over many generations, which may render lower genetic affinity between the cultured and wild populations. We also hypothesize that the founder broodstocks for the domestication program may have originated from more than one shrimp breeding company, and wild populations of different geographical localities. A lack of significant differentiation among domesticated populations may suggest the probability of a relatively short domestication history or genetically closely related populations .P. monodon [Lack of gene flow between domesticated and wild populations is not unexpected when the confined environment of shrimp hatcheries and farms were taken into consideration. Moreover, our experimental design have defiled the potential occurrence of escapees from farms to the natural environment, given that all individuals were only obtained from populations, which were isolated completely from each other with a minimum of 500 km apart (MT and MS). The genetic structuring pattern being impacted by farm escapees was not uncommon, and has been reported in  monodon ,95. Howe monodon .Haliotis midae) [The genetic homogeneity between the domesticated populations may be due to the artificial selection of favorable traits or adaptation to similar aquaculture practices. Aquaculture practices have been observed to reduce genetic variability in farmed reared stocks of other aquatic species ,98,99. Ds midae)  and scals midae) , inhabits midae) ,104,105.The effect of random genetic drift is more accentuated in smaller sample size . UnequalUnique genes also persists in differentiating the domesticated and wild populations, which can be explained by selection of differentially favored alleles, holding particular reference to genetic improvement program in captive breeding environment . The eviP. monodon. mtDNA has been widely used in population genetic studies to reconstruct phylogenetic relationships and analyze population structure [P. monodon broodstocks to adapt to strong artificial selection during domestication process. The putatively adaptive SNP loci identified by the pcadapt approach also identifies genomic regions associated with the strong artificial selection during the domestication process over temporal scales. Of the 4983 putatively adaptive SNPs, only 50 genes were encoded successfully annotated through BLAST analysis. We have an array of uncharacterized proteins, with their functions are not known. Moreover, it was also observed that five mitochondrial genes  were mutated among different populations of tructure , due to tructure ,116,117.tructure . In facttructure ,120. Vartructure , and is tructure , developtructure , biologitructure  and fitntructure , and puttructure . Naturaltructure ,128. Destructure , developtructure  and immutructure ,132. HowP. monodon across the Indo-Pacific region. We suggested that similar founder populations, artificial selection regimes of desired commercial traits and local adaptive process to similar aquaculture practices have dramatically reduced the genetic heterogeneity of the domesticated stocks. Despite being geographically isolated, we denote that the patterns of genetic homogeneity in wild populations maybe strongly influenced by hydrographic conditions and ecological niches which are expected to increase the adaptive processes of the populations towards their natural habitats. Genetic differences between wild and domesticated populations presented in this study can be likely explained by a number of unique genes within the putatively adaptive SNP loci. These genes were found to be significantly associated with the mitochondrial genome. Our results from KEGG pathway analysis further reinforce the potential selective force which have diverged both groups. We noted the presence of polymorphism in the mitochondrial regions, notably those that are related to the energy production, metabolic functions, respiration regulation and developmental rates. The combination of these functional properties of peptide encoded by mitogenome are linked to various environmental parameters, and this likely acting to promote the genetic isolation of domesticated populations from their wild. Separated by geographical distance and various selection breeding programs, these two groups may each develop local adaptation to ecological niches, biological traits and demographic histories found within their thriving habitats. Taken together, this study has demonstrated the applicability of SkimSeq in a highly duplicated genome of penaeid shrimp, P. monodon specifically, across a range of genetic backgrounds and geographical distributions. This ultra-low sequencing coverage has enabled the sequencing of low number of individuals to achieve either similar or perhaps more powerful coverage, but with lower cost than other traditional GBS methods. In future work, larger sample sizes from a larger number of populations collected from wider distribution range should be included within a similar comparative framework, to validate if this specific trend represents a general rule associated with the genetic divergence of P. monodon broodstocks.We observed a pronounced genetic divergence between the wild and domesticated broodstock populations of


The free-living flatworm Macrostomum mirumnovem is a neopolyploid species whose genome underwenta recent Whole Genome Duplication (WGD). In the result of chromosome fusions of the ancient haploidchromosome set, large metacentric chromosomes were formed. In addition to three pairs of small metacentrics,the current karyotype of M. mirumnovem contains two pairs of large metacentric chromosomes, MMI1 and MMI2.The generation of microdissected DNA libraries enriched for DNA repeats followed by DNA probe preparation andfluorescent in situ hybridization (FISH) were performed. The DNA probes obtained marked chromosome regionsenriched for different DNA repeats in the M. mirumnovem chromosomes. The size and localization of these regionsvaried in different copies of large chromosomes. They varied even in homologous chromosomes, suggesting theirdivergence due to genome re-diploidization after a WGD. Besides the newly formed chromosome regions enrichedfor DNA repeats, B chromosomes were found in the karyotypes of the studied specimens of M. mirumnovem. TheseB chromosomes varied in size and morphology. FISH with microdissected DNA probes revealed that some Bs hada distinct DNA content. FISH could paint differently B chromosomes in different worms and even in the same sample.B chromosomes could carry a bright specific fluorescent signal or could show no fluorescent signal at all. In lattercases, the specific FISH signal could be absent even in the pericentromeric region of the B chromosome. Possiblemechanisms of B chromosome formation and their further evolution are discussed. The results obtained indicatean important role that repetitive DNAs play in genome re-diploidization initiating a rapid differentiation of largechromosome copies. Taking together, karyotype peculiarities  and some phenotypic features of M. mirumnovem  make this species a perspective model in the studies of genomic andkaryotypic evolution in species passed through a recent WGD event. Comparative genomics opened up new possibilities for studiesof mechanisms of whole-genome duplication (WGD), as