Journal articles: 'Multilocus DNA analyses'

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Koehler, Andreas, Helge Karch, Thomas Beikler, Thomas F. Flemmig, Sebastian Suerbaum, and Herbert Schmidt. "Multilocus sequence analysis of Porphyromonas gingivalis indicates frequent recombination." Microbiology 149, no. 9 (September 1, 2003): 2407–15. http://dx.doi.org/10.1099/mic.0.26267-0.

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In this study, the genetic relationship of 19 Porphyromonas gingivalis isolates from patients with periodontitis was investigated by multilocus sequence analysis. Internal 400–600 bp DNA fragments of the 10 chromosomal genes ef-tu, ftsQ, hagB, gpdxJ, pepO, mcmA, dnaK, recA, pga and nah were amplified by PCR and sequenced. No two isolates were identical at all 10 loci. Phylogenetic analyses indicated a panmictic population structure of P. gingivalis. Split decomposition analysis, calculation of homoplasy ratios and analyses of clustered polymorphisms all indicate that recombination plays a major role in creating the genetic heterogeneity of P. gingivalis. A standardized index of association of 0·0898 indicates that the P. gingivalis genes analysed are close to linkage equilibrium.

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Murphy, Nicholas P., Rachael A. King, and Steven Delean. "Species, ESUs or populations? Delimiting and describing morphologically cryptic diversity in Australian desert spring amphipods." Invertebrate Systematics 29, no. 5 (2015): 457. http://dx.doi.org/10.1071/is14036.

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Cryptic species are frequently being discovered in refugial habitats, such as desert springs and groundwater systems. Unfortunately, many of these taxa remain as unnamed entities years after their initial discovery. Recent advances in the use of molecular data and coalescent analyses allow DNA-based delimitation of species to move from single locus, tree-based methods to multilocus coalescent analyses. This study compares two DNA-based approaches to delimit species of putatively cryptic freshwater amphipods (Chiltoniidae) from desert springs in central Australia. In addition, a morphometric analysis of 11 characters was undertaken to determine whether the DNA-delimited species were morphologically distinguishable. The single locus method results in identification of lineages that are not supported as species under the multilocus coalescent analyses. We conclude that Wangiannachiltonia guzikae King, 2009, as currently circumscribed, represents six genetically distinct amphipod species, and we describe and name these species despite no clear diagnosable morphological differences. Critically, all of these newly recognised species have extremely limited distributions, which increases the biodiversity significance of their desert spring habitat.

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Zeng, Nian-Kai, Li-Ping Tang, Yan-Chun Li, Bau Tolgor, Xue-Tai Zhu, Qi Zhao, and Zhu L. Yang. "The genus Phylloporus (Boletaceae, Boletales) from China: morphological and multilocus DNA sequence analyses." Fungal Diversity 58, no. 1 (July 29, 2012): 73–101. http://dx.doi.org/10.1007/s13225-012-0184-7.

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O'Donnell, Kerry, Stacy Sink, María Mercedes Scandiani, Alicia Luque, Analía Colletto, Marisa Biasoli, Lisandro Lenzi, et al. "Soybean Sudden Death Syndrome Species Diversity Within North and South America Revealed by Multilocus Genotyping." Phytopathology® 100, no. 1 (January 2010): 58–71. http://dx.doi.org/10.1094/phyto-100-1-0058.

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Sudden death syndrome (SDS) of soybean has become a serious constraint to the production of this crop in North and South America. Phenotypic and multilocus molecular phylogenetic analyses, as well as pathogenicity experiments, have demonstrated that four morphologically and phylogenetically distinct fusaria can induce soybean SDS. Published molecular diagnostic assays for the detection and identification of these pathogens have reported these pathogens as F. solani, F. solani f. sp. glycines, or F. solani f. sp. phaseoli, primarily because the species limits of these four pathogens were only recently resolved. In light of the recent discovery that soybean SDS and Phaseolus and mung bean root rot (BRR) are caused by four and two distinct species, respectively, multilocus DNA sequence analyses were conducted to assess whether any of the published molecular diagnostic assays were species-specific. Comparative DNA sequence analyses of the soybean SDS and BRR pathogens revealed that highly conserved regions of three loci were used in the design of these assays, and therefore none were species-specific based on our current understanding of species limits within the SDS–BRR clade. Prompted by this finding, we developed a high-throughput multilocus genotyping (MLGT) assay which accurately differentiated the soybean SDS and two closely related Phaseolus and mung BRR pathogens based on nucleotide polymorphism within the nuclear ribosomal intergenic spacer region rDNA and two anonymous intergenic regions designated locus 51 and 96. The single-well diagnostic assay, employing flow cytometry and a novel fluorescent microsphere array, was validated by independent multilocus molecular phylogenetic analysis of a 65 isolate design panel. The MLGT assay was used to reproducibly type a total of 262 soybean SDS and 9 BRR pathogens. The validated MLGT array provides a unique molecular diagnostic for the accurate identification and molecular surveillance of these economically important plant pathogens.

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Huang, Chien-Hsun, Chih-Chieh Chen, Jong-Shian Liou, Ai-Yun Lee, Jochen Blom, Yu-Chun Lin, Lina Huang, and Koichi Watanabe. "Genome-based reclassification of Lactobacillus casei: emended classification and description of the species Lactobacillus zeae." International Journal of Systematic and Evolutionary Microbiology 70, no. 6 (June 1, 2020): 3755–62. http://dx.doi.org/10.1099/ijsem.0.003969.

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Taxonomic relationships between Lactobacillus casei , Lactobacillus paracasei and Lactobacillus zeae have long been debated. Results of previous analyses have shown that overall genome relatedness indices (such as average nucleotide identity and core nucleotide identity) between the type strains L. casei ATCC 393T and L. zeae ATCC 15820T were 94.6 and 95.3 %, respectively, which are borderline for species definition. However, the digital DNA‒DNA hybridization value was 57.3 %, which was clearly lower than the species delineation threshold of 70 %, and hence raised the possibility that L. casei could be reclassified into two species. To re-evaluate the taxonomic relationship of these taxa, multilocus sequence analysis (MLSA) based on the concatenated five housekeeping gene (dnaJ, dnaK, mutL, pheS and yycH) sequences, phylogenomic and core genome multilocus sequence typing analyses, gene presence and absence profiles using pan-genome analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling analysis, cellular fatty acid compositions, and phenotype analysis were carried out. The results of phenotypic characterization, MLSA, whole-genome sequence-based analyses and MALDI-TOF MS profiling justified an independent species designation for the L. zeae strains, and supported an emended the description of the name of Lactobacillus zeae (ex Kuznetsov 1956) Dicks et al. 1996, with ATCC 15820T (=DSM 20178T=BCRC 17942T) as the type strain.

