HERC family emerged >595 million years ago and has undergone gene duplication and gene loss events throughout its evolution. The structural topology of the RCC1-like domain and HECT domains from all HERC paralogs is highly conserved among evolutionarily diverse vertebrates despite low sequence homology. Functional analyses showed that the human small HERCs exhibit different degrees of antiviral activity toward HIV-1 and that HERC5 provides the strongest inhibition. Notably, coelacanth HERC5 inhibited simian immunodeficiency virus (SIV), but not HIV-1, particle production, suggesting that the antiviral activity of HERC5 emerged over 413 million years ago and exhibits species- and virus-specific restriction. In addition, we showed that both HERC5 and HERC6 are evolving under strong positive selection, particularly blade 1 of the RCC1-like domain, which we showed is a key determinant of antiviral activity. These studies provide insight into the origin, evolution, and biological importance of the human restriction factor HERC5 and the other HERC family members.In humans, homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing protein 5 (HERC5) is an interferon-induced protein that inhibits replication of evolutionarily diverse viruses, including human immunodeficiency virus type 1 (HIV-1). To better understand the origin, evolution, and function of HERC5, we performed phylogenetic, structural, and functional analyses of the entire human small-HERC family, which includes HERC3, HERC4, HERC5, and HERC6. We demonstrated that the IMPORTANCE Intrinsic immunity plays an important role as the first line of defense against viruses. Studying the origins, evolution, and functions of proteins responsible for effecting this defense will provide key information about virus-host relationships that can be exploited for future drug development. We showed that HERC5 is one such antiviral protein that belongs to an evolutionarily conserved family of HERCs with an ancient marine origin. Not all vertebrates possess all HERC members, suggesting that different HERCs emerged at different times during evolution to provide the host with a survival advantage. Consistent with this, two of the more recently emerged HERC members, HERC5 and HERC6, displayed strong signatures of having been involved in an ancient evolutionary battle with viruses. Our findings provide new insights into the evolutionary origin and function of the HERC family in vertebrate evolution, identifying HERC5 and possibly HERC6 as important effectors of intrinsic immunity in vertebrates. Vertebrates possess multiple defense mechanisms to inhibit the replication of viruses. This defense system is largely composed of specialized hematopoietic cells that react nonspecifically to pathogens (innate immunity), an antibody-dependent and cell-mediated response (adaptive immunity), and core cellular effector proteins called restriction factors (intrinsic immunity). Restriction factors are considered to be the front line of defense against viral infection, since their activity typically does not require virus-triggered signaling or intercellular communication . The imp3–Retroviridae, Orthomyxoviridae, Flaviviridae, Togaviridae, Herpesviridae, Poxviridae, Arteriviridae, and Pneumoviridae (–28–32–32–Interferon-stimulated gene 15 (ISG15) and/or its conjugation to newly translated proteins (referred to as ISGylation) exhibits broad antiviral activity toward evolutionarily diverse viruses, including those belonging to the families oviridae –31. The idae –28–, 32–36. By virtue of their RCC1-like domains, HERCs also belong to the phylogenetically widespread RCC1 superfamily of proteins , 39. The32–https://genome.ucsc.edu]) and NCBI gene and protein sequence databases for the presence of small HERC gene members. The oldest small-HERC member is HERC4, which is present in one of the only surviving lineages of jawless fish, sea lampreys (originating ∼595 million years ago [mya]) , just after the small-HERC family expanded with the duplication and divergence of HERC4 , giving rise to two different chromosomal HERC loci in most vertebrates, where the HERC3-HERC5-HERC6 locus is flanked by FAM13A and PIGY-PYURF and HERC4 by MYPN and SIRT1  . To bettngements . This alngements . A singlof HERC4 . HERC3 and SIRT1 .HERC family expanded further after the divergence of cartilaginous fish (∼430 mya), with the emergence of HERC6, which is present in most jawed vertebrates with an apparent absence in platypus and some fish  and bird  species (HERC family occurred after the divergence of ray-finned fish (∼413 mya), with the likely duplication of HERC6, giving rise to HERC5 , metatherian  mammals , and rodents    43, 45), 45HERC HERC sequences showed segregation of the small HERC genes into four major clusters consisting of HERC3, HERC4, HERC5, and HERC6 . Notably, coelacanth and lizard HERC5 sequences clustered on their own, showing more sequence similarity to HERC6 genes than to other HERC5 genes, possibly indicating that these genes are actually HERC6 or a hybrid of HERC5 and HERC6. Similar tree topologies regarding the main branching were predicted using several tree-generating algorithms . Consistent with the approximate emergence times of the small-HERC family members shown in HERC4 is the oldest of the HERC paralogs, followed by HERC3, HERC6, and then HERC5.Phylogenetic analysis of nd HERC6 . Most HEH structural measure derived from alignment of the predicted tertiary structures of RCC1-like domains (https://bmm.crick.ac.uk/~populus/) and showed that each of the RCC1-like domains of HERC3 to -6 adopted the characteristic β-propeller structure of the superfamily, despite their low sequence homology  constructs for the ability to knock down endogenous HERC mRNA and protein levels. As shown in HOS-CD4-CXCR4 cells were cotransfected with plasmids carrying HERC shRNA and HIV-1 R9 (a full-length replication-competent NL4-3 derivative). After 72 h of replication (∼2 or 3 rounds), quantitative Western blot analysis of cell lysates or virions produced from cells showed that cells knocked down for HERC5 expression exhibited a significant increase in Gag particle production (∼8-fold), whereas HERC3, -4, and -6 released modestly more virions into the supernatant than the control cells (∼2-fold) . The amo53–To test the effect of increased HERC expression on single-round HIV-1 particle production, human 293T cells, which do not support multiround replication, were cotransfected with plasmids carrying HIV-1 (R9) and either empty vector, HERC3, HERC4, HERC5 or HERC6. As expected, HERC5 potently inhibited HIV-1 particle production . HERC3 oWe previously showed that human HERC5 blocked nuclear export of Rev-dependent HIV-1 RNA . To detecis-acting element called the Rev-response element (RRE) located within an HIV-1 intron  and Rev-independent  constructs, as previously described , 58. HIV1 intron . HIV-1 m1 intron . Success1 intron  and F. THERC5, we tested the ability of coelacanth HERC5 to inhibit HIV-1 virus production. To assess potential virus-specific effects, we also tested the antiviral activity toward another, related nonhuman retrovirus, simian immunodeficiency virus (SIV) , which is thought to be at least 32,000 years older than HIV-1  or blade 1 (H6:H5blade1) from human HERC5 with the corresponding region in human HERC6. We then measured the abilities of these HERC6 mutants to inhibit HIV-1 particle production. As shown in HERC family has an ancient marine origin, where HERC4 emerged at least 595 mya and expansion of the family occurred sometime after the divergence of jawed vertebrates from jawless vertebrates (∼476 to 595 mya). Elephant sharks are among the oldest and most slowly evolving jawed vertebrates and have accumulated a small number of chromosomal rearrangements  comprise a substantial portion of vertebrate genomes and appear to have an ancient marine origin, with evidence of ERV sequences found in the genomes of elephant shark, coelacanth, and possibly lamprey , resulting in a smaller repertoire of immune genes than in humans . However, HERC3 and HERC4 do exhibit differential tissue-specific expression  supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% COHomo sapiens (human) (NP_055421.1), Gorilla gorilla gorilla (gorilla) (XP_004039158.1), Pan troglodytes (chimpanzee) (XP_517337.3), Nomascus leucogenys (gibbon) (XP_003265945.1), Papio anubis (baboon) (XP_003898995.1), Saimiri boliviensis boliviensis (squirrel monkey) (XP_003924058.1), Callithrix jacchus (marmoset) (XP_002745644.1), Bos taurus (cow) (NP_001077132.1), Ovis aries (sheep) (XP_004009758.1), Felis catus (cat) (XP_003985228.1), Canis lupus familiaris (dog) (XP_535653.3), Ailuropoda melanoleuca (giant panda) (XP_002913643.1), Equus caballus (horse) (XP_001496703.3), Callorhinchus_milii (elephant shark) (XM_007902532.1), Danio rerio (zebrafish) (NM_001145624.1), Takifugu rubripes (pufferfish) (XM_011612933.1), Oreochromis niloticus (tilapia) (XM_005456948.2), Latimeria chalumnae (coelacanth) (XM_006005071.2), Xenopus tropicalis (frog) (XM_002938630.3), Anolis carolinensis (lizard) (XM_008111037.2), Geospiza fortis (finch) (XM_005418124.1), Meleagris gallopavo (turkey) (XM_003205471.2), Gallus gallus (chicken) (XM_015276202.1), Monodelphis domestica (opossum) (XM_007495699.2), Mus musculus (mouse) (NM_028705.3), and Dasypus novemcinctus (armadillo) (XM_004480548.2); for HERC4, human (NP_071362.1), gorilla (XP_004049535.1), chimpanzee (XP_001167753.1), gibbon (XP_003258260.1), baboon (XP_003903924.1), squirrel (XP_003928728.1), marmoset (XP_002756356.1), cow (NP_001070362.2), sheep (XP_004021455.1), dog (XP_849808.1), panda (XP_002913788.1), horse (XP_001503636.1), elephant shark (XM_007897346.1), zebrafish (XM_005173035.3), pufferfish (XM_011602912.1), tilapia (XM_005473848.2), coelacanth (XM_005992449.2), frog (NM_001128650.1), lizard (XM_008115175.2), finch (XM_005428501.2), turkey (XM_010714265.1), chicken (XM_015278711.1), Ornithorhynchus anatinus (platypus) (XM_007667149.1), opossum (XM_016421882.1), mouse (NM_030114.2), armadillo (XM_012529817.1), and Petromyzon marinus (lamprey) ENSPMAG00000001626; for HERC5, human (NP_057407.2), chimpanzee (XP_003310459.1), gorilla (XP_004039179.1), marmoset (XP_002745648.1), baboon (XP_003898997.1), squirrel monkey (XP_003924055.1), gibbon (XP_003265940.1), horse (XP_001915115.2), giant panda (XP_002913645.1), sheep (XP_004009762.1), cow (NP_001095465.1), dog (XP_535652.3), cat (XP_003985249.1), coelacanth (XM_014498805.1), lizard (XM_008111035.2), and armadillo (XM_012520464.1); for HERC6, human (NP_060382.3), gorilla (XP_004039178.1), chimpanzee (XP_001160851.1), gibbon (XP_003265938.1), baboon (XP_003899001.1), squirrel (XP_003924053.1), marmoset (XP_002745681.1), cow (NP_001179573.1), sheep (XP_004010096.1), cat (XP_003985250.1), dog (XP_851549.1), horse (XP_001494887.1), pufferfish (XM_011618146.1), tilapia (XM_005474674.1), coelacanth (XM_014498807.1), frog (XM_002938624.2), lizard (XM_008111034.2), opossum (XM_007495704.1), mouse (NM_025992.2), and armadillo (XM_012520459.1).The MEGA 7.0 package was used for phylogenetic analysis . The amiCallorhinchus milii 6.1.3 GCF_000165045.1 (elephant shark), Danio rerio GRCz10 GCF_000002035.5 (zebrafish), Takifugu rubripes FUGU5 GCF_000180615.1 (pufferfish), Oreochromis niloticus Orenil1.1 GCF_000188235.2 (tilapia), Latimeria chalumnae LatCha1 GCF_000225785.1 (coelacanth), Xenopus tropicalis Xtropicalis_v7 GCF_000004195.2 (frog), Anolis carolinensis AnoCar2.0 GCF_0000090745.1 (lizard), Geospiza fortis GeoFor_1.0 GCF_000277835.1 (finch), Meleagris gallopavo Turkey_5.0 GCF_000146605.2 (Turkey), Gallus_gallus-5.0 GCF_000002315.4 (chicken), Ornithorhynchus_anatinus-5.0.1 GCF_000002275.2 (platypus), Monodelphis domestica MonDom5 GCF_000002295.2 (opossum), Dasypus novemcinctus Dasnovv3.0 GCF_000208655.1 (armadillo), Loxodonta africana Loxafr3.0 GCF_000001905.1 (elephant), Mus musculus GRCm38.p3 GCF_000001635.23 (mouse), Rattus norvegivcus Rnor_6.0 GCF_000001895.5 (rat), Elephantulus edwardii EleEdw1.0 GCF_000299155.1 (shrew), Chrysochloris asiatica ChrAsi1.0 GCF_000296735.1 (cape golden mole), Canis lupus familiaris CanFam3.1 GCF_000002285.3 (dog), and Homo sapiens GRCh38.p2 GCF_000001405.28 (human).Synteny maps were derived using the following reference assemblies: http://www.ncbi.nlm.nih.gov/tools/cobalt/) (Homo sapiens (human) (NP_055421.1), Gorilla gorilla gorilla (gorilla) (XP_004039158.1), Pan troglodytes (chimpanzee) (XP_517337.3), Nomascus leucogenys (gibbon) (XP_003265945.1), Papio anubis (baboon) (XP_003898995.1), Saimiri boliviensis boliviensis (squirrel monkey) (XP_003924058.1), Callithrix jacchus (marmoset) (XP_002745644.1), Bos taurus (cow) (NP_001077132.1), Ovis aries (sheep) (XP_004009758.1), Felis catus (cat) (XP_003985228.1), Canis lupus familiaris (dog) (XP_535653.3), Ailuropoda melanoleuca (giant panda) (XP_002913643.1), and Equus caballus (horse) (XP_001496703.3); for HERC4, human (NP_071362.1), gorilla (XP_004049535.1), chimpanzee (XP_001167753.1), gibbon (XP_003258260.1), baboon (XP_003903924.1), squirrel (XP_003928728.1), marmoset (XP_002756356.1), cow (NP_001070362.2), sheep (XP_004021455.1), dog (XP_849808.1), panda (XP_002913788.1), and horse (XP_001503636.1); for HERC5, human (NP_057407.2), chimpanzee (XP_003310459.1), gorilla (XP_004039179.1), marmoset (XP_002745648.1), baboon (XP_003898997.1), squirrel monkey (XP_003924055.1), gibbon (XP_003265940.1), horse (XP_001915115.2), giant panda (XP_002913645.1), sheep (XP_004009762.1), cow (NP_001095465.1), dog (XP_535652.3), and cat (XP_003985249.1); for HERC6, human (NP_060382.3), gorilla (XP_004039178.1), chimpanzee (XP_001160851.1), gibbon (XP_003265938.1), baboon (XP_003899001.1), squirrel (XP_003924053.1), marmoset (XP_002745681.1), cow (NP_001179573.1), sheep (XP_004010096.1), cat (XP_003985250.1), dog (XP_851549.1), and horse (XP_001494887.1). At least 2 independent sequences were available for human, sheep, baboon, marmoset, gibbon, and squirrel monkey. The following sequences were not independently validated: cat, dog, cow, horse, sheep, and giant panda. The identification of site-specific positive selection and purifying selection was generated using the Selecton server (http://selecton.tau.ac.il/index.html). The HERC5 phylogenetic tree was used in the Selecton analysis. Nested pairs of models  and a nonnested pair (M8a and MEC) were compared using the likelihood ratio test implemented in the Selecton program.Positive-selection analysis was performed as previously described . HERC secobalt/) . The folPlasmids encoding Flag-tagged HERC3, HERC4, HERC5, and HERC6 were created by first PCR amplifying the various HERC coding regions from their respective templates (as described above) using the following primers: for HERC3, forward, 5′ ACG TGA ATT CCA TGT TAT GTT GGG GAT ATT GG 3′, and reverse, 5′ ACG TGG TAC CTC AGG CCA AAC TAA ACC CTT CAT AGT TGT C 3′; for HERC4, forward, 5′ACG TGA ATT CTA TGT TGT GCT GGG GAA ATG C 3′, and reverse, 5′ ACG TTC TAG ATT ATA TTA AAC TGA AGC CTT CAT TGT G 3′; for HERC5, forward, 5′ AAT CGA GAT CTT ATG GAG CGC CGC AGC 3′, and reverse, 5′ TAT GCG GAT CCT CAG CCA AAT CCT CTG 3′; and for HERC6, forward, 5′ AGA TAA GAT CTT ATG TAC TTC TGT TGG GGC 3′, and reverse, 5′ TAG GAG ATA TCT TAT GAC TGT GTG AGC ATG 3′. The amplified products were then cloned into p3xFLAG-CMV10 using the following restriction enzymes: for HERC3, EcoRI and KpnI; for HERC4, EcoRI and XbaI; for HERC5, BglII and BamHI; and for HERC6, BglIII and EcoRV. The resulting plasmids were named pHERC3, pHERC4, pHERC5, and pHERC6.To generate pH 6:H5RLD, the HERC5 RCC1-like domain was PCR amplified from pHERC5 using the following primers: forward, 5′ GGA TGA CGA TGA CAA GAT GGA GCG CCG CAG CC 3′, and reverse, 5′ TAT GTT CCA GCA AAA ATT ATT AAC TCC TTT TCT GAG GTA TGG CTT TCA AG 3′. The backbone of pHERC6 was PCR amplified using the following primers: forward, 5′ TTT TTG CTG GAA CAT ATG CCA ACT TTG 3′, and reverse, 5′ CTT GTC ATC GTC ATC CTT GTA ATC GAT G 3′. The two amplified fragments were cloned using the fast cloning technique .To generate pH6:H5blade1, blade 1 of HERC5 (amino acids 1 to 100) was PCR amplified from pHERC5 using the following primers: forward, 5′ GGA TGA CGA TGA CAA GAT GGA GCG CCG CAG CCG CCC CAA CAG AAG TAC ATC TTG TCA TCG TCA TCC TTG TAA TCG ATG 3′, and reverse, 5′ GCT CCT TCC CGC AGC TCA CGT GGA TCT TCA TGT TCT TGC CCA GC 3′. The backbone of pHERC6 was PCR amplified using the following primers: forward, 5′ GCT GGG CAA GAA CAT GAA GAT CCA CAG CTG CGG GAA GGA GCA C 3′, and reverse, 5′ GGC TGC GGC GCT CCA TCT TGT CAT CGT CAT CCT TGT AAT CGA TG 3′. The two amplified fragments were cloned using the Gibson cloning technique per the manufacturer's instructions (New England Biolabs).To generate pH6:R10A, site-directed mutagenesis was performed on pHERC6 using the following primers: forward, 5′ TTC TGT TGG GGC GCC GAC TCC GCG GAG CTG CAG CGC CGG AGG 3′, and reverse, 5′ CCT CCG GCG CTG CAG CTC CGC GGA GTC GGC GCC CCA ACA GAA 3′. pH6:E67A was generated similarly using the following primers: forward, 5′ GCA GCG CGG GGA GCT GCC AGC ACC AAT TCA GGC ATT GGA AAC C 3′, and reverse, 5′ GGT TTC CAA TGC CTG AAT TGG TGC TGG CAG CTC CCC GCG CTG C 3′. pH6:R10A/E67A was generated similarly, except pH6:R10A was used as the template with the following primers: forward, 5′ GCA GCG CGG GGA GCT GCC AGC ACC AAT TCA GGC ATT GGA AAC C 3′, and reverse, 5′ GGT TTC CAA TGC CTG AAT TGG TGC TGG CAG CTC CCC GCG CTG C 3′. pR10G was generated similarly, using the following primers: forward, 5′ CGC CGA CTC CGG GGA GCT GCA 3′, and reverse, 5′ TGC AGC TCC CCG GAG TCG GCG 3′. pHERC3-ΔRLD was generated similarly, using the following primers: forward, 5′ GAC GAT GAC AAG ATG AGC TCA CCA CCA GAT GTT GAA G 3′, and reverse, 5′ CAT CTG GTG GTG AGC TCA TCT TGT CAT CGT CAT CCT TGT AAT CG 3′. pHERC4-ΔRLD was generated similarly, using the following primers: forward, 5′ ACG ATG ACA AGA TGA ATT GGT ACC CCT ATA ATG GGC AGT G 3′, and reverse, 5′ TAG GGG TAC CAA TTC ATC TTG TCA TCG TCA TCC TTG TAA TCG 3′. pHERC5-ΔRLD was generated previously . pHERC6-HERC3, HERC4, HERC5, and HERC6 were obtained from the following sources: HERC3 (NM_014606.2), HERC6 (NM_017912.3) (HERC5 (NP_057407.2) (HERC4 (NM_015601.3) was isolated from HeLa cells by first reverse transcribing total RNA and then PCR amplification of cDNA using the following primers: forward, 5′ACG TGA ATT CTA TGT TGT GCT GGG GAA ATG C 3′, and reverse, 5′ ACG TTC TAG ATT ATA TTA AAC TGA AGC CTT CAT TGT G 3′. All the constructs were verified by sequencing. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions unless otherwise indicated. Cotransfections of HERC plasmids with pR9 were performed at a ratio of 10:1 unless otherwise noted. Standard Western blot analyses were performed as previously described  , and HER57407.2) . HERC4 (escribed . DensitoSaccharomyces cerevisiae-based cloning of diverse HIV-1 strains (S. cerevisiae Hanson (MYA-906) (MATα ade6 can1 his3 leu2 trp1 URA3) was obtained from the American Type Culture Collection (ATCC). The yeast was grown at 30°C in appropriate medium , depending on the cloning step. Transformations/recombinations were performed using the lithium acetate (LiAc) method. Briefly, the linearized vector DNA (∼1 μg) and PCR product (∼3 μg) were added to competent cells at a 1:3 ratio, along with 50 μg of single-stranded salmon sperm carrier DNA  and sterile polyethylene glycol (50%)-TE -LiAc (100 mM). Following agitation for 30 min at 30°C, the yeast was heat shocked at 42°C for 15 min and plated on C-leu agar plates containing the appropriate selection.For the construction of pSIVmac239 (pREC_nfl_SIV239), the SIVmac239 Spx vector was obtained from the NIH AIDS Reagent Program. We previously constructed pREC_nfl_HIV and pCMV_cplt vectors for  strains . We deve strains . For yeaThe following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 monoclonal antibody (183-H12-5C) from Bruce Chesebro and Kathy Wehrly and anti-SIVmac p17 monoclonal antibody (KK59) from Karen Kent and Caroline Powell. Anti-FLAG and anti-hemagglutinin (HA) (clone 3F10) were purchased from Sigma, anti-myc and anti-β-actin were purchased from Rockland, anti-eGFP was purchased from Clontech, and anti-GAPDH  (clone 6C5) was purchased from EMD/Millipore. Anti-HERC3 (H00008916-B01P), anti-HERC4 (H00026091-A01), anti-HERC5 (H00051191-A01), and anti-HERC6 (H00055008-A01) were purchased from Abnova.TC) for each PCR. The gene-specific forward and reverse primer sets used were as follows: Gag- ; Rev . Quantification of endogenous HERC mRNA was run on the QuantStudio5 qPCR machine (Applied Biosystems) under the following cycling conditions: 2 min at 95°C and 40 cycles of 5 s at 95°C, 10 s at 60°C, and 20 s at 72°C. QuantStudio design and analysis desktop software (version 1.4) was used to determine the TC for each PCR. The primer pairs were as follows: HERC3 , HERC4 , HERC5 , HERC6 , GAPDH , and eGFP . To ensure no carryover of DNA into each total purified RNA sample, 3 μg of the purified RNA was used directly as the template without reverse transcription for qPCR, using the primer sets described above.Total RNA was extracted using the PureLink RNA minikit . Three micrograms of RNA was reverse transcribed to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase and oligo(dT) primers (Life Technologies). Prior to qPCR, the cDNA samples were diluted 1:5 with water. Each PCR mixture consisted of 10 μl of SYBR green master mix, 2 μl of gene-specific primers (1 μl of 10 μM forward primer and 1 μl of 10 μM reverse primer), 5 μl of diluted cDNA, and water to a total volume of 20 μl. For quantification of incompletely and fully spliced HIV RNAs, qPCR was run on the Rotor-Gene 6000 qPCR machine (Corbett Life Science) under the following cycling conditions: 10 min at 95°C and 40 cycles of 10 s at 95°C, 15 s at 60°C, and 20 s at 72°C. The Rotor-Gene 6000 series software (version 1.7) was used to determine the cycle threshold (P values and statistical tests used are mentioned where appropriate. P values of less than 0.05 were deemed significant.GraphPad Prism v6 was used for all statistical analyses mentioned in the text. The


Xueshuan Xinmaining Tablet (XXT) is a widely used traditional Chinese medicine for the treatment of stroke, chest pain, coronary heart disease, and angina pectoris caused by blood stasis. Having a multiple-component preparation, it is still far from meeting the requirements of modernization and standardization because its detailed chemical basis and action mechanism have not been clarified. In this work, the different batches of XXT samples were analyzed by HPLC and the typical sample was analyzed by UPLC-ESI-Q-TOF/MS to understand its chemical profiling. As a result, 77 chromatographic peaks were detected, among which 63 constituents were identified or tentatively characterized based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules and matching of empirical molecular formula with those of published components. This is the first systematic report on the chemical profiling of the commercial XXT products, which provides the sufficiently chemical evidence for the global quality evaluation of XXT products. Chuanxiong rhizoma), Huaihua (Sophorae flos), Danshen , Shuizhi (Hirudo), Maodongqing (Hairy holly root), Rengong Niuhuang , Rengong Shexiang (Moschus artifactus), Renshen Jingye Zongzaogan , Bingpian (Borneolum syntheticum), and Chansu (Bufonis venenum) + (ESI+) or [M+HCOO]− (ESI-) were detected in MS spectrum. In MS/MS spectrum, a series of fragmentation ions were observed due to the neutral loss of glucose, rhamnose, arabinose, or xylose  were unambiguously identified as ginsenoside Rg1, ginsenoside Re, ginsenoside Rb2, ginsenoside Rb3, 20S-ginsenoside F1, ginsenoside Rd, 20S-ginsenoside F2, and 20S-ginsenoside Rh2. Unknown ginsenoside saponins followed the similar dissociation pathways. For the ginsenosides of protopanaxadiol, the ions at m/z 407, 425, and 443 could be found in the positive ion mode. Meanwhile the ginsenosides of protopanaxatriol showed the ions at m/z 405, 423, and 441 in the positive ion mode.lose see . These f1 showed [M+Na]+ ion at m/z 823.4824 (C42H72O14Na) in the positive ion mode and [M+HCOO]− ion at m/z 845.4895 (C43H73O16) in the negative ion mode. The fragmentation ions at m/z 621.4368 and 603.4259 were produced from the protonated ion [M+H]+ by losing glucose and the further loss of H2O. In the negative MS/MS analysis, [M-H]− at m/z 799.4838 gave the characteristic ion at m/z 637.4307 [M-H-glc]−and 475.3773[M-H-2glc]−. Compounds 23 and 40 showed the quasimolecular ions at m/z 823 [M+Na]+ and 845 [M+HCOO]− in the full mass spectrum. Compound 23 has the same retention time and MS data as those of reference ginsenoside Rg1 and it was identified as ginsenoside Rg1. As for compound 40, although the fragmentation ions at m/z 405, 423, or 441 could not be detected in the MS/MS spectrum, it was temporarily identified as ginsenoside Rf.Ginsenoside Rg+ ion at m/z 969.5427 (C48H82O18Na) in the positive ion mode and [M+HCOO]− ion at m/z 991.5482 (C49H83O20) in the negative ion mode. The fragmentation ions at m/z 945.5428 [M-H]−, 799.4845 [M-H-rha]−, 783.4893 [M-H-glc]−, and 637.1430 [M-H-rha-glc]− were found in MS/MS spectrum in the negative ion mode. Compound 24 showed similar retention time and MS data as those of reference ginsenoside Re and it was identified as ginsenoside Re.Ginsenoside Re showed [M+Na]2, [M+Na]+ ion at m/z 1101.5868 (C53H90O22Na) in the positive ion mode and [M+HCOO]− ion at m/z 1123.5908 (C54H91O24) in the negative ion mode are shown in the full mass spectrum. The fragmentation ions at m/z 945.5425 [M-H-ara]− and 783.4889 [M-H-ara-glc]− were detected in MS2. Reference ginsenoside Rb3  and 683.4375 [M+HCOO]− (C37H63O11Na). In the negative MS/MS experiment, the fragmentation ion at m/z 475.3785 [M-H-glc]− was detected. Compound 49 showed similar retention time and MS data as those of reference 20S-ginsenoside F1.20 m/z 969.5379 [M+Na]+ and 991.5495 [M+HCOO]− in the full mass spectrum. The fragmentation ions at m/z 783.4892 [M-H-glc]−, 621.4368 [M-H-2glc]−, and 459.3842 [M-H-3glc]− were produced by losing glucose. Compound 53 was identified as ginsenoside Rd by displaying the same retention time and MS information.Reference ginsenoside Rd showed the adduct ions atS-ginsenoside F2, it showed the quasimolecular ions at m/z 807.4890 [M+Na]+ and 829.4949 [M+HCOO]−. The deprotonated ion peak at m/z 783.4881 [M-H]− produced the fragmentation ion at m/z 621.4355 [M-H-glc]− in MS2. Compounds 45, 61, 64, and 65 gave the same adduct ions [M+Na]+ in positive MS and [M+HCOO]− in negative MS, indicating the molecular formula as C42H72O13. Compound 45 was deduced as protopanaxatriol type and the latter three compounds were deduced as protopanaxadiol type on the base of the fragmentation rules described above. Among them, compound 61 has similar ions in full mass spectrum and MS/MS experiment as those of reference 20S-ginsenoside F2 and was unambiguously identified as 20S-ginsenoside F2. The deprotonated ion of 45 at m/z 783.4910 [M-H]− indicated the ion at m/z 475.3791 [M-H-rha-glc]−, supporting that 45 was ginsenoside Rg2. Compounds 64 and 65 displayed same MS data; however, unfortunately, MS2 information could not be detected and they were tentatively identified as 20R-ginsenoside Rg3 and 20S-ginsenoside Rg3.As for reference 202 showed the ions at m/z 1245.8995 [2M+H]+, 645.4348 [M+Na]+, and 667.4437[M+HCOO]−. Compound 74 was identified as 20S-ginsenoside Rh2 by comparing the retention time and MS data.Reference ginsenoside Rh 35, 36, and 37 have the same molecular formula of C48H82O19 and were tentatively characterized as 20S-glc-ginsenoside Rf, beads ginseng saponin F1, and notoginsenoside R3. They gave [M+Na]+ ions at m/z 985.5332, 985.5319, and 985.5339 in the positive ion mode and [M+HCOO]− ions at m/z 1007.5457, 1007.5457, and 1007.5459 in the negative ion mode. In their MS/MS experiments, the fragmentation ions of 35 at m/z 799.4843 and 637.4273 were produced from the ion at m/z 961.5391 [M-H]− by loss of glucose unit. Similarly, the fragmentation ions of 36 and 37 at m/z 799 and 637 were yielded. The retention order of three chromatographic peaks is determined according to the description in the literature [Compounds 42 and 43 are calculated as C41H70O13 based on the ions at m/z 793.4705 [M+Na]+ in the positive ion mode and 815.4818 [M+HCOO]− in the negative ion mode. Both compounds showed the fragmentation ions at m/z 405, 423, or 441 indicating that they are due to protopanaxatriol type. In MS/MS analysis, the ions at m/z 637.4326 [M-H-xyl]− and 475.3794 [M-H-xyl-glc]− for compound 42, as well as 637.4319 [M-H-ara]− and 475.3788 [M-H-ara-glc]− for compound 43, were detected from the deprotonated ions at m/z 769.4753 [M-H]− and 769.4758 [M-H]−, respectively. Compounds 42 and 43 were identified as notoginsenoside R2 and ginsenoside F3.The chemical formulas of compounds 54 showed the quasimolecular ions at m/z 939.5284 [M+Na]+ and 961.5390 [M+HCOO]−, indicating the molecular formula as C47H80O17. The fragmentation ions 783.4901 [M-H-xyl]− and 621.4363 [M-H-xyl-glc]− were detected, supporting that 54 was gypenoside IX.Compound 60 showed the quasimolecular ions at m/z 789.4742 [M+Na]+ and 811.4871 [M+HCOO]− and the molecular formula was calculated as C42H70O12. In the MS/MS analysis, the deprotonated ion at m/z 765.4792[M-H]− gave the ions at m/z 603.4261 [M-H-glc]− and 441.3730 [M-H-2glc]−. Further loss of H2O yielded the ions at m/z 423 and 405. It was identified as ginsenoside Rk1.Compound 34 showed [M+Na]+ ion at m/z 1117.5740 and [M+HCOO]− ion at m/z 1139.5891, suggesting the molecule formula of C53H90O23. Besides, it showed [M-H]− ion at m/z 1093.5826 and fragment ions at m/z 961 [M-H- xyl/ara]−, 799 [M-H-xyl/ara-glc]−, and 637 [M-H-xyl/ara-2glc]−, concerning consecutive loss of 132, 294, and 456. These data were in accordance with those of floral ginsenoside P, floranotoginsenoside D, notoginsenoside FT3, or yesanchinoside H [Compound 72 showed [M+Na]+ ion at m/z 789.4742 and [M+HCOO]− ion at m/z 811.4847, indicating the molecule formula as C42H70O12. Besides, the ions at m/z 603.4251 [M-H-162]− and 471.3466 [M-H-162-132]− in the MS/MS analysis suggested that the glycoside chain of 72 consisted of a molecule of glucose and a molecule of arabinose or xylose. It could be one of ilexoside A or ilexoside D described in the literature + and deprotonated ion at m/z 609.1461 [M-H]− as well as the ion at m/z 1219.3008 [2M-H]−. Quercetin, with UV λmax absorption at 253 and 356 nm, displayed the deprotonated ion at m/z 301.0376 [M-H]−. Four compounds  were determined as flavonoid derivatives due to the typical UV absorption. Especially, compound 9 was one of main constituents of XXT. Compounds 9 and 26 were unambiguously identified as rutin and quercetin based on the direct comparison of their UV spectra, and mass spectra with those of the authentic compounds. Compound 10 has UV λmax absorption at 264 and 343 nm. The molecular formula was calculated as C27H30O15 by the quasimolecular ion at m/z 595.1652 [M+H]+, 617.1472 [M+Na]+, and 593.1505 [M-H]−. Other fragment ions at m/z 449.1083 [M+H-rha]+ and 287.1055 [M+H-rha-glc]+ were detected. Therefore, compound 10 was identified as kaempferol-3-O-rutinoside. Compound 13 has UV λmax absorption at 252 and 339 nm. The molecular formula was calculated as C28H32O16 by the quasimolecular ion at m/z 625.1754 [M+H]+, 647.1578 [M+Na]+, and 623.1616 [M-H]−, suggesting it was isorhamnetin 3-O-rutinoside − ion at m/z 395.07949 was found. The MS/MS spectrum of [M-H]− exhibited an obvious fragment ion, [M-H-H2O]− at m/z 179.0338, and further loss of COOH obtained ion at m/z 135.0438. Compound 1 was identified as danshensu.Danshensu has UV λmax absorption at 323 nm. In the full mass spectrum, the deprotonated ion at m/z 353.0869 [M-H]− was found. The MS/MS spectrum of [M-H]− showed ions at m/z 191.0548, 179.0336, and 135.0438. Compound 2 was identified as neochlorogenic acid.Neochlorogenic acid has UV λmax absorption at 325 nm and showed the quasimolecular ion at m/z 353.0872 [M-H]− and 707.1482 [2M-H]−. The fragment ion at m/z 191.0553 was yielded by losing C9H6O3. Compound 4 was identified as chlorogenic acid.Chlorogenic acid has UV λmax absorption at 229, 278, and 310 nm. The [M-H]− ion at m/z 137.0236 was detected. In the MS/MS analysis, the ion at m/z 108.0204 was yielded by losing CHO and further losing oxygen produced from the ion at m/z 92.0256. Compound 5 was identified as protocatechuic aldehyde.Protocatechuic aldehyde has UV λmax absorption at 238 and 322 nm. It showed [M-H]− ion at m/z 179.0339 in the full mass spectrum and the ion at m/z 135.0438 by losing CO2 in the MS/MS experiment. Compound 7 was identified as caffeic acid.Caffeic acid has UV 11, 14, and 15) at m/z 515 [M-H]− and 517 [M+H]+ were easily located in the chromatogram of XXT, suggesting the molecular formula of C25H24O12. They were assigned as dicaffeoylquinic acids by comparison with retention times. Besides, they gave the same fragment ions with those of chlorogenic acid, such as m/z 191, 179, 173, and 135. in MS/MS experiments. The fragmentation pathways are in accordance with those described in the literature terature . Based o 12 has UV λmax absorption at 244 and 327 nm. The molecular formula was calculated as C27H22O12 on the basis of the quasimolecular ions at m/z 539.1149 [M+H]+ and 537.1031 [M-H]−. In the MS/MS experiment, it indicated the characteristic ion at m/z 339.0500 [M-H-C9H10O5]−, supporting that compound 12 was salvianolic acid H.Compound 17 has UV λmax absorption at 250 and 306 nm. It showed the ion at m/z 341.0666 [M+H]+. In the MS/MS analysis, the ions at m/z 295.0617 and 279.0659 were yielded by losing CH2O2 and CH2O3. Compound 17 was identified as salvianolic acid G.Compound 18 has UV λmax absorption at 251 and 317 nm. It showed [M-H]− ion at m/z 493.1134 and the molecular formula was calculated as C26H22O10. In the MS/MS analysis, the fragment ion at m/z 295.0604 [M-H-C9H10O5]− was detected, suggesting compound 18 was salvianolic acid A.Compound 32 showed the ions at m/z 567.1498 [M+H]+, 589.1320 [M+Na]+, and 565.1342 [M-H]− in MS spectrum, revealing molecular formula of C29H26O12. Besides, fragment ion at m/z 369.0971 [M-H-C9H10O5]− was observed. Compound 32 was identified as ethyl lithospermate.CompoundBufadienolides. The MS/MS behaviors of bufadienolides have been extensively described + ions was triggered by initial loss of 60 Da (HOAc). The elimination of CO was significant for bufadienolides with a 19-formyl group, and the 19-hydroxyl group could be characterized by the loss of 30 Da (HCHO). These fragmentation rules were applied to the identification of bufadienolides in XXT sample. As shown in 19), arenobufagin (25), bufotalin (41), bufalin (50), resibufogenin (55), and cinobufagin (56) by comparison with reference substances isolated from toad venom. The other two bufadienolides were tentatively identified as bufarenogin (20) and hellebrigenin (29) + ion at m/z 403.2486 was found and the fragment ions at m/z 385.2366 [M+H-H2O]+, 367.2262 [M+H-2H2O]+, and 349.2169 [M+H-3H2O]+ were detected in MS2.escribed , 18. BriBile acids. Bile acid derivatives are in lack of conjugated system and their UV absorption is not obvious. However, they always present high sensitivity in the negative ion mode. The deprotonated ion [M-H]− and adduct ion [M+HCOO]− were obviously detected in MS spectrum. The loss of side chain was commonly observed in MS2. Four bile acid derivatives were detected from XXT sample, two of which were unambiguously identified as ursodeoxycholic acid  and chenodeoxycholic acid  based on the direct comparison of reference substances. The other two compounds were tentatively identified as cholanic acid (57) and hyodeoxycholic acid (67). They are derived from the raw material, Rengong Niuhuang  [ifactus) . m/z 391.2851 [M-H]−, 437.2916 [M+HCOO]−, and 783.5778 [2M-H]− in the negative ion mode. In the positive ion mode, the ion at m/z 357.2807 [M+H-2H2O]+ was assigned as the loss of two molecules of H2O. The ion at m/z 321.2587 represented its side chain loss in MS2. Compound 58 was identified as UDCA.Ursodeoxycholic acid showed the ions at m/z 391.2872 [M-H]−, 437.2907[M+HCOO]−, and 783.5798 [2M-H]− in the negative ion mode. In the positive ion mode, the loss of H2O unit yielded [M+H-2H2O]+ ion at m/z 357.2809. The occurrence of the ion at m/z 321.2586 in MS2 was due to side chain loss. Compound 70 was identified as CDCA.CDCA showed the ions at 57 showed [M+Na]+ ion at m/z 431.2778 in positive mode, and [M-H]− ion at m/z 407.2803, [M+HCOO]− ion at m/z 453.2857, and [2M-H]− ion at m/z 815.5698 in the negative mode. The ions representing a series of H2O loss was observed, such as 373.2749 [M+H-2H2O]+ and 355.2649 [M+H-3H2O]+. It was tentatively characterized as cholanic acid.Compound 67 showed [M-H]− ion at m/z 391.2852, [M+HCOO]− ion at m/z 437.2903, and [2M-H]− at m/z 783.5792 in the full mass spectrum. The molecular formula was calculated as C24H40O4. It was tentatively deduced as hyodeoxycholic acid.CompoundQuinones derivatives. Quinones derivatives are another kind of active constituents from Danshen and they were easily detected in XXT sample. Compounds 63, 71, and 76 were identified as dihydrotanshinone, cryptotanshinone, and tanshinone II A by comparison with reference substances. They displayed similar fragmentation pathways concerning successive eliminations of H2O and CO molecules. Tanshinone IIA was used as an example to illustrate the fragmentation pathway of quinones constituents as shown in  31, 1-oxo tanshinone IIA 59, neotanshinone D 62, tetrahydrotanshinone I 66, methyltanshinonate 69, methylenetanshinquinone 73, and miltirone 77. + ion at m/z 207.1024. The above data were in accordance with those of 4-hydroxyl-3-butylphthalide in the literature [ 21 was tentatively assigned as 4-hydroxyl-3-butylphthalide. It could be derived from individual herb Chuanxiong rhizome [terature . Compoun rhizome .Among the identified compounds, most constituents were derived from the raw materials, Danshen and Renshen Jingye Zongzaogan, and a small proportion of compounds were considered from Huaihua, Maodongqing, Chuanxiong, Chansu, and Rengong Niuhuang. The other ingredients, Rengong Shexiang, Bingpian, and Shuizhi, were not characterized in the present HPLC and UPLC-QTOF/MS condition. This could be related to the prescription of raw materials and the manufacturing process employed. Usually, muscone, one of active constituents in Rengong Shexiang and borneol are detected by GC or GC-MS . They arIn this work, HPLC analysis was employed to find out the common chromatographic peak in various batches of XXT samples and UPLC-Q-TOF/MS was used for the identification of main constituents in the typical XXT sample. As a result, a total of 63 constituents including twenty saponins, four flavonoids, fifteen phenolic acids, eight steroids, four bile acids, ten quinones, and other two compounds were identified or tentatively characterized based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules and matching empirical molecular formula with those of published components. The present investigation clearly understood the nonvolatile constituents in XXT and provided good basis for further study on the active substances and quality control of this preparation.


Scientific Reports 10.1038/s41598-019-42959-4, published online 29 April 2019Correction to: In Figure 5 there is a missing axis label. The y-axis of the bottom graph should be labelled “Intensity (arb. units)” The correct Figure 5 appears below as Figure Additionally, this Article contains errors in Reference 44 which is incorrectly given as:Jpn. J. Appl. Phys. 56, 01AC06 (2016)Ito, T., Uchida, G., Nakajima, A., Takenaka, K. & Setsuhara, Y. Selective production of reactive oxygen and nitrogen species in the plasma-treated water by using a nonthermal high-frequency plasma jet. The correct reference is listed below as ref.