wellas its consequences after the large-scale changes of genomeinvolving almost all of the genes. The most of the existingspecies of plants and animals were resulted from at least oneround of WGD, more often a few WGD events . In differentphylogenetic lineages, these WGDs took place hundredsof MYA. Hence, the genomes of modern species are paleoploids,and they contain only traces of WGDs . Therefore, comparative analysis of their genomescan provide only limited knowledge about mechanisms ofpolyploid formation and further reorganization of the duplicatedgenome. There are two main mechanisms of WGD:autopolyploidy resulted in autotetraploid genome formation,and allopolyploidization, i. e. doubling of hybrid genomeafter interspecific hybridization. In the latter case, a new allopolyploidgenome contains both parental genomes, and it canretain many traits of its hybrid ancestor. The key role of WGDin genome evolution in animals was considered in the recentreview .WGD events occurred in genomes of different phylogeneticlineages . Many studies performed bydifferent research groups have uncovered that the number ofWGDs can distinguish for different phylogenetic lineages:for instance, in rotifer – 1 WGD, in the vast majority ofvertebrates – 2, in many plants – 3, in salmonids – 4 .The genome evolution of Brassica rapa contained alternatingrounds of multiplication events, including both whole-genomeduplication and even triplication . Someresearchers suggest that WGD creates conditions for followinglarge evolutionary transformations, despite other scientiststake a different view, suggesting post-WGD species are mostlyevolutionary dead-end . For many years the most of studies were devoted tothe analysis of polyploidy and WGD in plants that was linkedwith the limitations of the available methodological possibilities.Development of new methods in molecular genetics, improvement of NGS technologies have fundamentally changedthe WGD studies in animals .In a result, comparative genomics has arisen, and more andmore animal species continue to be involved in these studies. The limited number of speciesunderwent a recent WGD is a challenge in researchesdevoted to WGD and early-stage genome reorganization aftera WGD in animals .It should be noted that a large number of polyploid variantsare likely to be evolutionary dead-ends . They represent new, but less competitivevariants in comparison with their diploid ancestors. Nevertheless,traces of WGDs revealed in genomes of many existingspecies indicate to a significant contribution of WGD in theformation of new mechanisms of environmental adaptation. AlthoughWGD can be considered as a key event in the formation of newphylogenetic lineages, the possibilities of studying a WGDand the processes it triggers in the followed stages of genomeevolution are limited. The rapid progress in the development ofnew methods of molecular genetics and genomics has openedup new opportunities and perspectives in the evolutionarystudies. However, the number of perspective model organismsfor studying WGD remains extremely low. The involvementin studies of new species, whose genomes passed through arecent WGD, is one of the most urgent tasks. The group ofspecies of free-living flatworms of the genus Macrostomummay turn out to be one of such promising model species.Molecular cytogenetic analysis of the M. lignano chromosomesuncovered that the main traits of its genome and karyotypeorganization differ from that of the other Macrostomumspecies, and these peculiarities are linked with a recent WGDevent in its genome evolution .The karyotypes of many Macrostomum species consist of alow number of small metacentric chromosomes . It wassuggested that a basal chromosome number was 2n = 6, butdue to the WGD, the doubled chromosome number led to theincreased karyotype 2n = 12 in some Macrostomum species.Chromosome fusions of all chromosomes of one haploid chromosome set of ancestor have resulted in the decreasedchromosome number (from 12 to eight chromosomes) inthe M. lignano karyotype . Moreover, paralogous regions revealed in theM. lignano chromosomes accumulated some differencesthat more likely arose after a WGD event . The uncovered karyotype instability in M. lignanois considered to be a consequence of the ongoing rediploidizationprocesses in its genome. The karyotyping of newMacrostomumspecies led us to explore a new species, namelyM. mirumnovem arose through a recent independent roundof WGD. The genome evolution of this species accompaniedby intensive chromosome reshuffling resulted in chromosomefusions, structural chromosome rearrangements, formation ofnew repeat-enriched regions, and B chromosome formation. Based on the cytogenetic analysis ofthe M. mirumnovem chromosomes, we concluded that expansionand amplification of repetitive DNA play a significantrole in the reorganization of its karyotype. By generation ofmicrodissected DNA probes enriched with DNA repetitivesequences, we studied the distribution of these DNA repeatsin the M. mirumnovem chromosomes.Laboratory outbred culture of the free-living flatwormM. mirumnovem. The laboratory outbred culture of M. mirumnovem,kindly provided by Professor Lukas Schärer,was used in the current study. The culture of M. mirumnovemhas been cultivated under the laboratory conditions for threeyears. The first karyotyping was performed three months aftersampling the M. mirumnovem worms from natural populations. Already at this stage, a highlevel of karyotypic diversity was revealed. The most commonkaryotype consisted of nine chromosomes: the largest unpairedmetacentric (MMI1), a pair of large metacentrics (MMI2) abit smaller in size than the MMI1, and three pairs of smallmetacentric chromosomes MMI3–MMI5 .In the chromosomes thus formed, the active expansionof transposable elements (TEs) and amplification of DNAsequences probably led to the formation of numerous regionsenriched for repetitive sequences .Single-worm karyotyping, carried out before microdissectionof small metacentrics, showed an increase in the karyotypicdiversity among the worms of the laboratory culture, as wellas the formation of B chromosomes in it. In the current study,we used the metaphase plates with the 2n = 9 karyotype togenerate the microdissected DNA probes. For fluorescencein situ hybridization (FISH) metaphase chromosome slidesobtained from individual worms with different karyotypevariants were used.Preparation of metaphase chromosome plates. Regularkaryotyping of individual worms of the laboratory cultureof M. mirumnovem was done as was previously described. We checked at least ten metaphasespreads per each specimen. For microdissection, we preparedchromosome slides using a cell suspension technique that provides a better preservation of DNA in metaphase chromosomes.To make a suspension of fixed mitotic cells, we used100 mature worms, as was earlier described . Metaphase plates were spread on a wet surface ofcold coverslips (60 × 24 × 0.17 mm). Preparation of metaphasespreads on coverslips allowed us to use a maximum magnificationof the inverted microscope AXIOVERT10  during microdissection.Staining of metaphase chromosomes. For routine karyotypingthe metaphase chromosomes were stained with a fluorescentdye DAPI dissolved in the VECTASHIELD® mounting medium containingantifade .Metaphase chromosome slides prepared for microdissectionwere stained in a 2 % Giemsa solution  .Microscopic analysis of chromosome preparations wasperformed at the Center for the collective use of microscopicanalysis of biological objects of the SB RAS at the Institute ofCytology and Genetics SB RAS  usingan Axioplan 2 luminescent microscope (ZEISS) equipped witha CCD camera and a set of filters # 49, # 10 and # 15 (ZEISS).Registration and subsequent processing of microscopic imagesof chromosomes was performed using the ISIS4 software.Microdissection of metaphase chromosomes and generationof microdissected DNA probes. Microdissection ofmetaphase chromosomes was used for the generation ofthe DNA probes from the M. mirumnovem chromosomes aswas described earlier . To amplifythe DNA of the dissected chromosomal material, we used astandard variant of sequence-independent polymerase chainreaction (PCR) . The procedure ofmicrodissection from the selection of the metaphase plate tothe transfer of dissected material into a PCR tube with a reactionmixture were described in detail . In the current study, we collected two single copies ofsmall chromosomes.The difference in the preparation of DNA probes from thestandard protocol consisted in modifying the composition ofthe collection buffer, into which the dissected chromosomematerial was directly collected (~40 nl in the drawn-out siliconetip of the Pasteur pipette). The collection drop containeda proteinase and DNA fragmentation buffer  kit, Sigma-Aldrich,USA) and 0.1 % non-ionic detergent Triton X-100 . All subsequent stages of DNApreparation for amplification and directly DNA amplificationwere carried out according to the previously describedprotocol  with an increasednumber of PCR cycles (up to 35 cycles) at the last stage of thepreparation of a microdissected DNA library.Generation of microdissected DNA libraries involves twosteps: (1) the generation of DNA fragments  flanked by the correspondingsequences  and (2) theamplification of such DNA fragments in PCR. Depending onthe efficiency of obtaining the flanked DNA fragments in the first stage, there are two different variants for the DNA contentof the obtained DNA libraries. In the first one, the sequencesrepeated many times in the genome and a significant partof the unique and low repetitive sequences are involved inamplification. In the second one, the part of genomic DNAsequences involved in amplification was reduced and mostlyhighly repeated sequences appeared to be amplified. The representationof unique DNA sequences in the obtained microdissectedDNA libraries is significantly reduced with a decreasein the efficiency of generation of DNA fragments capable ofamplification at the first stage. In a result, the generated DNAlibraries are enriched with repetitive sequences.An increase in the number of PCR cycles at the secondstage of generation of microdissected DNA library allows toobtain the required amount of DNA product. The modificationof the collection buffer used in the first stage of generationDNA libraries led to the production of DNA libraries thatwere enriched in highly repeated sequences. The presencemostly of highly repetitive DNA in the obtained DNA librarieswas confirmed by the complete absence of a signal in mosteuchromatin regions of chromosomes after FISH with DNAprobes obtained on their basis.The resulting PCR product was labelled in 20 additionalPCR cycles in the presence of fluorochrome-conjugatednucleotides:Flu-12-dUTP [fluorescein-5(6)-carboxamidocaproyl-[52′-deoxyuridine-5′-triphosphate] or TAMRA-5-dUTP [tetramethylrhodamine-5(6)-] (Biosan) .GenomePlex® Whole Genome Amplification Reamplificationkit (WGA3)  was used for labelling.Fluorescent in situ hybridization on metaphase chromosomesof DNA probes with metaphase chromosomes ofM. mirumnovem was performed according to the previouslydescribed protocol , withoutsuppression of the repetitive DNA hybridization. DuringFISH, chromosome slides prepared from individual wormsof M. mirumnovem were analyzed. When FISH was carriedout with separate microdissected DNA probes (S3 or S4), aswell as when two-colour FISH with DNA probes S3 and S4,was performed ten or more specimens of M. mirumnovemwere studied.Generation of DNA probes from chromosomes of M. mirumnovemand FISH of the obtained DNA probes withmetaphase chromosomes. The S3 and S4 DNA probeswere obtained from single copies of two small metacentrics.FISH with the DNA probes on metaphase chromosomes ofM. mirumnovem revealed chromosome regions enriched forrepetitive sequences homologous to DNA in the DNA probesS3 and S4 . The regions painted by the DNA probesS3 or S4 will be called S3D- or S4D-regions, respectively,i. e. the regions detected by FISH using the DNA probes S3and S4. In our study, the definition of precise localization ofthese regions in metaphase chromosomes of M. mirumnovemposed a certain problem of the description based on theirmorphologyand differential staining.Fluorescent in situ hybridization with the S4 DNA probeon the large metacentric chromosomes, MMI1 and MMI2,gave bright fluorescence exactly in the region of their primaryconstriction, and additional signals in some regions of theirchromosome arms . In addition to FISH signals inlarge chromosomes, the DNA probe S4 gave an intense signalin the pericentromeric region of the chromosome MMI4. Insome specimens, the distal region of q-arm of MMI4 was alsointensively painted . The weak signals(on the edge of being able to detect them) were also detectedin the pericentromeric regions of the chromosomes MMI3and MMI5.Due to the small block of the pericentromeric structuralheterochromatin in the M. mirumnovem chromosomes, it ishardly possible to conclude whether the S4D-regions werelocated directly in the pericentromeric regions or in adjacenteuchromatin regions even in the large chromosomes MMI1and MMI2. Localization of FISH signal on the condensedmetaphase chromosomes MMI3–MMI5 often turned outto be even more complicated . However, itshould be noted that on less condensed chromosomes MMI3–MMI5 with a pronounced primary constriction, the FISHsignal of the DNA probe S4 was always localized in thisregion .Thus, taking into account the small size of the pericentromericheterochromatic regions, the question whether the S4Dregionsare located in the pericentromeric heterochromatin ofthe chromosomes or the proximal euchromatin of p- or q-arms,remains open. For simplicity, a further description of the obtainedresults, we will define these regions pericentromeric,recognizing some incorrectness of the use of this term.Given the specificity in the description of FISH results,we can conclude that the DNA probe S4 painted the pericentromericregions of all A-chromosomes. The intensity ofFISH signals in different chromosomes varied significantly,changing from the intense signal in the region of the primaryconstriction of some copies of chromosomes MMI1, MMI2,and MMI4 to the weak separate signals on both chromatidsof small metacentrics MMI3 and MMI5 . Thevariation in fluorescence intensity of the signal of the DNAprobe S4 in regions of different chromosomes, includingdifferentcopies of homologous chromosomes, was probablyderived from the different copy number of the correspondingrepeats in these regions. However, it cannot be ruled