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Fang, Wei, Min-wei Guo, Zhi-yong Ruan, Han Xue, Lai-fa Wang, Guo-zhong Tian, Chun-gen Piao, and Yong Li. "Multilocus sequence analysis of the genus Kurthia, and a description of Kurthia populi sp. nov." International Journal of Systematic and Evolutionary Microbiology 65, Pt_11 (November 1, 2015): 3788–93. http://dx.doi.org/10.1099/ijsem.0.000494.

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Four novel bacterial strains belonging to the genus Kurthia were isolated from the surface of a weevil of the family Curculionidae (strain 10y-14T), and from bark samples of hybrid poplar, Populus × euramericana (strains 6-3, 2-5 and 06C10-3-14), in Puyang, Henan Province, China. Phylogenetic analyses of the 16S rRNA gene and multilocus sequence analysis (MLSA) data showed that the four strains form a distinct cluster in the genus Kurthia, indicating that they all belong to a single taxon within the genus. DNA–DNA hybridization levels between strain 10y-4T and Kurthia huakuii LAM0618T and Kurthia massiliensis DSM 24639T were 58.31 and 53.92 %, respectively. This indicates that the four novel strains represent a species distinct from these two closely related species. The DNA G+C content of the novel strains was 42.1–42.6 %. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0.The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown phospholipid and unidentified aminophospholipids. The predominant menaquinones were MK-7 (90 %) and MK-6 (10 %). The major cell-wall amino acids were lysine, alanine, glutamic acid and glycine. On the basis of the MLSA and 16S rRNA gene sequence phylogenetic analyses, DNA–DNA reassociation values, DNA base composition, and biochemical and phenotypic characteristics, the four strains are considered to represent a novel species within the genus Kurthia, for which the name Kurthia populi sp. nov. is proposed. The type strain is 10y-14T ( = CFCC 11600T = KCTC 33522T).

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Thompson, Cristiane C., Vanessa E. Emmel, Erica L. Fonseca, Michel A. Marin, and Ana Carolina P. Vicente. "Streptococcal taxonomy based on genome sequence analyses." F1000Research 2 (March 1, 2013): 67. http://dx.doi.org/10.12688/f1000research.2-67.v1.

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The identification of the clinically relevant viridans streptococci group, at species level, is still problematic. The aim of this study was to extract taxonomic information from the complete genome sequences of 67 streptococci, comprising 19 species, by means of genomic analyses, multilocus sequence analysis (MLSA), average amino acid identity (AAI), genomic signatures, genome-to-genome distances (GGD) and codon usage bias. We then attempted to determine the usefulness of these genomic tools for species identification in streptococci. Our results showed that MLSA, AAI and GGD analyses are robust markers to identify streptococci at the species level, for instance,S. pneumoniae,S. mitis, andS. oralis. AStreptococcusspecies can be defined as a group of strains that share ≥ 95% DNA similarity in MLSA and AAI, and > 70% DNA identity in GGD. This approach allows an advanced understanding of bacterial diversity.

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GUPTA, BHAVNA, NALINI SRIVASTAVA, and APARUP DAS. "Inferring the evolutionary history of IndianPlasmodium vivaxfrom population genetic analyses of multilocus nuclear DNA fragments." Molecular Ecology 21, no. 7 (February 21, 2012): 1597–616. http://dx.doi.org/10.1111/j.1365-294x.2012.05480.x.

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Zarza, Eugenia, Elizabeth M. Connors, James M. Maley, Whitney L. E. Tsai, Peter Heimes, Moises Kaplan, and John E. McCormack. "Combining ultraconserved elements and mtDNA data to uncover lineage diversity in a Mexican highland frog (Sarcohyla; Hylidae)." PeerJ 6 (December 11, 2018): e6045. http://dx.doi.org/10.7717/peerj.6045.

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Molecular studies have uncovered significant diversity in the Mexican Highlands, leading to the description of many new endemic species. DNA approaches to this kind of species discovery have included both mitochondrial DNA (mtDNA) sequencing and multilocus genomic methods. While these marker types have often been pitted against one another, there are benefits to deploying them together, as linked mtDNA data can provide the bridge between uncovering lineages through rigorous multilocus genomic analysis and identifying lineages through comparison to existing mtDNA databases. Here, we apply one class of multilocus genomic marker, ultraconserved elements (UCEs), and linked mtDNA data to a species complex of frogs (Sarcohyla bistincta, Hylidae) found in the Mexican Highlands. We generated data from 1,891 UCEs, which contained 1,742 informative SNPs for S. bistincta and closely related species and captured mitochondrial genomes for most samples. Genetic analyses based on both whole loci and SNPs agree there are six to seven distinct lineages within what is currently described as S. bistincta. Phylogenies from UCEs and mtDNA mostly agreed in their topologies, and the few differences suggested a more complex evolutionary history of the mtDNA marker. Our study demonstrates that the Mexican Highlands still hold substantial undescribed diversity, making their conservation a particularly urgent goal. The Trans-Mexican Volcanic Range stands out as a significant geographic feature in Sarcohyla and may have acted as a dispersal corridor for S. bistincta to spread to the north. Combining multilocus genomic data with linked mtDNA data is a useful approach for identifying potential new species and associating them with already described taxa, which will be especially important in groups with undescribed subadult phenotypes and cryptic species.

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Mammella, Marco A., Frank N. Martin, Santa O. Cacciola, Michael D. Coffey, Roberto Faedda, and Leonardo Schena. "Analyses of the Population Structure in a Global Collection of Phytophthora nicotianae Isolates Inferred from Mitochondrial and Nuclear DNA Sequences." Phytopathology® 103, no. 6 (June 2013): 610–22. http://dx.doi.org/10.1094/phyto-10-12-0263-r.