Decline in driving skills begins in preclinical AD, when an older adult remains cognitively normal, but the underlying disease process has begun. Preclinical AD is detectable among cognitively normal individuals using molecular biomarkers: positron emission tomography (PET) imaging and cerebrospinal fluid (CSF). The aim of this prospective, longitudinal study is to determine whether naturalistic driving behavior using in-vehicle dataloggers can distinguish older adults with (n=36) and without preclinical AD (n=134). Driving data was calculated as mean/month for several variables  for participants followed between one to 46 months. Using stepwise logistic regression, the area under the receiver operating curve (AUC) and 95% confidence interval for these five variables was 0.73 (0.63-0.79) in distinguishing those with and without preclinical AD via amyloid imaging. When age, gender, race, and education were added, the model improved: 0.80 (0.72-0.88). Finally, when apolipoprotein ε4 allele (APOε4), obtained via blood or saliva, was added to the model, accuracy improved: 0.84 (0.77-0.89). Similar results were found using CSF biomarker tau/Aβ42: AUCs (95% CI) were 0.68 (0.58-0.79) for driving variables alone, 0.77 (0.69-0.86) for driving variables and demographics, and 0.87 (0.80-0.94) driving variables, demographics, and apolipoprotein ε4 allele. These promising findings suggest that naturalistic driving behavior can predict those with and without preclinical AD. The AUC is further improved with demographics and APOε4, an easily obtainable genetic biomarker. This model may be used in clinical/research settings as a screen or adjunct for diagnostics and prognostics purposes.


Rhodococcus erythropolis X5 is a psychrotrophic (cold-adapted) hydrocarbon-degrading bacterium, as it showed effective n-alkane destruction at low positive temperatures. Here, the genome of strain X5 was completely sequenced; it consists of a 6,472,161-bp circular chromosome (62.25% GC content) and a 526,979-bp linear plasmid, pRhX5-526k (62.37% GC content). Rhodococcus erythropolis X5 is a psychrotrophic (cold-adapted) hydrocarbon-degrading bacterium, as it showed effective n-alkane destruction at low positive temperatures. Here, the genome of strain X5 was completely sequenced; it consists of a 6,472,161-bp circular chromosome (62.25% GC content) and a 526,979-bp linear plasmid, pRhX5-526k (62.37% GC content). Rhodococcus erythropolis X5 (VKM Ac-2532D) was isolated from oil-polluted soil (R. erythropolis X5 is a psychrotrophic (cold-adapted) hydrocarbon-degrading bacterium, as it showed effective n-alkane destruction at low positive temperatures . Sequencing was performed using a MinIon sequencer (Oxford Nanopore Technologies [ONT]) at the Center of Analytical and Genetic Engineering Research . A library was prepared with the ONT ligation sequencing kit . Guppy v3.2.4 software was used for base calling, which yielded a total of 301 Mbp, distributed in 33,708 reads. Reads with a Q score of >10 were used for further analysis. Additionally, the same DNA sample was sequenced with an Illumina MiSeq platform using a MiSeq reagent kit v3 (2 × 300 bp). A paired-end library for sequencing was prepared with the MuSeek library preparation kit . The Nanopore reads were assembled into 2 contigs using Canu assembler v1.8 (Genomic DNA was isolated from a fresh culture biomass (a colony) of R. erythropolis. For the average nucleotide identity (ANI) analysis, we used all available GenBank data on the R. erythropolis representatives and on Rhodococcus sp. strains. Average nucleotide identity parameter analysis revealed that the closest phylogenetic relatives of strain X5 are Rhodococcus sp. strain 008 (GenBank accession number CP012749), Rhodococcus sp. strain NJ-530 (CP034152), R. erythropolis CCM2595 (CP003761), and Rhodococcus sp. strain YL-1 (CP017299).The X5 genome consists of a 6,472,161-bp circular chromosome (62.25% GC content) and a 526,979-bp linear plasmid, pRhX5-526k (62.37% GC content). Chromosome circularization and plasmid linearity were specified by Canu. Also, there were no reads overlapping with plasmid ends. The strain was previously identified as R. erythropolis X5 bears 5 copies of alkane hydroxylase-encoding gene alkB. These genes are essential for the degradation process of alkanes with a chain length of C12 to C20  v4.6 , which i2 to C20 , 10. In 2 to C20 , 12. ApaCP044283 and CP044284, BioSample number SAMN12818508, BioProject number PRJNA573614, and SRA accession number PRJNA573614.This genome project has been deposited in the NCBI database under GenBank accession numbers


Elaeagnus macrophylla were analyzed via conserved DNA-derived polymorphism molecular markers. A total of 289 discernible loci were obtained from 102 individuals via fifteen primers, and 100% of the loci were polymorphic. The observed number of alleles was 1.9654, and the effective number of alleles was 1.2604. Nei’s genetic diversity index was 0.1724 on average, and Shannon’s information index was 0.2869, indicating that Elaeagnus macrophylla had lower levels of genetic diversity than those reported for its continental relatives and other continental species. The average percentage of polymorphic loci was 42.1%, and the maximum and minimum were 80.97% and 14.88%, respectively, which were associated with the Nanji Island and Liugong Island populations, respectively. The populations of Elaeagnus macrophylla were highly differentiated. Cluster analysis revealed that the similarity between the tested samples was related to their geographical location, that the samples from the same island tended to cluster together, and that there was no cross-clustering between samples. The Nanji Island and Da Rushan populations differentiated into two subpopulations. Last, we detected no correlation between genetic distance and geographic distance between populations .The genetic diversity and genetic structure of five natural populations of the island and coastal endangered plant species  Elaeagnus macrophylla is an endangered evergreen shrub species of East Asian coastal areas and islands. It is distributed in the Shandong, Zhejiang, and Jiangsu Provinces of China, mainly on offshore islands and in coastal lowlands  molecular markers (Neolitsea sericea based on random amplified polymorphic DNA (RAPD) molecular markers  have shown that CDDP markers have many advantages, including convenience, low cost, and rich polymorphism, and can effectively mark sequences of target traits (Solanum dulcamara (Chrysanthemum (Paeonia suffruticosa (Vaccinium vitis-idaea (Rosa rugosa (E. macrophylla.The conserved DNA-derived polymorphism (CDDP) method is based on a single primer amplification reaction, with primers designed to target conserved sequences of plant functional genes, mostly transcription factors such as WRKYs, MYBs, MADs, ERFs, KNOXs, and ABP1. Because of the strong conservation of some sequences of plant DNA, CDDP molecular marker technology can be used across different species. Studies of rice . Only one individual each was found on Lingshan Island and Putuo Island. After the samples were collected, silica gel was used to quickly dry the specimens, after which they were stored at 20 °C.A total of 102 individual leaf samples were collected from 7 islands and offshore sites  from AprE. macrophylla via the modified cetyltrimethylammonium bromide (CTAB) method  method . The qua2O, 1 µl of 30 ng/µl DNA template, and 1.0 µl of 10 pmol/µl primers . A standard PCR thermocycler  was used and the PCR program was as follows: an initial denaturation step at 94 °C for 3 min; 35 annealing cycles of 94  °C for 1 min, 50 °C for 1 min and 72 °C for 2 min; and a final extension of 72  °C for 5 min. The PCR products were subsequently stored at 4 °C. The products were then electrophoresed on a 2% agarose gel (110 V and 110 mA) for 1.5–2 h; a DL2000 marker was used as a size marker. The electrophoresis results were imaged and recorded by a gel imaging system. All amplification procedures were repeated at least twice to ensure the repeatability of the experiment.The DNA from one sample per population was selected to screen 21 CDDP primers  synthes. PCR wasWe used POPGEN v.1.32  to compuA dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA) clustering procedure in NTSYS-pc 2.10e software . The relK values) was set from 1 to 10, with 10 independent runs for each K. The contribution of the accessions to the genotypes was calculated on the basis of a 105 iteration burn-in period and 105 iteration sampling period. The optimal number of K clusters was then identified according to the methods of The genetic structure of populations was further assessed via the Bayesian clustering approach implemented in STRUCTURE v.2.3.4 . The numE. macrophylla was high.The DNA of 102 samples was amplified with 15 primers, yielding 289 bands, and the fragment length was between 500 and 2,000 bp . The numAt the population level, the PPL ranged from 14.88% to 80.28%, with an average of 48.928%, whereas it was 96.54% at the species level. The Na ranged from 1.1488 to 1.8028, while the Ne varied from 1.0739 to 1.2410. H varied from 0.0446 to 0.1580, with an average of 0.1149, and I ranged from 0.0690 to 0.2613, with an average of 0.1848. At the species level, H and I were 0.1724 and 0.2869, respectively . The Na,The Ht and Hs were 0.1706 and 0.1149, respectively, as calculated by POPGEN v.1.32 software. The Gst was 0.3263, indicating that 67.37% of the variation was within the populations and that 32.63% of the variation occurred between the populations. A certain degree of genetic differentiation was observed between the populations. The Nm was 1.0325, indicating that there was some  genetic exchange between populations.E. macrophylla, the genetic distance was between 0.0490 and 0.1443 .The applied measure of genetic similarity was used to construct UPGMA dendrograms . The cluThe UPGMA clustering map provided a clear division of the 102 samples . NotablyE. macrophylla best fit three genetic groups, and when K = 5, the delta K value was also large (K = 3 (K = 5 (K = 5: a northwestern group (indicated in yellow) and a southeastern group (indicated in blue). The DRS population differentiated into two subpopulations when K = 3 and K = 5: Nos. 9–18 and Nos. 19–28, respectively.The results of the Bayesian clustering analysis of the genetic structure showed that the populations of so large . When K e   (Elaeagnus mollis)   and Paeo 0.2682)  estimate mollis) , the geniversity . E. macrlination . The Ht is sweet . The frutinction . Island tinction .E. macrophylla were 0.1706 and 0.1149, respectively. Compared with endangered and Chinese secondary protected plants  studies   . This ge > 0.15) . Furtherulations .E. macrophylla displayed little gene flow (Nm = 1.0325), and the UPGMA clustering analysis of samples revealed no hybridization among individuals from different localities. Structure analysis (K = 5) revealed that most of the populations had a simple pedigree, and the genetic exchange between each pair of populations was low. These results were mainly due to the geographical isolation of the islands (mainly barriers posed by seawater), which limited the range of dispersal by pollen- and seed-dispersing birds ; it is difficult for small pollinators to spread pollen across islands separated by vast seas. In addition to pollen, seeds play an important role in the spread of gene flow. One important aspect of seed movement is its role in the initial founding of a population  of N. sericea were dispersed within a range of approximately 480 m (Hakdongri on Keojae Island) and 680 m (Naechori on Oenaro Island), respectively, from their maternal plants . After possible causes such as the prevention of gene flow were excluded, the reason may be related to differences in habitats within the population. The NJD nature reserve has numerous islands and reefs with meandering shorelines, headlands, and numerous bays. There are many types of coastal beaches, such as