out that insome cases such differences may be based on the differencesinhomology level between repetitive sequences in these regionsand the DNA of the S4 probe. It should be noted that additionallyto specific signals in the regions of A-chromosomes, theDNA probe S4 weakly painted some B-chromosomes .Intense FISH signals of the DNA probe S3 localized onlyin small chromosome MMI4 and in large metacentric chromosomes(MMI1 and MMI2). Furthermore, two-colour FISHwith the DNA probes S3 and S4 showed that the large chromosomesare enriched for different highly repeated sequences.Moreover, the revealed S3D- and S4D-regions were not colocalized. Even in the proximal region ofMMI4, where the S3D- and S4D-regions were localized close to each other, no co-localized FISH signals of the DNA probesS3 and S4 were observed.In one specimen, a transition of the S3D-region to the distalregion of the p-arm of MMI4 was revealed, which is visible onlow condensed chromosomes . It is likely that thistransition could be resulted from an inversion involving oneof the breakpoints located between the S3D and S4D regions.The obtained FISH results indicated that different repetitivesequences belonging to MMI4 were involved in DNA amplificationduring generation of the DNA probes S3 and S4.The low-intense FISH signals of the DNA probe S3 (on theedge of the possibility of their detection) were revealed in theregions of small chromosomes MMI3 and MMI5. Eithertherevealed regions probably contained a relatively low copynumber of DNA repeats homologous to that of the DNAprobe S3, or DNA repeats in them had a low homology levelwith the DNA of the probe S3. We should note that the DNAprobe S3 intensively painted the regions in the arms of somecopies of large metacentrics . Someof these regions were painted even more intensively than theproximal part of MMI4.Summarizing data on FISH with the DNA probes S3 andS4 it is possible to state that two different types of largechromosomeswere revealed in the karyotypes of studiedspecimens of M. mirumnovem. One of them was characterizedby the presence of strong hybridization signals in their arms,while another showed the level of specific FISH signal intensitycomparable to registered ones in the regions of smallchromosomes MMI3 and MMI5 .Possible mechanisms for the formation of regions enrichedfor repetitive sequences and their following evolution.The question of the molecular mechanisms providingthe variability of the regions enriched for DNA repeats in sizeand location remains open. However, it is necessary to takeinto account that chromosomes MMI1 and MMI2 containextended regions homologous to the euchromatic regions ofMMI4 . Besides variable S3D- andS4D-regions  observed inthe MMI1 and MMI2, the S3D- and S4D-regions were alsodetected in the chromosome MMI4, but only their traceswere found in the MMI3 and MMI5. Probably, the fusionsof ancestral chromosomes that formed the large metacentricswere accompanied by amplification of DNA repeats derivedfrom MMI4. Further expansion and amplification of repetitiveDNA sequences could lead to the divergence of the formedlarge metacentrics. The distribution of such regions withinthe chromosome could also occur as a result of inversions,as it was shown in case of transition of the S3D-region inthe chromosome MMI4 in one of the analyzed specimens ofM. mirumnovem .Based on the results obtained in our study, it is not possibleto estimate what kind of repetitive sequences are present inthe microdissected DNA probes. In future, the answer to thisquestion can be obtained by cloning DNA fragments fromthe generated DNA libraries and subsequent FISH with DNAprobes prepared from these DNA fragments. Previously,this approach was successfully applied to analyze the DNAcomposition of the B chromosome of the locust species, Podisma kanoi . An alternative way forstudying the DNA composition of the microdissected DNAlibraries is their NGS sequencing. Earlier, this approachwas applied to determine the DNA composition of Bs of theKorean field mouse Apodemus peninsulae from East Asia,the yellow-necked mouse Apodemus f lavicolis, and a smallsupernumerarymarker chromosome in humans . However, the latter experimental design impliesfollowing verification of the results of NGS sequencing usingDNA probes generated based on the obtained sequences, sincea certain amount of DNA always contaminate microdissectedDNA libraries. Additionally, for a successful and efficientinterpretation of the data derived from the microdissectedlibrary sequencing, it is desirable to have a reference genomeassembly or at least genome draft of the studied species. In the case of M. mirumnovem, itis problematic due to the prominent karyotype and genomeinstability, and therefore it requires consideration of alternativeapproaches.B chromosomes of M. mirumnovem. In addition to the regionsof A chromosomes, the DNA probe S4 gave specific signalin some Bs . These results allow usto propose one of the scenarios for the formation of such Bs.They could be originated from the pericentromeric region ofone of the A chromosomes as a result of chromosome breaks,leading to the arising of a small additional chromosome.At the beginning, this chromosome (or proto-B chromosome)consisted of the pericentromeric region of the ancestral A chromosome,and the subsequent amplification of repetitive DNAsequences homologous to DNA of the microdissected DNAprobes and other different DNA repeats. The uneven and notintense specific fluorescence from the DNA probe S4 on theB indicates the presence of such different DNA repeats inthe B chromosome. It is worth noting that these DNA repeatscan derived both from the pericentromeric region of the ancestralA chromosome and from different chromosome regions.For instance, some Bs gave no specific signal after FISH withthe DNA probe S4. Moreover, specific FISH signal was absenteven in the pericentromeric regions of such B chromosomes, which indicates the amplification of differenttypes of DNA repeats during the formation of the Bs, and,probably,their different origin.Using microdissected DNA probes generated by the modifiedprotocol and containing predominantly highly repeated DNAsequences, we identified the regions enriched in repetitiveDNA sequences in the M. mirumnovem chromosomes. Wehave shown that enrichment of such chromosomal regionsfor DNA repeats could vary substantially. The formationand following changes of these regions in large metacentricsMMI1 and MMI2 could lead to differentiation of the copies ofhomologous chromosomes. Additionally, the obtained resultsindicate that the DNA content in different copies of B chromosomescould differ among individuals of M. mirumnovem.The repetitive DNA sequences homologous to DNA repeatsfrom the proximal regions of A chromosomes took part in theformation of at least some B chromosomes.The authors declare no conflict of interest.Barker M.S., Husband B.C., Pires J.C. Spreading winge and flyinghigh: the evolutionary importance of polyploidy after a century ofstudy. Am. J. Bot. 2016;103(7):1-7. DOI 10.3732/ajb.1600272.Bugrov A.G., Karamysheva T.V., Perepelov E.A., Elisaphenko E.A.,Rubtsov D.N., Warchalowska-Sliwa E., Tatsuta H., Rubtsov N.B.DNA content of the B chromosomes in grasshopper Podisma kanoiStorozh. . Chromosome Res. 2007;15(3):315-326. DOI 10.1007/s10577-007-1128-z.Comparative Genomics. Sankoff D., Nadeau J.H. (Eds.). Kluwer AcademicPubl., 2000. DOI 10.1007/978-94-011-4309-7.Dehal P., Boore J.L. Two rounds of whole genome duplication in theancestral vertebrate. 