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Genetic variation within the heterothallic cosmopolitan plant pathogen Phytophthora nicotianae was determined in 96 isolates from a wide range of hosts and geographic locations by characterizing four mitochondrial (10% of the genome) and three nuclear loci. In all, 52 single-nucleotide polymorphisms (SNPs) (an average of 1 every 58 bp) and 313 sites with gaps representing 5,450 bases enabled the identification of 50 different multilocus mitochondrial haplotypes. Similarly, 24 SNPs (an average of 1 every 69 bp), with heterozygosity observed at each locus, were observed in three nuclear regions (hyp, scp, and β-tub) differentiating 40 multilocus nuclear genotypes. Both mitochondrial and nuclear markers revealed a high level of dispersal of isolates and an inconsistent geographic structuring of populations. However, a specific association was observed for host of origin and genetic grouping with both nuclear and mitochondrial sequences. In particular, the majority of citrus isolates from Italy, California, Florida, Syria, Albania, and the Philippines clustered in the same mitochondrial group and shared at least one nuclear allele. A similar association was also observed for isolates recovered from Nicotiana and Solanum spp. The present study suggests an important role of nursery populations in increasing genetic recombination within the species and the existence of extensive phenomena of migration of isolates that have been likely spread worldwide with infected plant material.

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Komaki, Hisayuki, and Tomohiko Tamura. "Reclassification of Streptomyces hygroscopicus subsp. glebosus and Streptomyces libani subsp. rufus as later heterotypic synonyms of Streptomyces platensis." International Journal of Systematic and Evolutionary Microbiology 70, no. 7 (July 1, 2020): 4398–405. http://dx.doi.org/10.1099/ijsem.0.004279.

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We investigated the taxonomic relationships among Streptomyces hygroscopicus subsp. glebosus , Streptomyces libani subsp. rufus and Streptomyces platensis . The three species formed a single clade in the phylogenetic trees based on 16S rRNA gene sequence and multilocus sequence analyses. Digital DNA–DNA hybridization using whole genome sequences suggested that S. hygroscopicus subsp. glebosus , S. libani subsp. rufus and S. platensis belong to the same genomospecies. Previously reported phenotypic data also supported this synonymy. Therefore, S. hygroscopicus subsp. glebosus and S. libani subsp. rufus should be reclassified as later heterotypic synonyms of S. platensis .

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Eriksen, Kirsten T., Dorte Haubek, and Knud Poulsen. "Intragenomic recombination in the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans." Microbiology 151, no. 10 (October 1, 2005): 3371–79. http://dx.doi.org/10.1099/mic.0.28193-0.

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The highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans is strongly associated with aggressive periodontitis in adolescents of African descent. DNA fingerprinting using the frequently cutting restriction enzyme MspI and multilocus sequence typing (MLST) showed that five strains of this clone were genetically virtually identical, although ribotyping of the six rrn genes and EcoRI RFLP analysis of the seven IS150-like elements revealed differences. PCR analyses demonstrated that these multi-copy sequences are subject to intragenomic homologous recombination, resulting in translocations or large inversions. The genome rearrangements were reflected in differences among 25 strains representing the JP2 clone in DNA fingerprinting using the rare-cutting restriction enzyme XhoI and resolved by PFGE. XhoI DNA fingerprinting provides a tool for studying local epidemiology, including transmission of this particularly pathogenic clone of A. actinomycetemcomitans.

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Donahoo, R. S., J. B. Jones, G. H. Lacy, V. K. Stromberg, and D. J. Norman. "Genetic Analyses of Xanthomonas axonopodis pv. dieffenbachiae Strains Reveal Distinct Phylogenetic Groups." Phytopathology® 103, no. 3 (March 2013): 237–44. http://dx.doi.org/10.1094/phyto-08-12-0191-r.

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A comprehensive analysis of 175 Xanthomonas axonopodis pv. dieffenbachiae strains isolated from 10 Araceae hosts was done to identify pathogen variation. The strains were subjected to repetitive extragenic palindromic sequence polymerase chain reaction and four major phylogenetic clusters were generated. A subset of 40 strains isolated from Anthurium, Dieffenbachia, and Syngonium was further defined by amplified fragment length polymorphism and fatty acid methyl ester analysis and the same four phylogenetic clusters were observed. Comparison of representative strains in the first three clusters using DNA-DNA hybridization and multilocus sequence analysis supports the previous reclassification of strains in cluster I, including the X. axonopodis pv. dieffenbachiae pathovar reference strain (LMG695), to X. citri. Our research findings indicate that strains in cluster I, isolated primarily from anthurium, probably represent an undescribed pathovar. Other phylogenetic subclusters consisting primarily of strains isolated from xanthosoma and philodendron in clusters III and IV, respectively, may yet represent other undescribed species or pathovars of Xanthomonas.

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Gonçalves, Micael F. M., Tânia F. L. Vicente, Ana C. Esteves, and Artur Alves. "Neptunomyces aureus gen. et sp. nov. (Didymosphaeriaceae, Pleosporales) isolated from algae in Ria de Aveiro, Portugal." MycoKeys 60 (October 31, 2019): 31–44. http://dx.doi.org/10.3897/mycokeys.60.37931.

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A collection of fungi was isolated from macroalgae of the genera Gracilaria, Enteromorpha and Ulva in the estuary Ria de Aveiro in Portugal. These isolates were characterized through a multilocus phylogeny based on ITS region of the ribosomal DNA, beta-tubulin (tub2) and translation elongation factor 1 alpha (tef1-α) sequences, in conjunction with morphological and physiological data. These analyses showed that the isolates represented an unknown fungus for which a new genus, Neptunomycesgen. nov. and a new species, Neptunomyces aureussp. nov. are proposed. Phylogenetic analyses supported the affiliation of this new taxon to the family Didymosphaeriaceae.

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M. Lambert, David, and Craig D. Millar. "DNA science and conservation." Pacific Conservation Biology 2, no. 1 (1995): 21. http://dx.doi.org/10.1071/pc950021.