mudflats, gravel beaches, and rocky reefs, and NJD is at the intersection of the Taiwan Warm Current and the Jiangsu and Zhejiang Coastal Currents. The flow system is complex, so the habitat is complex  was significantly greater than that of island species with relatively small populations   NJD (0.1580)>DRS (0.1290)>DGD (0.1222)>LS (0.1208)>LGD (0.0446), with an average value of 0.1149. The average genetic diversity index of the NJD population is much greater than that of the LGD population. The values of the other three populations are relatively similar. The NJD population is the largest, the DRS, DGD, and LS populations are similar in size, and the LGD population is declining. Human disturbances such as excessive logging, habitat destruction, and the introduction of exotic species are considered to be the main causes endangering island species  . The ave species , which a species . NJD is E. macrophylla.Am in situ conservation method was proposed because the conservation of sufficient natural population numbers and sizes to prevent a reduction in genetic diversity is urgently needed. The best strategy for in situ conservation of genetic diversity during an endemic is the preservation of natural habitat . In thisElaeagnus macrophylla using conserved DNA-derived polymorphism markers to investigate the distribution and genetic variation. The results showed that conserved DNA-derived polymorphism markers can be effectively used to study the genetic diversity of Elaeagnus macrophylla populations and revealed that Elaeagnus macrophylla populations have low genetic diversity and high genetic differentiation. The low levels of gene flow between populations are the main cause of the high levels of genetic differentiation. On the basis of these findings, some conservation measures for Elaeagnus macrophylla are proposed.The present study is the first genetic investigation of 10.7717/peerj.8498/supp-1Supplemental Information 10-1 matrix obtained from the results of electropherogram.Click here for additional data file.10.7717/peerj.8498/supp-2Supplemental Information 2It can be seen that the three primer amplification bands are clear and specific and can be used for experiments.Click here for additional data file.10.7717/peerj.8498/supp-3Supplemental Information 3It can be seen that the MYB1, MYB2, ERF1 amplified bands are clear and specific and can be used for experiments.Click here for additional data file.10.7717/peerj.8498/supp-4Supplemental Information 4It can be seen that the WRKY-F1, WRKY-R1 amplified bands are clear and specific and can be used for experiments. Although the WRKY-R2 has a strip, the strip is weak and therefore not used.Click here for additional data file.10.7717/peerj.8498/supp-5Supplemental Information 5It can be seen that the WRKY-R3 amplified band is clear and specific, and can be used for experiments. WRKY-R2B, WRKY-R3B is not effective, so it is not used.Click here for additional data file.10.7717/peerj.8498/supp-6Supplemental Information 6It can be seen that KNOX-2, KNOX-3, MADS-1 amplified bands are clear and specific and can be used for experiments.Click here for additional data file.10.7717/peerj.8498/supp-7Supplemental Information 7It can be seen that the ABP1-1, ABP1-3 amplified bands are clear and specific and can be used for experiments. ABP1-2 did not amplify the band and therefore could not be used.Click here for additional data file.10.7717/peerj.8498/supp-8Supplemental Information 8It can be seen that the MADS-4 amplification band is clear and specific and can be used for experiments. Although the MADS-2 has a band, it is not effective in use, and the MADS-3 does not amplify the band, so it is not used.Click here for additional data file.10.7717/peerj.8498/supp-9Supplemental Information 9Amplification results of WRKY-F1 on LGD1-8 and DRS9-20.Click here for additional data file.10.7717/peerj.8498/supp-10Supplemental Information 10Amplification results of WRKY-F1 on DRS21-28, NJD1-12.Click here for additional data file.10.7717/peerj.8498/supp-11Supplemental Information 11Amplification results of WRKY-F1 on NJD13-NJD32.Click here for additional data file.10.7717/peerj.8498/supp-12Supplemental Information 12Amplification results of WRKY-F1 on NJD 33, LS 1-13, PTD1, LSD1.Click here for additional data file.10.7717/peerj.8498/supp-13Supplemental Information 13Amplification results of WRKY-F1 on DGD1-13.Click here for additional data file.10.7717/peerj.8498/supp-14Supplemental Information 14Amplification results of WRKY-F1 on DGD14-26.Click here for additional data file.10.7717/peerj.8498/supp-15Supplemental Information 15Amplification results of WRKY-R1 on LGD1-8 and DRS9-20.Click here for additional data file.10.7717/peerj.8498/supp-16Supplemental Information 16Amplification results of WRKY-R1 on DRS21-28, NJD1-12.Click here for additional data file.10.7717/peerj.8498/supp-17Supplemental Information 17Amplification results of WRKY-R1 on NJD13-32.Click here for additional data file.10.7717/peerj.8498/supp-18Supplemental Information 18Amplification results of WRKY-R1 on NJD 33, LS1-13, PTD1, LSD1.Click here for additional data file.10.7717/peerj.8498/supp-19Supplemental Information 19Amplification results of WRKY-R1 on DGD1-20.Click here for additional data file.10.7717/peerj.8498/supp-20Supplemental Information 20Amplification results of WRKY-R1 on DGD21-26.Click here for additional data file.10.7717/peerj.8498/supp-21Supplemental Information 21Amplification results of WRKY-R3 on LGD1-8, DRS9-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-22Supplemental Information 22Amplification results of WRKY-R3 on DRS21-28, NJD1-12 samples.Click here for additional data file.10.7717/peerj.8498/supp-23Supplemental Information 23Amplification results of WRKY-R3 on DRS21-28, NJD13-32 samples.Click here for additional data file.10.7717/peerj.8498/supp-24Supplemental Information 24Amplification results of WRKY-R3 on NJD33, LS1-13, PTD1, LSD1 samples.Click here for additional data file.10.7717/peerj.8498/supp-25Supplemental Information 25Amplification results of WRKY-R3 on DGD1-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-26Supplemental Information 26Amplification results of WRKY-R3 on DGD21-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-27Supplemental Information 27Amplification results of MYB1 on LGD1-8, DRS9-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-28Supplemental Information 28Amplification results of MYB1 on DRS21-28, NJD1-12 samples.Click here for additional data file.10.7717/peerj.8498/supp-29Supplemental Information 29Amplification results of MYB1 on NJD13-33 samples.Click here for additional data file.10.7717/peerj.8498/supp-30Supplemental Information 30Amplification results of MYB1 on NJD33, LS1-13, PTD1, LSD1 samples.Click here for additional data file.10.7717/peerj.8498/supp-31Supplemental Information 31Amplification results of MYB1 on DGD1-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-32Supplemental Information 32Amplification results of MYB1 on DGD21-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-33Supplemental Information 33Amplification results of MYB2 on LGD1-8, DRS9-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-34Supplemental Information 34Amplification results of MYB2 on DRS21-28, NJD1-12 samples.Click here for additional data file.10.7717/peerj.8498/supp-35Supplemental Information 35Amplification results of MYB2 on NJD13-32 samples.Click here for additional data file.10.7717/peerj.8498/supp-36Supplemental Information 36Amplification results of MYB2 on NJD33, LS1-13, PUD1, LSD1 samples.Click here for additional data file.10.7717/peerj.8498/supp-37Supplemental Information 37Amplification results of MYB2 on DGD1-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-38Supplemental Information 38Amplification results of MYB2 on DGD21-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-39Supplemental Information 39Amplification results of ERF1 on LGD1-8, DRS9-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-40Supplemental Information 40Amplification results of ERF1 on DRS21-28, NJD1-12 samples.Click here for additional data file.10.7717/peerj.8498/supp-41Supplemental Information 41Amplification results of ERF1 on NJD13-32 samples.Click here for additional data file.10.7717/peerj.8498/supp-42Supplemental Information 42Amplification results of ERF1 on NJD33, LS1-13, PTD1, LSD1 samples.Click here for additional data file.10.7717/peerj.8498/supp-43Supplemental Information 43Amplification results of ERF1 on DGD1-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-44Supplemental Information 44Amplification results of ERF1 on LS1-6, DGD21-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-45Supplemental Information 45Amplification results of ERF2 on LGD1-8, DRS9-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-46Supplemental Information 46Amplification results of ERF2 on DRS21-28, NJD1-12 samples.Click here for additional data file.10.7717/peerj.8498/supp-47Supplemental Information 47Amplification results of ERF2 on NJD13-32 samples.Click here for additional data file.10.7717/peerj.8498/supp-48Supplemental Information 48Amplification results of ERF2 on NJD33, LS1-13, PTD1, and LSD1 samples.Click here for additional data file.10.7717/peerj.8498/supp-49Supplemental Information 49Amplification results of ERF2 on DGD1-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-50Supplemental Information 50Amplification results of ERF2 on DGD21-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-51Supplemental Information 51Amplification results of ERF3 on LGD1-8, DRS9-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-52Supplemental Information 52Amplification results of ERF3 on DRS21-28, NJD1-12 samples.Click here for additional data file.10.7717/peerj.8498/supp-53Supplemental Information 53Amplification results of ERF3 on NJD13-32 samples.Click here for additional data file.10.7717/peerj.8498/supp-54Supplemental Information 54Amplification results of ERF3 on NJD33, LS1-13, PTU1, LSD1, and DGD24-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-55Supplemental Information 55Amplification results of ERF3 on DGD1-23 samples.Click here for additional data file.10.7717/peerj.8498/supp-56Supplemental Information 56Amplification results of ERF3 on DGD24-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-57Supplemental Information 57KNOX-1 amplification results for LGD1-8, DRS9-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-58Supplemental Information 58KNOX-1 amplification results for DRS21-28, NJD1-12 samples.Click here for additional data file.10.7717/peerj.8498/supp-59Supplemental Information 59KNOX-1 amplification results for NJD13-32 samples.Click here for additional data file.10.7717/peerj.8498/supp-60Supplemental Information 60KNOX-1 amplification results for NJD33, LS1-13, PUD1, LSD1 samples.Click here for additional data file.10.7717/peerj.8498/supp-61Supplemental Information 61KNOX-1 amplification results for DGD1-20 samples.Click here for additional data file.10.7717/peerj.8498/supp-62Supplemental Information 62KNOX-1 amplification results for DGD21-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-63Supplemental Information 63KNOX-2 amplification results for LGD1-8, DRS9-23 samples.Click here for additional data file.10.7717/peerj.8498/supp-64Supplemental Information 64KNOX-2 amplification results for DRS24-28, NJD1-18 samples.Click here for additional data file.10.7717/peerj.8498/supp-65Supplemental Information 65KNOX-2 amplification results for NJD19-33, LS1-6 samples.Click here for additional data file.10.7717/peerj.8498/supp-66Supplemental Information 66KNOX-2 amplification results for LS7-13, PTD1, LSD1and DGD1-13 samples.Click here for additional data file.10.7717/peerj.8498/supp-67Supplemental Information 67KNOX-2 amplification results for DGD14-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-68Supplemental Information 68KNOX-3 amplification results for LGD1-8, DRS9-23 samples.Click here for additional data