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New evidence for genome-wideduplications at the origin of vertebrates using an amphioxus gene setand completed animal genomes. Genome Res. 2003;13:1056-1066.DOI 10.1101/gr.874803.Schärer L., Brand J.N., Singh P., Zadesenets K.S., Stelzer C.-P., ViktorinG. A phylogenetically informed search for an alternative Macrostomummodel species with notes on taxonomy, mating behavior,karyology, and genome size. J. Zool. Syst. Evol. Res. 2020;58:41-65.DOI 10.1111/jzs.12344.Soltis D.E., Segovia-Salcedo M.C., Jordon-Thaden I., Majure L.C.,Miles N.M., Mavrodiev E.V., Mei W., Cortez M.B., Soltis P.S.,Gitzendanner M.A. Are polyploids really evolutionary dead-ends(again)? A critical reappraisal of Mayrose et al. New Phytol. 2014;202(4):1105-1117. DOI 10.1111/nph.12756.Wendel J.F. Genome evolution in polyploids. Plant Mol. Biol. 2000;42:225-249. DOI 10.1023/A:1006392424384.Zadesenets K.S., Ershov N.I., Berezikov E., Rubtsov N.B. 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Salmonella, especially those located on mobile genetic elements, are part of the problem. This study used phenotypic and genomic methods to identify antimicrobial resistance and resistance genes, as well as the plasmids that bear them, in Salmonella isolates obtained from poultry in Nigeria. Seventy-four isolates were tested for susceptibility to eleven commonly used antimicrobials. Plasmid reconstruction and identification of resistance and virulence genes were performed with a draft genome using in silico approaches in parallel with plasmid extraction. Phenotypic resistance to ciprofloxacin (50.0%), gentamicin (48.6%), nalidixic acid (79.7%), sulphonamides (71.6%) and tetracycline (59.5%) was the most observed. Antibiotic resistance genes (ARGs) detected in genomes corresponded well with these observations. Commonly observed ARGs included sul1, sul2, sul3, tet (A), tet (M), qnrS1, qnrB19 and a variety of aminoglycoside-modifying genes, in addition to point mutations in the gyrA and parC genes. Multiple ARGs were predicted to be located on IncN and IncQ1 plasmids of S. Schwarzengrund and S. Muenster, and most qnrB19 genes were carried by Col (pHAD28) plasmids. Seventy-two percent (19/24) of S. Kentucky strains carried multidrug ARGs located in two distinct variants of Salmonella genomic island I. The majority of strains carried full SPI-1 and SPI-2 islands, suggesting full virulence potential.Antimicrobial resistance is a global public health concern, and resistance genes in  Salmonella is an important pathogen affecting the poultry industry and is of high zoonotic importance [Salmonella isolates have been encountered with high frequency in many parts of the world, including Europe [portance . In the portance . In mostportance . Sadly, g Europe  and sub-g Europe , making Salmonella and are important in the development and spread of resistance. AMR might be transmitted to humans via direct contact, or through the food chain and the environment [Salmonella in food animals to humans via mobile genetic elements (MGEs), such as plasmids, integrons and transposons harboring resistance genes [Salmonella within both human and animal hosts [Enterobacteriaceae [The development of antimicrobial resistance (AMR) has been attributed to the overuse and misuse of antimicrobials both in veterinary and human medicine, especially in low- and middle-income countries. In Nigeria, overuse of gentamicin, tetracycline, sulphonamides and other antibiotics has been reported due to a lack of a regulatory framework to enforce the law . Food-prironment . Resistace genes . These mal hosts . Certainal hosts , and someriaceae .Salmonella in sub-Saharan Africa [Salmonella in the region, particularly in poultry production, and it is currently unknown whether the so-called epidemic resistance plasmids are harbored in strains of Salmonella. This information is essential to control the spread of AMR, as well as to monitor the emergence of new resistant clones. Thus, in this study, we determine the occurrence of AMR in Salmonella strains from poultry in Nigeria and determine an association between resistance genes and mobile plasmids using whole genome sequence data. Additionally, we assess the pathogenic potential of the strains by identifying Salmonella pathogenicity islands and other virulence factors.Previous studies have investigated the prevalence of AMR in n Africa ,13,14,15Salmonella belonging to 23 serovars obtained by random sampling in 165 poultry farms in Nigeria was tested , S. Muenster (75.0%) and S. Isangi (62.5%) were MDR  and DNA topoisomerase  conferring resistance to nalidixic acid and ciprofloxacin were observed in 70.3% and 35.1% of isolates, respectively. The most commonly predicted resistance genes were combinations of aac (6′)-Iaa and aph (3′)-Ia,b found in 95.0% of isolates, which confer resistance to kanamycin. Thirty-two isolates carrying the aac (6′)-Iaa gene were phenotypically susceptible to kanamycin. Genome analysis revealed that 13 of these isolates had stop codons inserted at a specific position of the promoter region, while 19 possessed several stop codons at different positions of the promoter (tet(A), tet(M)), sulphonamides  and gentamycin (aac (3)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6′)-IIa, aac (3)-Id), respectively (blaTEM), trimethoprim  and chloramphenicol   . Surprisp = 0.0001), ampicillin , tetracycline  and gentamicin  resistance was observed. Only moderate yet significant agreement was observed for chloramphenicol  and ciprofloxacin  resistance, and fair agreement was found for nalidixic acid resistance , while no agreement was observed for kanamycin  resistance  values were used as cut-offs, there was a reduction in the level of concordance between the phenotype and prediction of resistance genes . In ordeS. Schwarzengrund (2/4) and S. Muenster (3/4) were predicted to have IncN and IncQ1 plasmids, while S. Takoradi (3/6), S. Larochelle (4/4) and S. Isangi (6/8) were predicted to have IncL, Col (pHAD28)/Col (Ye4449) and Col (pHAD28) replicons, respectively. Strains of S. Kentucky showed variation in the plasmid replicons (11/24 positive for replicons) with the presence of IncFIIpCRY, ColpVC and IncM1 replicons , ColpVC and IncFIIpCRY as the most commonly found replicons. eplicons . Plasmideplicons . PlasmidqnrB19. In the remaining 12 strains, no resistance genes were predicted to be located on the putative plasmids.Among the 30 strains with identified plasmid replicons, eight strains contained plasmids predicted to carry multiple antimicrobial resistance genes, while ten strains carried plasmids where the only predicted resistance gene was S. Schwarzengrund were aligned with a reference sequence (CP028173.1) using BLASTN. There was 99.9% identity over 86.0% query cover of these plasmids and the reference sequence . The resistance genes were flanked by a repeat region, followed by transposase (Tn3). The transposase was identified to be homologous to an NCBI reference sequence (pfam01526). Resistance genes harbored by these