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A wide array of DNA-based genetic techniques are now available for the study of many problems in conservation biology. Either directly or indirectly, such techniques are becoming increasingly available to scientists and managers alike. Although these technologies are generally known to conservationists, there is a need to clearly outline the principal characteristics of such genetic tools and to detail how they can most appropriately be used in the management of wildlife species. The essential characteristics of mitochondrial and chloroplast restriction fragment analyses are detailed, together with discussions of single locus nuclear restriction fragment length polymorphisms (RFLPs), multilocus DNA fingerprinting, microsatellite DNA, randomly amplified polymorphic DNAs (RAPDs), and DNA sequence variation. We also give relevant information about the development times for these techniques, their relative costs, and the quality of tissue required. In addition, we discuss which conservation problems are appropriate to each of these methods and give examples of their application and potential use in relation to New Zealand organisms. The following problems are considered: sex assignment; parentage and kinship; migration; species, population and strain identification; forensic applications; genetic effects of population bottlenecks; disease identification; feeding preferences; philopatry; pest control; and understanding population extinction. Finally, we suggest that both microsatellite and minisatellite DNA techniques have particular advantages over many other currently-available techniques and conclude that these two approaches are applicable to a wide range of the conservation problems.

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VENEGAS, J., S. ORTIZ, S. MUNOZ, and A. SOLARI. "Molecular karyotype and schizodeme analyses of Trypanosoma cruzi stocks from Chilean triatomines." Parasitology 115, no. 1 (July 1997): 41–46. http://dx.doi.org/10.1017/s0031182097001066.

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Forty-one Trypanosoma cruzi stocks isolated from the Chilean vectors Triatoma infestans and Triatoma spinolai were characterized by pulse-field gel electrophoresis and Southern blotting with the cruzipain gene, and by schizodeme analysis of kinetoplast DNA with EcoRI and Msp I. Seven parasite groups were found by molecular karyotype which correlate with schizodeme and multilocus enzyme electrophoresis, supporting the concept of clonal propagation for Trypanosoma cruzi. A predominant T. cruzi stock was isolated from domiciliary T. infestans in several geographical areas of Chile. In contrast, other frequently found genotypes were circulating in the sylvatic and domestic transmission cycles of specific geographical areas. The greatest heterogeneity of T. cruzi stocks was found among sylvatic T. spinolai where at least 4 genotypes were obtained from a sample of 16 T. cruzi stocks.

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McDermott, J. M., B. A. McDonald, R. W. Allard, and R. K. Webster. "Genetic variability for pathogenicity, isozyme, ribosomal DNA and colony color variants in populations of Rhynchosporium secalis." Genetics 122, no. 3 (July 1, 1989): 561–65. http://dx.doi.org/10.1093/genetics/122.3.561.

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Abstract Samples of Rhynchosporium secalis were collected from two experimental barley populations known to carry a diverse array of alleles for resistance to this fungal pathogen. Classification of 163 isolates for four putative isozyme systems, a colony color dimorphism and 20 ribosomal DNA restriction fragment length variants revealed 49 different multilocus phenotypes (haplotypes). The six most common haplotypes differed significantly in pathogenicity. Genetic analyses of the data indicated that effective population sizes of the fungus were very large, that the effects of genetic drift were small, and that negligible recombination occurred in the populations studied. Frequency dependent selection was suggested as an explanation for the maintenance of variation in pathogenicity in the fungus.

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Glass, Mindy B., Arnold G. Steigerwalt, Jean G. Jordan, Patricia P. Wilkins, and Jay E. Gee. "Burkholderia oklahomensis sp. nov., a Burkholderia pseudomallei-like species formerly known as the Oklahoma strain of Pseudomonas pseudomallei." International Journal of Systematic and Evolutionary Microbiology 56, no. 9 (September 1, 2006): 2171–76. http://dx.doi.org/10.1099/ijs.0.63991-0.

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C6786, the clinical isolate of the ‘Oklahoma’ strain of Pseudomonas (now Burkholderia) pseudomallei, was originally isolated in 1973 from a wound infection resulting from a farming accident in Oklahoma, USA. Environmental isolates C7532 and C7533 from the Oklahoma accident site were found to match C6786. These three isolates and a clinical isolate originally identified as B. pseudomallei that was recovered from a person in Georgia, USA, involved in an automobile accident were characterized by biochemical, 16S rRNA gene sequencing, multilocus sequence typing and DNA–DNA hybridization analyses. Results indicated that these strains comprise a novel species. The name Burkholderia oklahomensis sp. nov. is proposed, with strain C6786T (=LMG 23618T=NCTC 13387T=CCUG 51349T) as the type strain.

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Shilov, I. A., E. N. Kislin, O. P. Malyuchenko, and P. N. Kharchenko. "A Technology for Genetic Identification of Varieties and Wild Forms of Grapes Based on Multilocus Microsatellite Analysis." Biotekhnologiya 37, no. 3 (2021): 85–95. http://dx.doi.org/10.21519/0234-2758-2021-37-3-85-95.

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The genotyping technology has been developed based on 9 microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VVMD28, VVMD25, VVMD32, VrZAG62, VrZAG79). It can be used for efficient, accurate and fast identification of grape varieties and forms (genus Vitis). The proposed approach includes a multiplex PCR of all loci followed by electrophoretic analysis of DNA fragments in one capillary of a genetic analyzer. The application of an additional length standard, an allelic ladder, consisting of all possible DNA fragments of the analyzed microsatellite loci, is one of the key features of the technology, which ensures the accuracy and reproducibility of the results. The advantage of the proposed technology is the ability to standardize and automate the procedure using 96-well plates, which opens up the possibility of conducting mass analyses. As a result of the study of varieties and forms of Vitis genus species, genetic passports were obtained, according to which a dendrogram was constructed, reflecting the genetic relationship of the studied samples. The developed technology makes it possible to distinguish varieties and wild forms of grapes; it can be used for their identification and determination of genetic distance between them, as well as for assessment of planting material and protection of breeders' rights. Vitis, grapes, microsatellites, SSR, genotyping, multiplex PCR, DNA fragment analysis The work was carried out within the framework of the state assignment on the topic "Development of New Technologies for Genetic Analysis of Forms of Agricultural Plants to Accelerate and Control the Selection Process" (project no. 0574-2019-0003).