file.10.7717/peerj.8498/supp-69Supplemental Information 69KNOX-3 amplification results for DRS24-28, NJD1-18 samples.Click here for additional data file.10.7717/peerj.8498/supp-70Supplemental Information 70KNOX-3 amplification results for NJD19-33, PTD1, LSD1, LS1-4 samples.Click here for additional data file.10.7717/peerj.8498/supp-71Supplemental Information 71KNOX-3 amplification results for LS5-13, DGD1-13 samples.Click here for additional data file.10.7717/peerj.8498/supp-72Supplemental Information 72KNOX-3 amplification results for DGD14-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-73Supplemental Information 73Amplification results of MADS-1 on LGD1-8, DRS9-23 samples.Click here for additional data file.10.7717/peerj.8498/supp-74Supplemental Information 74Amplification results of MADS-1 on DRS24-28, NJD1-18 samples.Click here for additional data file.10.7717/peerj.8498/supp-75Supplemental Information 75Amplification results of MADS-1 on NJD19-NJD33, PTD1, LSD1, LS1-4 samples.Click here for additional data file.10.7717/peerj.8498/supp-76Supplemental Information 76Amplification results of MADS-1 on LS5-13, DGD1-13 samples.Click here for additional data file.10.7717/peerj.8498/supp-77Supplemental Information 77Amplification results of MADS-1 on DGD14-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-78Supplemental Information 78Amplification results of MADS-4 on LGD1-8 and DRS9-23 samples.Click here for additional data file.10.7717/peerj.8498/supp-79Supplemental Information 79Amplification results of MADS-4 on DRS24-28, NJD1-18 samples.Click here for additional data file.10.7717/peerj.8498/supp-80Supplemental Information 80Amplification results of MADS-4 on NJD19-33, PUD1, LSD1, LS1-4 samples.Click here for additional data file.10.7717/peerj.8498/supp-81Supplemental Information 81Amplification results of MADS-4 on LS5-13, DGD1-13 samples.Click here for additional data file.10.7717/peerj.8498/supp-82Supplemental Information 82Amplification results of MADS-4 on DGD14-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-83Supplemental Information 83ABP1-1 amplification results for LGD1-8, DRS9-23 samples.Click here for additional data file.10.7717/peerj.8498/supp-84Supplemental Information 84ABP1-1 amplification results for DRS24-28, NJD1-18 samples.Click here for additional data file.10.7717/peerj.8498/supp-85Supplemental Information 85ABP1-1 amplification results for NJD19-33, LS1-4 samples.Click here for additional data file.10.7717/peerj.8498/supp-86Supplemental Information 86ABP1-1 amplification results for LS5-13, DGD1-13 samples.Click here for additional data file.10.7717/peerj.8498/supp-87Supplemental Information 87ABP1-1 amplification results for DGD14-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-88Supplemental Information 88ABP1-3 amplification results for LGD1-8, DRS9-23 samples.Click here for additional data file.10.7717/peerj.8498/supp-89Supplemental Information 89ABP1-3 amplification results of DRS24-28, NJD1-18 samples.Click here for additional data file.10.7717/peerj.8498/supp-90Supplemental Information 90ABP1-3 amplification results for NJD19-33, PTD1, LSD1, LS1-4 samples.Click here for additional data file.10.7717/peerj.8498/supp-91Supplemental Information 91ABP1-3 amplification results for LS5-13, DGD1-13 samples.Click here for additional data file.10.7717/peerj.8498/supp-92Supplemental Information 92ABP1-3 amplification results for DGD14-26 samples.Click here for additional data file.10.7717/peerj.8498/supp-93Supplemental Information 93When K=3, the value of delat K is the largest and best fit three genetic groups. When K=5, the value of delat K is also large, so we analyze the cases of K=3 and K=5.Click here for additional data file.10.7717/peerj.8498/supp-94Supplemental Information 94Nm represents the gene flow, and Gst represents the genetic differentiation coefficient.Click here for additional data file.10.7717/peerj.8498/supp-95Supplemental Information 95Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-96Supplemental Information 96Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-97Supplemental Information 97Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-98Supplemental Information 98Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-99Supplemental Information 99Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-100Supplemental Information 100Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-101Supplemental Information 101Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-102Supplemental Information 102Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-103Supplemental Information 103Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-104Supplemental Information 104Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-105Supplemental Information 105Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-106Supplemental Information 106Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-107Supplemental Information 107Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-108Supplemental Information 108Two repetitions per sample.Click here for additional data file.10.7717/peerj.8498/supp-109Supplemental Information 109Raw data format when generating a sample cluster map.Click here for additional data file.10.7717/peerj.8498/supp-110Supplemental Information 110Generate raw data for population clustering analysis.Click here for additional data file.


Xanthomonas sp. strain ATCC PTA-13101, which was isolated from rice. The 44-kbp Pagan genome contains direct terminal repeats and contains 59 genes, 27 of which have a predicted function. Pagan is most closely related to Xanthomonas phage phi Xc10 and Xylella phage Prado.The T7-like podophage Pagan infects  Xanthomonas sp. strain ATCC PTA-13101, which was isolated from rice. The 44-kbp Pagan genome contains direct terminal repeats and contains 59 genes, 27 of which have a predicted function. Pagan is most closely related to Xanthomonas phage phi Xc10 and Xylella phage Prado.The T7-like podophage Pagan infects  Xanthomonas consists of many diverse species of Gram-negative, rod-shaped phytopathogenic gammaproteobacteria , and phages were propagated via the soft agar overlay method described previously (www.bioinformatics.babraham.ac.uk/projects/fastqc). Sequence reads were then trimmed using the FASTX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/). The genome was assembled through SPAdes v3.5.0 (using default parameters) with a sequencing coverage of 6.3-fold  , mixed freshwater collected in Vidor, TX, and infects the rice-associated eviously , 7. Full6.3-fold . Next, txy-pub/) . Structuxy-pub/) –14. Geney-pub/) ––17. Like-pub/) ––, 19. Rho-pub/) ––. progres-pub/) ––. Phage m-pub/) ––.Xanthomonas genome average of 64% G+C content (Xanthomonas phage phi Xc10 (GenBank accession number MF375456) and Xylella phage Prado (accession number KF626667), with 93.46% and 69.55% nucleotide sequence identity and 53 and 48 shared proteins, respectively (The 44,448-bp podophage Pagan genome has a coding density of 96.95% and contains 59 predicted genes but no tRNAs. The genome of Pagan displayed a G+C content of 62.3%, near the host  content . Pagan iectively . These sMK903278, BioProject accession number PRJNA222858, SRA accession number SRR8892144, and BioSample accession number SAMN11408680.The genome sequence and associated data for phage Pagan were deposited under GenBank accession number


Klebsiella pneumoniae, a Gram-negative pathogen whose multidrug-resistant strains are a public health issue. Here, we describe the annotation of the 157,741-bp Magnus genome and its similarity to other myophages.Bacteriophage Magnus infects  Klebsiella pneumoniae, a Gram-negative pathogen whose multidrug-resistant strains are a public health issue. Here, we describe the annotation of the 157,741-bp Magnus genome and its similarity to other myophages.Bacteriophage Magnus infects  Klebsiella pneumoniae is an opportunistic, Gram-negative enteric bacterium. Carbapenemase-producing K. pneumoniae (KPC) strains are resistant to carbapenem antibiotics and are an emerging cause of nosocomial and systemic human infections (fections . Phage tK. pneumoniae strain 39427 (BioSample number SAMN06218024) (www.bioinformatics.babraham.ac.uk/projects/fastqc). Quality-controlled reads were trimmed using the FASTX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/) and then assembled into a single contig of circular assembly at 146-fold coverage using SPAdes v3.5.0   collected at a wastewater treatment facility in Austin, TX, against  v3.5.0 68. Termin v3.5.0 6 and BLAS v3.5.0 6, 12. Bioxy-pub/) , 14. Unlxy-pub/) .Magnus is a myophage that has a 157,741-bp genome and a coding density of 92%, with 212 predicted protein-coding genes, 71 of which have a predicted function. The Magnus genome also contains 5 tRNA genes . Its G+C content was determined to be 46.3%. It was predicted by PhageTerm that Magnus uses a headful packaging system .Klebsiella phage 0507KN21 (GenBank accession number AB797215) (Serratia phage vB_Sru_IME250 (GenBank accession number KY073123) and Shigella phage Ag3 (GenBank accession number FJ373894), all classified within the Ackermannviridae. Additionally, Magnus contains at least 27 genes with significant amino acid similarity to type phages T4 (GenBank accession number NC_000866) and K (GenBank accession number KF766114), including core structural and replication proteins. The large terminase gene (NCBI accession number QEG07939) harbors an intein element.By BLASTp and progressiveMauve analyses, Magnus has 75.15% nucleotide identity to, and shares 146 similar proteins with, B797215) . Magnus MN045230, BioProject accession number PRJNA222858, SRA accession number SRR8869229, and BioSample accession number SAMN11360383.The genome sequence and associated data for phage Magnus were deposited under GenBank accession number


Psychrobacter spp. Analysis of Arctic psychrophilic Psychrobacter sp. DAB_AL32B genome content provided an insight into its overall stress response, and genes conferring protection against various life-limiting factors  were recognized and described. Moreover, it was revealed that the strain carries a large plasmid pP32BP2. Its replication system was used for the construction of two novel shuttle vectors (pPS-NR—Psychrobacter-Escherichia coli-specific plasmid and pPS-BR—Psychrobacter-various Proteobacteria-specific plasmid) of an increased carrying capacity, which may be used for genetic engineering of Psychrobacter spp.Cold-active bacteria are currently of great interest in biotechnology, and their genomic and physiological features have been extensively studied. One of the model psychrotolerant bacteria are The online version of this article (10.1007/s00203-018-1595-y) contains supplementary material, which is available to authorized users. Psychrobacter spp. belong to the Moraxellaceae family (Gammaproteobacteria). Bacteria of this genus are frequently isolated from various cold environments, including seawater, ice, permafrost and Arctic and Antarctic ornithogenic soils. Some strains  are considered to be opportunistic pathogens, as they are occasionally isolated from human patients, as well as from infected animals , 77 genomes (including 65 drafts) and an additional 65 plasmid sequences of Psychrobacter spp. are available (22nd June 2018). Analysis of the Psychrobacter genomes revealed the presence of various adaptation mechanisms allowing their survival in extremely cold environments. Hence, psychrophilic Psychrobacter strains are model, cold-active bacteria useful for studying bacterial adaptation to extreme conditions  a pABW1 vector containing a ColE1-type replication system. Therefore, the broad host range vector pBBR1 MCS-2 with pBBR1-type replication system was tested. Surprisingly, this replication system was also found to be inactive in tested Psychrobacter spp. These negative results encouraged us to construct novel, shuttle Psychrobacter–Escherichia coli-specific vectors.It was previously reported