plasmids were sul2, tet(A), tet(R), aac(3)-II, aph (3”)-I, aph (6)-Ic, qnrB19 and blaTEM . There was 99.9% identity with 94.0% query cover between replicons and reference sequence . The plasmid harbored sul2, tet(A), tet(R), aph(3″)-I and aph (6)-Ic resistance genes ), hypothetical proteins, a relaxase and replication protein (RepA)-encoding genes. The remaining three plasmids with genes responsible for multidrug resistance were one IncM1 plasmid and one IncHI2/IncHI2A plasmid from S. Kentucky and one IncHI2/IncHI2A plasmid from S. Isangi.Similarly, all three IncQ1 plasmids from ce genes  which weqnrB genes from six strains of S. Isangi were aligned with reference plasmid CP025247.1 . These small-size plasmids harbor genes which reduce susceptibility to quinolones. Other genes located on these plasmids are the psp operon transcriptional activator, a putative replicase, hypothetical proteins and an uncharacterized Bsu1883a protein (qnrB genes were four strains of S. Larochelle. Col (pHAD28) plasmids harboring 025247.1 . There w protein . The remS. Kentucky and S. Isangi carried a multidrug efflux pump (Mdtl) and small multidrug-resistant genes (smr). Plasmids of the IncHIA/IncHI2A type in one strain of S. Kentucky were shown to harbor a resistance gene against a macrolide (mphA) , while one strain each of e (mphA) . S. Kentucky carried plasmids bearing no resistance genes. These included four small ColpVC plasmids, which contained replication origin and hypothetical protein genes only. An IncFIIpCRY replicon was present in four strains and was mapped to plasmids which carried traG, ccdB toxin and virB genes. Furthermore, three IncL plasmids from S. Takoradi harbored genes encoding phage proteins, traI and traY in addition to other encoded genes (Eight isolates of ed genes . These ped genes .aac (3)-Id, aadA7, aph (3″)-Ib, aph (6′)-Id, tet(A), sul1) on specific contigs of 19 strains of S. Kentucky. From NCBI BLAST, twelve of these strains had these genes located on a region that is homologous to the approximately 20 kb Salmonella genomic island 1 variant SGI1-K in S. Kentucky strain SRC73 (GenBank accession no. AY463797.8) with 100% coverage and 99.9% identity. Alignment of this cluster I with a reference strain is shown in S. Kentucky strain 161,365 Salmonella genomic island (GenBank accession no. CP043664.1) with 95% coverage and 100% identity. Alignment of this cluster II with a reference is shown in intI-encoding integrase, yid genes, mercury resistance modules (mer genes), tra genes involved in conjugative transfer, insertion sequences and other genes involved protein transport and metabolism  including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-8, SPI-13, SPI-14 and centisome 63 pathogenicity island (C63PI). C63PI was present in all strains. Thirty-three strains carried SPI-1, SPI-2 and SPI-3 genomic islands, while seven strains had SPI-1 and SPI-2 only, eleven strains had SPI-1 and SPI-3 only, one strain had SPI-2 and SPI-3 only, four strains had SPI-3 only and eight strains had SPI-1 only. The draft genomes of three S. Kentucky strains, two S. Isangi strains, two S. Telekebir strains, one S. Muenster strain, one S. Abadina strain and one S. Larochelle strain completely lacked the three SPIs. Some variation in gene presence or absence in SPI gene profiles of strains showing the same serovar was observed; however, most strains of a serovar shared identical SPI profiles  and the phoP and phoQ regulatory genes. Strains were observed to have different profiles of SPI-1 and SPI-2 effectors genes . This included all four strains of S. Schwarzengrund, three of S. Muenster, five of S. Takoradi, two of S. Chester and one each of S. Ituri, S. Give, S. Poona and S. Telekebir. The complete virulence gene profile of each strain is shown in Other common virulence factors in the virulence factor database (VFDB) shared by most serovars included fimbria and non-fimbrial adherence determinants, a macrophage-inducible gene , one of the most common causes of foodborne disease and an important cause of mortality worldwide [Salmonella is endowed with virulence factors that allow systemic propagation leading to severe infections, particularly in immunocompromised patients, and it has the ability to rapidly acquire antimicrobial resistance genes from related and unrelated bacteria, especially intestinal commensals [Salmonella from poultry in Nigeria, the largest poultry producer in sub-Saharan Africa [Salmonella may also be problematic because such genes may spread to other bacteria; to assess the likelihood of this, we analyzed whole genome sequencing data to determine whether resistance genes were carried on transferrable plasmids.Antimicrobial resistance (AMR) in zoonotic and foodborne pathogens is considered a serious threat to public health . The raporldwide . Salmonemmensals . In the n Africa . We usedS. Kentucky, S. Isangi, S. Schwarzengrund, S. Muenster and S. Telekebir showed MDR. All strains were predicted to have at least one ARG. A different degree of concordance was observed between predictions of ARGs and resistance to a specific class of antimicrobial. The level of concordance reduced when ECOFF was used as a cut-off point compared to when clinical breakpoints were used. This could be attributed to the fact that ECOFF values for disk diffusion methods are often set higher than the cut-off values for clinical breakpoints, and there is the likelihood of classifying a resistant (non-wild-type) strain as sensitive (wild-type). Misclassification of resistance could also be attributed to the fact that clinical breakpoints include intermediate classification ranges that reflect the concentration range for which isolates may contain resistance determinants but do not present zone diameters above the resistance threshold [All strains were resistant to at least one antimicrobial, and more than 50% displayed MDR; in particular, strains of serovars hreshold . Zone dihreshold . Anotherhreshold . FurtherblaTEM genes were only detected in three strains of S. Schwarzengrund and one strain of S. Kentucky. This is considerably less frequent than reports from other sub-Saharan countries [Resistance to ampicillin was rare, and ountries ,22. Prodaac (3)-Ia, aac(3)-IIa, aac(3)-Iva, aac(6′)-IIa and aac (3)-Id) was concordant with phenotypic resistance, while there was slight to no agreement with occurrence of aac (6′)-Iaa, aph(3′)-Ia,b and resistance to kanamycin, tobramycin or amikacin [aac (6)-Iaa are chromosomally located genes in Salmonella, and they may be rendered silent by mutation in the promoter [aph(3″)-Ib, aph (6′)-Id, aadA1, aadA5) that confer resistance to streptomycin and spectinomycin [S. Kentucky, S. Isangi and S. Schwarzengrund displayed resistance to aminoglycosides both phenotypically and genotypically. This finding corroborates several reports demonstrating the presence of multiple aminoglycoside resistance genes in Salmonella serovars [Forty-eight percent and 50% of strains were phenotypically resistant to gentamicin and kanamycin, respectively. Prediction of resistance genes to gentamicin (amikacin . aac (6)promoter . In accoinomycin  were alsserovars ,25,26.qnrB19 or qnrS1 genes with or without the presence of point mutations in DNA gyrase and topoisomerase. The qnrB19 and qnrS1 genes