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Silva, Claudia, Pablo Vinuesa, Luis E. Eguiarte, Esperanza Martínez-Romero, and Valeria Souza. "Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications." Applied and Environmental Microbiology 69, no. 2 (February 2003): 884–93. http://dx.doi.org/10.1128/aem.69.2.884-893.2003.

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ABSTRACT The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed.

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Boldo, X. M., L. Villa-Tanaca, G. Zuniga, and C. Hernandez-Rodriguez. "Genetic Diversity among Clinical Isolates of Candida glabrata Analyzed by Randomly Amplified Polymorphic DNA and Multilocus Enzyme Electrophoresis Analyses." Journal of Clinical Microbiology 41, no. 10 (October 1, 2003): 4799–804. http://dx.doi.org/10.1128/jcm.41.10.4799-4804.2003.

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Gavora, J. S., R. W. Fairfull, B. F. Benkel, W. J. Cantwell, and J. R. Chambers. "Prediction of Heterosis from DNA Fingerprints in Chickens." Genetics 144, no. 2 (October 1, 1996): 777–84. http://dx.doi.org/10.1093/genetics/144.2.777.

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Abstract To assess the value of DNA fingerprints for the prediction of heterosis in chickens, retrospective analyses of data from three crossbreeding experiments and DNA fingerprints (DFP) of parental strains were conducted using two minisatellite and one middle-repetitive DNA probes. DFP bands were assessed on pooled DNA samples of 10-15 individuals per parental genetic group. The number of DFP bands evaluated in the experiments ranged from 81 to 139. The probes varied in their predictive value, but predictability of heterosis generally increased with multiple probes. Highly significant correlations (0.68-0.87) between band sharing ratios (SH) and heterosis were found in 25 crosses of White Leghorns in the first egg production cycle for age at sexual maturity, egg production, and mature body weight: traits with heterosis of 10% or more of the means. Regressions on SH explained 78.4% of the variation in heterosis in age at sexual maturity, 60.2% in egg production and 46.4% in mature body weight. For “broiler” traits with heterosis of <1%, none of the correlations, based on 13 crosses, were significant. It was concluded that multilocus probe DFP of pooled DNA samples show promise as predictors of heterosis.

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HONEGGER, Rosmarie, Undine ZIPPLER, Sandra SCHERRER, and Paul S. DYER. "Genetic diversity in Xanthoria parietina (L.) Th. Fr. (lichen-forming ascomycete) from worldwide locations." Lichenologist 36, no. 6 (November 2004): 381–90. http://dx.doi.org/10.1017/s002428290401477x.

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Specimens of Xanthoria parietina were collected from worldwide locations and ascospore discharge used to establish axenic cultures of the mycobiont. DNA was extracted and RAPD-PCR fingerprinting of 59 isolates was successfully achieved, resulting in 58 unique fingerprints. 110 multilocus RAPD markers were generated and used to construct a dendrogram. Two main groups were distinguished (75% bootstrap support): the first comprising samples from the Iberian Peninsula, the Balearic and Canary Islands; the second comprising all other worldwide samples including isolates from throughout Europe and North America. Samples from Australia and New Zealand clustered with the second group except one additional, phenotypically distinct specimen, not belonging to X. parietina, which formed an outgroup. However, comparative DNA sequence analyses are required to verify this interpretation.

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Peever, Tobin L., and Michael G. Milgroom. "Genetic structure of Pyrenophora teres populations determined with random amplified polymorphic DNA markers." Canadian Journal of Botany 72, no. 7 (July 1, 1994): 915–23. http://dx.doi.org/10.1139/b94-116.

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The genetic structure of Pyrenophora teres, an ascomycete fungus that causes net blotch of barley, was examined using random amplified polymorphic DNA (RAPD) markers. Twenty-seven random oligonucleotide primers were screened against DNA from 16 isolates of P. teres of diverse geographic origin. Five primers gave scorable, reproducible DNA products (bands) suitable for population genetic studies. Genetic analyses of bands produced by two of the primers revealed single locus segregation in three of four crosses, indicating that these RAPDs can be interpreted as alleles at genetic loci. Allele frequencies were determined for 10 putative RAPD loci from five primers in 22–35 isolates of P. teres sampled from each of five geographically separated populations in Canada, Germany, and the U.S.A. Eight RAPD loci were polymorphic in at least one population and two loci were monomorphic in all five populations. Variation in allele frequencies (allelic diversity) among the five populations was partitioned into within- and among-population components using Nei's GST. A GST value of 0.46 was obtained among all populations indicating that approximately 46% of the total genetic variability detected was due to differentation among populations compared with 54% within populations. A GST value of 0.33 was obtained among the North American populations only. From five to nine multilocus genotypes were found in each population. Nine genotypes occurred exclusively in the German population and four exclusively in the New York population. The other populations had one or two unique genotypes. Gametic disequilibrium values (nonrandom associations of RAPD loci) were calculated among all pairs of polymorphic loci within each population. Eleven of 49 values were significantly different from 0 (P < 0.05); 8 of the 11 significant gametic disequilibrium values occurred in the New York population. Highly significant gametic disequilibrium was detected between the same two RAPD loci in three different populations, suggesting that these loci are genetically linked. Two different multilocus analyses revealed that the genetic structures of the Alberta, North Dakota, and German populations but not the New York population were consistent with random sexual reproduction occurring in these populations. Key words: polymerase chain reaction, population genetics, fungi, genetic differentiation.

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Sandoval-Denis, Marcelo, Wijnand J. Swart, and Pedro W. Crous. "New Fusarium species from the Kruger National Park, South Africa." MycoKeys 34 (June 1, 2018): 63–92. http://dx.doi.org/10.3897/mycokeys.34.25974.