that plasmids carrying ColE1- and p15a-type replication systems were stably maintained in E. coli-specific replication system originating from the pMB1 plasmid, i.e., high copy number replicon) or pWSK29  plasmids, and carrying replication systems of the pP43BP3 and pP43BP4 plasmids originating from psychrophilic Psychrobacter sp. DAB_AL43B, were reported , based on the pBGS18  was obtained. The genome analyses provided brief insights into the stress adaptation mechanisms of the strain. Moreover, we found that the strain carries a large, approximately 60-kb, plasmid. Although this plasmid was not fully assembled during genome drafting, its replication system was used for the construction of two novel Psychrobacter-specific vectors that are suitable for cloning of large DNA fragments.In this study, the draft genome sequence of Psychrobacter spp.) or 37 °C (Escherichia coli DH5α). The medium was solidified by the addition of 1.5% (w/v) agar. Where necessary, the media were supplemented with X-gal, IPTG and antibiotics: kanamycin (20 µg/ml for Psychrobacter spp. or 50 µg/ml for E. coli) and rifampin (50 µg/ml).The bacterial strains and plasmids used in this study are listed in Table The CTAB/lysozyme method was used for isolation of genomic DNA .ACCGGTGCGAACCACTGTGAGTATTG-3′ and 32B_REPR—5′-ATACCGGTTTAATTCTATCGCCCGCCTG-3′. To obtain the PCR product, the following conditions were applied: 35 cycles  preceded by 3-min denaturation at 95 °C and followed by 2-min extension at 72 °C.Plasmid DNA was isolated using a GeneMATRIX Plasmid Miniprep DNA Purification Kit  and alkaline lysis method  was specified as taxon separating from other bacterial taxa.Genome completeness was assessed by the presence/absence of bacterial orthologs according to the OrthoDB database using BUSCO  available at the Ribosomal Database Project (RDP) website (Nawrocki and Eddy P. adeliensis DSM 15333T (HE654007.1), P. aestuarii SC35 (EU939718.1), P. alimentarius JG-100 (AY513645.1), P. allis E2 (JX122558.1), P. aquaticus CMS 56 (NR_042206.1), P. aquimaris SW-210 (AY722804.1), P. arcticus 273-4 (NR_075054.1), P. arenosus R7T (AJ609273.1), P. celer SW-238 (NR_043225.1), P. ciconiae 176/10 (KM486054.1), P. cryohalolentis K5 (NR_075055.1), P. faecalis DSM 14664 (NR_118025.1), P. fjordensis BSw21516B (GQ358940.1), P. fozii NF23 (NR_025531.1), P. frigidicola DSM 12411 (NR_042222.1), P. fulvigenes KC 40 (NR_041688.1), P. glacialis DD43 (AJ539102.1), P. glaciei BIc20019 (NR_148850.1), P. glacincola DSM 12194 (NR_042076.1), P. halophilus DD2 (AJ539103.1), P. immobilis ATCC 43116 (NR_118808.1), P. jeotgali YKJ-103 (NR_025205.1), P. luti NF11 (NR_025532.1), P. lutiphocae IMMIB L-1110 (NR_044602.1), P. marincola KMM 277 (NR_025458.1), P. maritimus Pi2-20 (NR_027225.1), P. meningitidis SBA4 (KR091838.1), P. muriicola 2pS (NR_114669.1), P. namhaensis SW-242 (AY722805.1), P. nivimaris 88/2–7 (AJ313425.1), P. oceani 4k5 (AB910522.1), P. okhotskensis MD17 (NR_024806.1), P. pacificensis NBRC 103191 (NR_114238.1), P. pasteuri CIP110853 (KY292376.1), P. phenylpyruvicus ATCC 23333 (NR_118815.1), P. piechaudii CIP110854 (KY292375.1), P. piscatorii T-3-2 (NR_112807.1), P. piscidermidis 45 (FJ613616.1), P. pocilloporae S6-60 (KT444699.2), P. proteolyticus HAMBI 2948 (LT899990.1), P. psychrophilus BBDP29 (DQ337513.1), P. pulmonis CCUG 46240 (NR_118026.1), P. salsus DD 48 (NR_042166.1), P. sanguinis 13983 (HM212668.1), P. submarinus KMM 225 (AJ309940.1), P. urativorans DSM 14009T (AJ609555.1), P. vallis CMS 39 (AJ584832.1), P. cibarius JG-219 (AY639871.1), Psychrobacter sp. DAB_AL32B (JF714884.1) and Psychrobacter sp. DAB_AL43B (JF714885.1).Phylogenetic analysis was performed based on the comparison of partial 16S rRNA gene sequences of the DAB_AL32B strain, DAB_AL43B strain and strains representing 48 Psychrobacter sp. DAB_AL32B draft genome sequence has been deposited in the GenBank (NCBI) database under accession numbers NEXU01000001-NEXU01000218 and the sequences of vectors constructed in this study under accession numbers MH539767 (pPS-BR) and MH539768 (pPS-NR).The Psychrobacter spp. genomes available in the NCBI database (the average length of the Psychrobacter genome is 3.07 Mb). An assembly of the DAB_AL32B draft genome resulted in 37 scaffolds composed of 218 contigs. The average GC content is 41.9%, which is typical for Psychrobacter spp. . An automatic annotation of the DAB_AL32B genome performed applying the NCBI Prokaryotic Genome Annotation Pipeline resulted in 2,799 predicted genes with an average length of 973.43 bp, which covers about 80.08% of the genome. Additionally, 41 tRNA genes were identified .The estimated size of the DAB_AL32B genome is 3,211,529 bp, which is similar to the size of other Gammaproteobacteria BUSCO set (containing 452 BUSCO groups), was performed. The gene set predicted within the DAB_AL32B genome contains 94.3% of genes present in an applied BUSCO set (92.5% complete and 1.8% fragmented genes), which suggests that the genome assembly is complete or very close to completeness.To estimate the completeness of the obtained draft genome, a BUSCO analysis, using Psychrobacter genomes, with only short fragments homologous to genomes of phylogenetically closely related Moraxella and Acinetobacter. Other two contigs (contig00048 and contig00081) exhibit similarity to Psychrobacter genomes in majority of their fragments, while the remaining fragments are either homologous to Acinetobacter, Moraxella, and other unrelated bacteria, or not homologous to any sequence in the nr database. However, BLASTx search revealed significant similarity to proteins encoded within most of these fragments to Psychrobacter and Acinetobacter proteins. For three other contigs  the best BLAST hits were sequences from either Acinetobacter or Moraxella; however, they showed comparable similarity to Psychrobacter genomes. One contig (contig00082) was mistakenly automatically classified as a contaminant, because of a non-significant hit to zebrafish genome in low-complexity region. As a result of above analysis, these 15 contigs were recategorized as non-contaminant sequences. Seven other contigs  exhibited no significant homology to sequences available in the GenBank database , therefore it is unclear if these are contaminants or not. Finally, only nine contigs   may be considered as potential contaminants, as they do not show similarities to neither Psychrobacter sequences nor closely related bacteria.To examine if obtained draft genome is contaminated with other genomic sequences, an analysis using Taxoblast was performed . This analysis revealed that 30 out of 218 contigs were classified as possible contaminants or did not exhibit significant homology to sequences available in the GenBank database. These contigs were manually examined. Putative prophage was found within contig00038. Seven contigs  mostly exhibited homology to Psychrobacter frigidicola DSM 12411. As several new Psychrobacter species have been described since the last analysis, we performed another analysis with dataset extended on 14 new Psychrobacter-type strains  in NCBI database . Unfortunately, the genome of P. frigidicola DSM 12411 or some other representative of this species, is not publicly available. The phylogenomic analysis revealed that the DAB_AL32B strain is most related with Psychrobacter sp. DAB_AL43B . However, to predict if these strains may be considered as representatives of novel Psychrobacter species, the calculation of the ANI value with P. frigidicola is needed.For more precise classification of DAB_AL32B at the species level, the ANI analysis, based on whole-genome comparison between various Psychrobacter sp. DAB_AL32B had to cope not only with low temperatures, but also increased ultraviolet (UV) radiation, osmotic pressure and oxidative stress. The genes involved in the response to oxidative, osmotic, and cold shock encoded within the DAB_AL32B genome are listed in Table Various adaptive mechanisms enable survival of psychrophilic bacteria in Arctic. Identification and analysis of genes encoding various stress response proteins of psychrophiles is important for studying their biology and adaptation, as well as for proper recognition of their biotechnological potential , lipids cross-linking with proteins and disturbances to membrane structure affecting its fluidity are observed  formed in the cell. Increased ROS formation in cells may occur as a result of (i) depletion of the UV-protective ozone layer in the Arctic region , peroxidase (1), superoxide dismutase (1), peroxiredoxin (2), rubredoxin (1) and glutaredoxins (3) Table .2+). Hydroxyl radicals are then produced via Fenton reaction (Touati 2+ ions) and formation of very stable complexes with DNA , decreasing gene expression and membrane fluidity  from the environment or synthesise them in elevated quantities. Compatible solutes increase the stability of proteins and cell membranes without interfering with cellular function , cryptic plasmid pP32BP1  within the N-terminal part of this protein and winged helix DNA-binding domain (residues 1–136 and 140–233). Upstream to the repA gene, the putative origin of replication (oriV) was found . It is composed of five 20-bp-long direct repeats DR1.1-1.5 (5′-ATAACACTAAATGATGTGGG-3′) followed by a pair of 19-bp long inverted repeats, IR1 and IR2   of pP32BP2 shows 76% identity with RepB protein (GenBank: AFD62164) of pP62BP1 of parS, were found. ParA contains the ParA domain (residues 4-182) and shows 93% identity to ParA-like protein (GenBank: ABE76262) encoded within the plasmid 1 of Psychrobacter cryohalolentis K5. Within the ParB protein, the ParG domain, specific to the ParB-like proteins of, e.g., Salmonella enterica plasmid TP228 and Pseudomonas alcaligenes plasmid pRA2, was found (Hayes Psychrobacter urativorans R10.10B. The putative parS centomer-like sequence is located upstream to the parA gene. It consists of eight direct repeats DR2.1-2.8 (5′-(A/T)ATACTCA-3′) , and centromer-like sequence nd Hayes . The prePsychrobacter-specific cloning vectors. Currently, all available Psychrobacter-specific vectors are based on replication systems of small (not exceeding 6.4 kb) plasmids, which may exclude their application as molecular tools for cloning of large genetic modules. Since Psychrobacter spp. recently became a model bacterium representing cold-active microorganisms  plasmid pP32BP2, encouraged us to use this system for the construction of novel Psychrobacter spp., were constructed . Its functionality was confirmed by introducing it  into two Psychrobacter spp. strains, DAB_AL12R and DAB_AL43BR. Vector stability was tested in both these strains, and after approximately 30 generations of growth without antibiotic selection pressure, plasmid pPS-NR was found in 21% and 61% of the host cells, respectively . The vector was successfully introduced via triparental mating into Psychrobacter sp. DAB_AL12R and DAB_AL43BR. To analyse the carrying capacity of the pPS-BR vector, we cloned and successfully introduced to Psychrobacter spp. two relatively large restriction fragments of the DAB_AL32B genome, of a length of 5.8 kb (approx. the size of the vector) and 12.7 kb (approx. two times bigger than the vector), respectively.For the construction of this vector, the PCR-amplified (1.8-kb) DNA fragment containing the replication system of the pP32BP2 plasmid was cloned within PfoI site of the pBBR1 MCS-2 vector. This resulted in construction of a unique Psychrobacter spp. and E. coli (pPS-NR) or various Proteobacteria (pPS-BR), (iii) the system enabling mobilization to conjugal transfer, (iv) aphA gene-conferring resistance to kanamycin, (v) multiple cloning site (MCS) and (vi) a selection marker (lacZ’ gene) enabling blue-white screening of clones in E. coli  two replication systems, which enable their replication in oli Fig. . The obtBelow is the link to the electronic supplementary material.Supplementary material 1 (DOCX 308 KB)Supplementary material 2 (DOCX 50 KB)Supplementary material 3 (DOCX 31 KB)