encode transferable (fluoro) quinolone resistance mechanisms that are responsible for reduced susceptibility to quinolones [gyrA) and topoisomerase C (parC) genes in most members of Enterobacteriaceae [gyrA results in resistance to nalidixic acid only [S. Kentucky, S. Isangi, S. Schwarzengrund and S. Muenster strains showed phenotypic and genotypic resistance to quinolones. These findings corroborate an increasing number of reports revealing that S. Kentucky, S. Schwarzengrund, S. Isangi and S. Muenster from livestock carry qnrB19 and multiple mutations in parC and gyrA genes [Fifty percent and 79.7% of isolates showed phenotypical resistance to ciprofloxacin and nalidixic acid, respectively. Predictions by ResFinder demonstrated that most of these strains carried either inolones  and whicinolones . Clinicaeriaceae . An incrcid only . In our rA genes ,30,31. sul1, sul2 and sul3 and dfrA14, dfrA15 and dfrA17, respectively [S. Isangi, S. Kentucky, S. Schwarzengrund and S. Muenster strains showed phenotypic and genotypic resistance to sulphonamides, while only S. Kentucky and S. Isangi carried resistance genes against trimethoprim. Phenotypic resistance to chloramphenicol and tetracycline was mostly observed in strains of S. Isangi, S. Kentucky and S. Schwarzengrund, which also carried genes encoding resistance to these two classes of antimicrobials. Interestingly, genes that confer resistance to macrolides (mef(B), mph(A), erm(B)) were located in one strain each of S. Kentucky, S. Chester and S. Larochelle, while a resistance gene to fosfomycin (fosA7) was found in S. Menston. This finding is indeed worrisome because these are broad-spectrum antimicrobials that are mostly used as a drug of last resort in treating human infections. Others have also showed increased resistance to macrolides [S. Larochelle was found to be phenotypically resistant to meropenem; however, genomic analysis did not reveal any genes known to encode carbapenem resistance. The lack of genotypic detection could be due the presence of other mechanisms of carbapenem resistance such as overproduction of AmpC β-lactamase, mutation in porin or drug efflux [ompR porin gene, which could be responsible for the phenotype observed [blaKPC [Fifty-three (71.6%) strains were phenotypically resistant to sulphonamides, while only 12% of strains showed resistance to trimethoprim. Likewise, 44.6% and 8.1% of strains were predicted to have sulphonamides and trimethoprim resistance genes, respectively. As previously reported, resistance to folic acid inhibitors is one of the most frequent mechanisms of bacterial resistance to sulphonamides and trimethoprim due to the acquisition of dihydropteroate synthase and a reductase enzyme encoded by the genes ectively . Predomicrolides  and fosfcrolides  in NTS. g efflux . Interesobserved . Other s [blaKPC ,38. Despite reports of high usage of colistin in poultry in Nigeria , no geneSeveral approaches were used to reconstruct plasmids from whole genome sequence data. This work was conducted in parallel with plasmid extraction. Even though there are limitations in using short-read sequences in plasmid identification, it has been recommended for plasmid analysis . StudiesS. Schwarzengrund, S. Muenster and S. Isangi. Especially the IncQ1 of S. Shwarzengrund and IncN of S. Muenster were found to have multiple resistance genes , tet(R), aph(3”)-I and aph (6)-Ic) flanked by a repeat region with the Tn3 transposase. These resistance genes were likely acquired by transposition into this plasmid. In addition to transposase, the plasmid also harbored conjugative transfer genes, demonstrating mobility potential. However, a conjugation assay would be needed to provide insight into the transferability of the resistance genes. Reports of multiple antimicrobial resistance genes, including blaTEM, carried on similar but not identical plasmids in S. Enteritidis have previously been reported [S. Typhimurium [S. Isangi. These are small plasmids, and our study confirms that they harbor qnrB19 genes, which have been associated with plasmid-mediated reduced susceptibility to quinolones in Salmonella Hadar [dmt), a multidrug efflux pump (mdtl) and a small multidrug resistance (smr) efflux transporter and are considered the first line of bacterial defense against antimicrobials, by decreasing the intracellular level of the drug [Plasmid replicons were detected in 40% of strains representing seven of the 23 serovars detected. The high proportion of strains where plasmid replicons were not detected was surprising compared to other studies ,23. Therreported ,23,43,44himurium . Col -Id, aadA7, aph (3″)-Ib, aph (6′)-Id, tet(A) and sul1 that confer resistance to gentamicin, spectinomycin, streptomycin, tetracycline and sulphonamide, respectively. The set of genes present in these strains is, however, different from that reported from a previous study [Salmonella [S. Kentucky strains were circulating in the study area.Notably, most (91.7%) variants , while 2variants . The islus study , which mS. Kentucky, the serovars found in poultry in Nigeria have not been well characterized, and little is known about their potential to cause human disease. SPIs are involved in Salmonella adherence, invasion, replication and survival within the host cells [S. Abadina, S. Birmingham and S. Poona lacked all these major pathogenicity islands and may therefore be less pathogenic. SPI-1 encodes a Type III secretion system (TSSS) considered essential for Salmonella invasion and for growth in the intestine [Salmonella serovars depend on induction of inflammation to grow in the gut of chickens remains to be investigated, and it may be that serovars such as S. Abadina, S. Birmingham and S. Poona have adapted to grow in the gut environment in the absence of inflammation. Phenotypes associated with the presence of T3SS encoded by genes of Salmonella pathogenicity island 2 (SPI-2) are considered essential for the development of systemic disease including proliferation of Salmonella in host macrophages [phoP/phoQ regulatory genes which are present in most Gram-negative bacteria were also located in all of our strains. These genes control SPI-2 and, consequently, the expression of virulence genes located in this island [2+ concentrations and for long-term attachment to host cells [Salmonella strains [Salmonella and has been reported to have a nucleotide composition that is significantly different from that of SPI-1 [With the exception of st cells . We founst cells . SPIs 1,ntestine ,52. The ntestine , and thentestine . Whetherrophages . SPI-2 ws island . Most stst cells ,56. In a strains ,57. C63Pof SPI-1 .Salmonella serovars carry different types of chaperone-usher fimbriae, and such fimbriae have been proposed as an additional mediator for allowing some serovars to colonize and persist in different hosts/environments [Salmonella pathogenesis [csg, bcf, fim, lpf, pef, saf, sti-k and tcf). There were, however, variations in the specific sub-types of these genes between serovars, and all strains lacked the sef and stg fimbrial genes. Interestingly, some strains  were also observed to possess the type six secretion system (T6SS), which might have been acquired through horizontal gene transfer. There seems to be little information available about the