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Three new Fusarium species, F.convolutans, F.fredkrugeri, and F.transvaalense (Ascomycota, Hypocreales, Nectriaceae) are described from soils collected in a catena landscape on a research supersite in the Kruger National Park, South Africa. The new taxa, isolated from the rhizosphere of three African herbaceous plants, Kyphocarpaangustifolia, Melhaniaacuminata, and Sidacordifolia, are described and illustrated by means of morphological and multilocus molecular analyses based on sequences from five DNA loci (CAL, EF-1 α, RPB1, RPB2 and TUB). According to phylogenetic inference based on Maximum-likelihood and Bayesian approaches, the newly discovered species are distributed in the Fusariumbuharicum, F.fujikuroi, and F.sambucinum species complexes.

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Bustamante, Danilo E., Manuel Oliva, Santos Leiva, Jani E. Mendoza, Leidy Bobadilla, Geysen Angulo, and Martha S. Calderon. "Phylogeny and species delimitations in the entomopathogenic genus Beauveria (Hypocreales, Ascomycota), including the description of B. peruviensis sp. nov." MycoKeys 58 (September 9, 2019): 47–68. http://dx.doi.org/10.3897/mycokeys.58.35764.

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The genus Beauveria is considered a cosmopolitan anamorphic and teleomorphic genus of soilborne necrotrophic arthropod-pathogenic fungi that includes ecologically and economically important species. Species identification in Beauveria is difficult because of its structural simplicity and the lack of distinctive phenotypic variation. Therefore, the use of multi-locus sequence data is essential to establish robust species boundaries in addition to DNA-based species delimitation methods using genetic distance, coalescent, and genealogical concordance approaches (polyphasic approaches). In this regard, our study used multilocus phylogeny and five DNA-based methods to delimit species in Beauveria using three molecular makers. These polyphasic analyses allowed for the delimitation of 20–28 species in Beauveria, confirming cryptic diversity in five species (i.e. B. amorpha, B. bassiana, B. diapheromeriphila, and B. pseudobassiana) and supporting the description of B. peruviensis as a new taxon from northeastern Peru. The other five species were not evaluated as they did not have enough data (i.e. B. araneola, B. gryllotalpidicola, B. loeiensis, B. medogensis, and B. rudraprayagi). Our results demonstrate that the congruence among different methods in a polyphasic approach (e.g. genetic distance and coalescence methods) is more likely to show reliably supported species boundaries. Among the methods applied in this study, genetic distance, coalescent approaches, and multilocus phylogeny are crucial when establishing species boundaries in Beauveria.

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Hirota, Kikue, Keiko Yamahira, Kenji Nakajima, Yoshinobu Nodasaka, Hidetoshi Okuyama, and Isao Yumoto. "Pseudomonas toyotomiensis sp. nov., a psychrotolerant facultative alkaliphile that utilizes hydrocarbons." International Journal of Systematic and Evolutionary Microbiology 61, no. 8 (August 1, 2011): 1842–48. http://dx.doi.org/10.1099/ijs.0.024612-0.

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A psychrotolerant, facultatively alkaliphilic strain, HT-3T, was isolated from a sample of soil immersed in hot-spring water containing hydrocarbons in Toyotomi, Hokkaido, Japan. 16S rRNA gene sequence-based phylogeny suggested that strain HT-3T is a member of the genus Pseudomonas and belongs to the Pseudomonas oleovorans group. Cells of the isolate were Gram-negative, aerobic, straight rods, motile by a single polar flagellum. The strain grew at 4–42 °C, with optimum growth at 35 °C at pH 7, and at pH 6–10. It hydrolysed Tweens 20, 40, 60 and 80, but not casein, gelatin, starch or DNA. Its major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 65.1 mol%. The whole-cell fatty acid profile consisted mainly of C16 : 0, C16 : 1ω9c and C18 : 1ω9c. Phylogenetic analyses based on gyrB, rpoB and rpoD sequences revealed that the isolate could be discriminated from Pseudomonas species that exhibited more than 97 % 16S rRNA gene sequence similarity and phylogenetic neighbours belonging to the P. oleovorans group including the closest relative of the isolate, Pseudomonas alcaliphila. DNA–DNA hybridization with P. alcaliphila AL15-21T revealed 51±5 % relatedness. Owing to differences in phenotypic properties and phylogenetic analyses based on multilocus gene sequence analysis and DNA–DNA relatedness data, the isolate merits classification in a novel species, for which the name Pseudomonas toyotomiensis sp. nov. is proposed. The type strain is HT-3T ( = JCM 15604T = NCIMB 14511T).

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GALIÀ-CAMPS, CARLES, LEILA CARMONA, ANDREA CABRITO, and MANUEL BALLESTEROS. "Double trouble. A cryptic first record of Berghia marinae Carmona, Pola, Gosliner, & Cervera 2014 in the Mediterranean Sea." Mediterranean Marine Science 21, no. 1 (April 27, 2020): 191. http://dx.doi.org/10.12681/mms.20026.

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AbstractIn 2014, Berghia marinae Carmona, Pola, Gosliner & Cervera, 2014 from Senegal was described along with the revision of the genus Berghia Trinchese, 1877. In this study, we establish a second record for the senegalese species B. marinae in the Mediterranean Sea, 4,000 Km away from its type location. The morphological mismatch from the original description hampered its identification, and thus, a molecular approach was needed. Multilocus phylogenetic trees were inferred from Maximum-likelihood and Bayesian analyses based on partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes, and the nuclear gene histone-3. Species delimitation analyses were performed to support the phylogenetic results and a new morphological description is provided complementing earlier information on this barely known species.

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Vallender, Rachel, Jean-Philippe Gagnon, and Irby Lovette. "An Intergeneric Wood-Warbler Hybrid (Mniotilta varia × Dendroica coronata) and Use of Multilocus DNA Analyses to Diagnose Avian Hybrid Origins." Wilson Journal of Ornithology 121, no. 2 (June 2009): 298–305. http://dx.doi.org/10.1676/08-050.1.

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Prodöhl, Paulo A., Andrew F. Walker, Rosaleen Hynes, John B. Taggart, and Andrew Ferguson. "Genetically monomorphic brown trout (Salmo trutta L.) populations, as revealed by mitochondrial DNA, multilocus and single-locus minisatellite (VNTR) analyses." Heredity 79, no. 2 (August 1997): 208–13. http://dx.doi.org/10.1038/hdy.1997.144.

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Killer, J., I. Sedláček, V. Rada, J. Havlík, and J. Kopečný. "Reclassification of Bifidobacterium stercoris Kim et al. 2010 as a later heterotypic synonym of Bifidobacterium adolescentis." International Journal of Systematic and Evolutionary Microbiology 63, Pt_11 (November 1, 2013): 4350–53. http://dx.doi.org/10.1099/ijs.0.054957-0.

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The taxonomic position of Bifidobacterium stercoris Eg1T ( = JCM 15918T) based on comparative 16S rRNA gene and hsp60 sequence analyses was found to be controversial, as the strain showed high similarity to the type strain of Bifidobacterium adolescentis , CCUG 18363T. Therefore, the relationship between the two species was investigated by a taxonomic study that included, in addition to re-evaluation of the 16S rRNA gene sequence, determination of DNA–DNA binding and multilocus sequence analysis (MLSA) of housekeeping genes encoding the DNA-directed RNA polymerase B subunit (rpoC), putative xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp), elongation factor EF-G (fusA), 50S ribosomal protein L2 (rplB) and DNA gyrase B subunit (gyrB). Comparative 16S rRNA gene sequence analysis showed relatively high similarity (98.9 %) between B. stercoris KCTC 5756T and B. adolescentis ATCC 15703T. MLSA revealed close relatedness between B. stercoris KCTC 5756T and B. adolescentis CCUG 18363T, with 99.3–100 % similarity between the rpoC, xfp, fusA, rplB and gyrB gene sequences. In addition, relatively high dnaJ1 gene sequence similarity of 97.7 % was found between the strains. Similar phenotypes and a high DNA–DNA binding value (78.9 %) confirmed that B. stercoris and B. adolescentis are synonymous. Based on these results, it is proposed that the species Bifidobacterium stercoris Kim et al. 2010 should be reclassified as a later heterotypic synonym of Bifidobacterium adolescentis Reuter 1963 (Approved Lists 1980).

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O'Donnell, Kerry, Deanna A. Sutton, Nathan Wiederhold, Vincent A. R. G. Robert, Pedro W. Crous, and David M. Geiser. "Veterinary Fusarioses within the United States." Journal of Clinical Microbiology 54, no. 11 (September 7, 2016): 2813–19. http://dx.doi.org/10.1128/jcm.01607-16.

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Multilocus DNA sequence data were used to assess the genetic diversity and evolutionary relationships of 67Fusariumstrains from veterinary sources, most of which were from the United States. Molecular phylogenetic analyses revealed that the strains comprised 23 phylogenetically distinct species, all but two of which were previously known to infect humans, distributed among eight species complexes. The majority of the veterinary isolates (47/67 = 70.1%) were nested within theFusarium solanispecies complex (FSSC), and these included 8 phylospecies and 33 unique 3-locus sequence types (STs). Three of the FSSC species (Fusarium falciforme,Fusarium keratoplasticum, andFusariumsp. FSSC 12) accounted for four-fifths of the veterinary strains (38/47) and STs (27/33) within this clade. Most of theF. falciformestrains (12/15) were recovered from equine keratitis infections; however, strains ofF. keratoplasticumandFusariumsp. FSSC 12 were mostly (25/27) isolated from marine vertebrates and invertebrates. Our sampling suggests that theFusarium incarnatum-equisetispecies complex (FIESC), with eight mycoses-associated species, may represent the second most important clade of veterinary relevance withinFusarium. Six of the multilocus STs within the FSSC (3+4-eee, 1-b, 12-a, 12-b, 12-f, and 12-h) and one each within the FIESC (1-a) and theFusarium oxysporumspecies complex (ST-33) were widespread geographically, including three STs with transoceanic disjunctions. In conclusion, fusaria associated with veterinary mycoses are phylogenetically diverse and typically can only be identified to the species level using DNA sequence data from portions of one or more informative genes.

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Ducey, Thomas F., Brent Page, Thomas Usgaard, Monica K. Borucki, Kitty Pupedis, and Todd J. Ward. "A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of Listeria monocytogenes." Applied and Environmental Microbiology 73, no. 1 (November 3, 2006): 133–47. http://dx.doi.org/10.1128/aem.01453-06.

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ABSTRACT Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.

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McDowell, Jan R., and John E. Graves. "Population structure of striped marlin (Kajikia audax) in the Pacific Ocean based on analysis of microsatellite and mitochondrial DNA." Canadian Journal of Fisheries and Aquatic Sciences 65, no. 7 (July 2008): 1307–20. http://dx.doi.org/10.1139/f08-054.

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Genetic variation was surveyed at five microsatellite loci and the mitochondrial control region (819 bp) to test for the presence of genetic stock structure in striped marlin ( Kajikia audax ) collections taken from seven locations throughout the Pacific Ocean. Temporal replicates separated by 9 years were taken off Japan, and three temporal samples spanning 11 years were collected off the coast of eastern Australia. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences showed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for microsatellite markers (FST = 0.013, P < 0.001). Mitochondrial control region sequences were not different across all samples (ΦST = –0.01, P = 0.642). Analyses of molecular variance (AMOVA) revealed significant genetic differentiation between geographic regions for both microsatellite and mitochondrial markers. These results suggest the presence of genetically discrete populations within the Pacific Ocean and are supported by both the results of tagging studies, which show limited dispersal, and the presence of geographically separated spawning grounds.

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Jensen, Anders, Tomonori Hoshino, and Mogens Kilian. "Taxonomy of the Anginosus group of the genus Streptococcus and description of Streptococcus anginosus subsp. whileyi subsp. nov. and Streptococcus constellatus subsp. viborgensis subsp. nov." International Journal of Systematic and Evolutionary Microbiology 63, Pt_7 (July 1, 2013): 2506–19. http://dx.doi.org/10.1099/ijs.0.043232-0.