association of these serotypes with T6SS. T6SS systems in Salmonella are encoded from the horizontally acquired islands, SPI-6 or SPI-19 [Salmonella pathogenicity; SPI-19 plays a role in killing competing bacteria in the intestine [S. Muenster, S. Chester and S. Aberdeen. Reports have shown that ronments ,59. Thesogenesis . All strr SPI-19 ,61. Repontestine , and it S. Isangi, S. Takoradi and S. Larochelle strains were identified to have virulence genes not possessed by the other strains in our study; virulence genes that have not been reported in these serotypes previously. This included genes that encode for toxin  production, serum resistance (rck), quorum sensing (luxS) and biofilm formation (adeG) [cdtB, pltA, pltB) [S. Schwarzengrund, S. Chester, S. Muenster, S. Ituri, S. Give, S. Takoradi and S. Telekebir to this list. The rck gene is known to confer serum resistance to complement killing by binding human complement factor H and may also contribute to autoaggregation [Interestingly, three n (adeG) ,64,65. AA, pltB) , and theregation . The preSalmonella isolates belonging to 23 serovars obtained from a previous study were analyzed [https://cge.cbs.dtu.dk/services/SPAdes/), and assembled contigs were used to assign serovars to strains using in silico typing resources (SeqSero2 and SISTR). Sequence quality based on total length, N50, contig size and Guanine-Cytosine (GC)content was used to select for strains to be included in the study. Draft whole genome sequences of strains are available from the European Nucleotide Archive under study accession number PRJEB37477 with individual strain accession numbers. Detailed information of strains is shown in Seventy-four draft genomes of analyzed . Isolateanalyzed . StrainsSalmonella enterica subsp. enterica serovar Typhimurium ATCC 14,028 was used for quality control purposes. Inhibition zone data were entered into WHONET version 5.6 configured with the tested antimicrobials. Isolates were categorized as sensitive, intermediate or resistant using CLSI clinical breakpoints and CLSI guidelines for disc diffusion. For comparison, available ECOFF values of antibiotics in EUCAST (www.EUCAST.org) were used to classify isolates as wild-type (WT) or non-wild-type (NWT). In this study, WTs are considered as sensitive, while NWTs are referred to as resistant. Strains that were resistant to at least one drug in at least three different antimicrobial classes were defined as multidrug-resistant (MDR) according to [Strains were phenotypically tested for susceptibility to 11 commonly used antimicrobials representing eight classes of antimicrobials by the Kirby–Bauer disk diffusion test using Clinical and Laboratory Standards Institute (CLSI) protocols . Antimicrding to .https://cge.cbs.dtu.dk/services/ResFinder/). This online platform uses a curated database of resistance genes to identify acquired antimicrobial resistance genes and chromosomal mutations in total or partial sequences. MEGA X v.10.2.2 was used for the alignment of the aac (6)-Iaa gene to identify changes in the sequences that could explain the lack of kanamycin phenotypic expression in 32 isolates carrying the gene [A draft assembled genome was used for prediction using ResFinder v.3.2 with default settings (90% selected percentage identity threshold and 60% selected minimum coverage length), available at the Centre for Genomic Epidemiology (the gene .https://cge.cbs.dtu.dk/services/SPAdes/) was used to assemble raw reads into different contigs containing chromosomal and extra-chromosomal sequences. Subsequently, draft genomes were submitted to PlasmidFinder v.2.0 available at the Centre for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/PlasmidFinder/), using default settings (95% selected minimum threshold for percentage identity and 60% selected minimum coverage length). The pipeline uses sequence similarity of replicons to predict putative plasmids. In the second step, raw reads of strains that harbored plasmid replicons were used to extract plasmid contigs using PlasmidSPAdes v.3.13.0. The algorithm in PlasmidSPAdes is able to predict contigs that belong to a plasmid and assign those contigs into components; putative plasmids from the same incompatibility group are assigned into same component [https://rast.nmpdr.org/) for annotation and detection of resistant genes and were again submitted to PlasmidFinder v.2.0 to verify previous predictions. In the third step, putative plasmids were used to search for the most similar sequences in the NCBI database using nucleotide BLAST (www.ncbi.nlm.nih.gov). Homologous reference sequences (.gbk) with query strains were downloaded from GenBank. Lastly, to confirm in silico plasmid prediction, plasmid extraction was performed using the [E. coli V517 [E. coli 39R861 [A three-step procedure was used to reconstruct plasmids . In the omponent . The quaomponent . Plasmidsing the  protocololi V517  and E. ci 39R861  were usehttps://server.gview.ca/guide). Non-resistance plasmids were aligned using progressive MAUVE v.2.4.0 [S. Kentucky strains, which were not located on plasmids, were BLASTed to NCBI to detect the genomic location of resistance genes. These contigs were aligned with NCBI references using EasyFig v.2.2.3 [To align and display the location of resistant genes on the plasmid, BLAST ring analysis of annotated plasmids with the NCBI reference plasmids was performed in GView v.1.7 server ) was used to identify SPIs using default settings (95% identity threshold and 60% selected minimum coverage length). To further identify virulence factors, draft genomes were submitted to the virulence factor database . VFanalyzer v.2019 uses orthologous groups to predict virulence genes by conducting iterative BLAST sequence similarity searches. The Phenotypic and genotypic resistance data were stored in Microsoft Excel 2016  and later exported to SPSS version 26  for inferential statistics. Cohen’s kappa statistic was used to assess the level of concordance between phenotypic resistance and in silico gene predictions. Cohen’s kappa is a statistic measure that is used to evaluate the level of agreement between two test protocols, assigning a kappa value between zero and one. Values ≤ 0 indicate no agreement; 0.01–0.20 none to slight; 0.21–0.40 fair; 0.41–0.60 moderate; 0.61–0.80 substantial; and values in the range of 0.81–1.00 indicate values with an almost perfect agreement .S. Kentucky, S. Schwarzengrud, S. Muenster and S. Isangi circulating in commercial poultry farms in Nigeria. Resistance genes were found to be harbored on plasmids of several incompatibility groups, which have previously not been associated with these particular serovars. Two major sub-populations of S. Kentucky strains with two variants of Salmonella genomic islands, each carrying multiple resistance genes, were identified. The fact that resistance genes were found on mobile elements increases the public health risks, as resistances may not only be transferred from animals to human through infection with Salmonella, but also through horizontal spread of plasmids involving intermediate bacterial hosts. Most of the strains were found to possess virulence genes that are essential for human infections, indicating zoonotic potential.This study demonstrated that MDR was common in strains of