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The Anginosus group of the genus Streptococcus has been the subject of much taxonomic confusion, which has hampered the full appreciation of its clinical significance. The purpose of this study was to critically re-examine the taxonomy of the Anginosus group, with special attention to β-haemolytic, Lancefield group C strains, using multilocus sequence analysis (MLSA) combined with 16S rRNA gene sequence and phenotypic analyses. Phylogenetic analysis of concatenated sequences of seven housekeeping genes previously used for examination of viridans streptococci distinguished seven distinct and coherent clusters in the Anginosus group. Analyses of 16S rRNA gene sequences and phenotypic characters supported the MLSA clustering and currently recognized taxa of the Anginosus group. Single gene analyses showed considerable allele sharing between species, thereby invalidating identification based on single-locus sequencing. Two novel clusters of β-haemolytic, Lancefield group C strains within the Streptococcus constellatus and Streptococcus anginosus species and isolated from patients with sore throat showed sufficient phylogenetic distances from other clusters to warrant status as novel subspecies. The novel cluster within S. anginosus was identified as the previously recognized DNA homology cluster, DNA group 2. The names S. anginosus subsp. whileyi subsp. nov. (type strain CCUG 39159T = DSM 25818T = SK1267T) and S. constellatus subsp. viborgensis subsp. nov. (type strain SK1359T = CCUG 62387T = DSM 25819T) are proposed.

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Rodríguez, Miguel, José Carlos Reina, Victoria Béjar, and Inmaculada Llamas. "Paenibacillus lutrae sp. nov., A Chitinolytic Species Isolated from A River Otter in Castril Natural Park, Granada, Spain." Microorganisms 7, no. 12 (December 2, 2019): 637. http://dx.doi.org/10.3390/microorganisms7120637.

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A highly chitinolytic facultative anaerobic, chemoheterotrophic, endospore-forming, Gram-stain-positive, rod-shaped bacterial strain N10T was isolated from the feces of a river otter in the Castril Natural Park (Granada, Spain). It is a slightly halophilic, motile, catalase-, oxidase-, ACC deaminase- and C4 and C8 lipase-positive strain. It is aerobic, respiratory and has a fermentative metabolism using oxygen as an electron acceptor, produces acids from glucose and can fix nitrogen. Phylogenetic analysis of the 16S rRNA gene sequence, multilocus sequence analysis (MLSA) of 16S rRNA, gyrB, recA and rpoB, as well as phylogenomic analyses indicate that strain N10T is a novel species of the genus Paenibacillus, with the highest 16S rRNA sequence similarity (95.4%) to P. chitinolyticus LMG 18047T and <95% similarity to other species of the genus Paenibacillus. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANIb) were 21.1% and <75%, respectively. Its major cellular fatty acids were anteiso-C15:0, C16:0, and iso-C15:0. G + C content ranged between 45%–50%. Using 16S rRNA phylogenetic and in silico phylogenomic analyses, together with chemotaxonomic and phenotypic data, we demonstrate that type strain N10T (= CECT 9541T =LMG 30535T) is a novel species of genus Paenibacillus and the name Paenibacillus lutrae sp. nov. is proposed.

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Marcelletti, Simone, and Marco Scortichini. "Definition of Plant-Pathogenic Pseudomonas Genomospecies of the Pseudomonas syringae Complex Through Multiple Comparative Approaches." Phytopathology® 104, no. 12 (December 2014): 1274–82. http://dx.doi.org/10.1094/phyto-12-13-0344-r.

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A total of 34 phytopathogenic strain genomes belonging to the Pseudomonas syringae species complex and related species, including many pathotype strains, were assessed using average nucleotide identity (ANI) analysis. Their taxonomic relationships were consistently confirmed by the tetranucleotide frequency correlation coefficient (TETRA) values, multilocus sequence typing analysis (MLSA) performed with seven housekeeping genes, using both maximum likelihood and Bayesian methods, and split consensus network analyses. The ANI, MLSA, and split consensus analyses provided consistent and identical results. We confirmed the occurrence of the well-demarcated genomospecies inferred sensu Gardan et al. using DNA-DNA hybridization and ribotyping analyses. However, some P. syringae strains of the pathovars morsprunorum and lachrymans were placed in different genomospecies in our analyses. Genomospecies 1, 2, 4, 6, and 9 resulted well demarcated, whereas strains of genomospecies 3 and 8 had ANI values between 95 and 96% in some cases, confirming that this threshold reveals very closely related species that might represent cases of splitting entities or the convergence of different species to the same ecological niche. This study confirms the robustness of the combination of genomic and phylogenetic approaches in revealing taxonomic relationships among closely related bacterial strains and provides the basis for a further reliable demarcation of the phytopathogenic Pseudomonas species. Within each species, the pathovars might represent distinct ecological units. The possibility of performing extensive and standardized host range and phenotypic tests with many strains of different pathovars can assist phytobacteriologists for better determining the boundaries of these ecological units.

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Skrodenytė-Arbačiauskienė, V., S. Radžiutė, V. Stunžėnas, and V. Būda. "Erwinia typographi sp. nov., isolated from bark beetle (Ips typographus) gut." International Journal of Systematic and Evolutionary Microbiology 62, Pt_4 (April 1, 2012): 942–48. http://dx.doi.org/10.1099/ijs.0.030304-0.

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Gram-negative-staining bacteria that were resistant to monoterpene myrcene (7-methyl-3-methylene-1.6-octadiene, C10H16, at concentrations of up to 10 µl ml−1 in TSB) were isolated from the gut contents of adult bark beetles Ips typographus (Coleoptera, Scolytidae). The beetles were collected from the bark of Norway spruce (Picea abies) in Lithuania. Bark beetles feed on conifers, which produce myrcene among many other defensive compounds. It has been suggested that the micro-organisms present within the beetles’ guts could be involved in their resistance towards this plant defensive compound. The most resistant bacterial strains were isolated and characterized by phenotypic assays as well as fatty acid analysis, 16S rRNA gene sequencing, multilocus sequence analyses (MLSA) based on the rpoB, atpD and infB genes and DNA–DNA hybridization. Biochemical characterization indicated that the bacteria belonged to the family Enterobacteriaceae . Phylogenetic analyses of the 16S rRNA gene sequences and MLSA of the novel strains revealed that they belonged to the genus Erwinia , but represented a novel species. The dominant cellular fatty acids were C16 : 0 and C17 : 0 cyclo. The DNA G+C content was 49.1 mol%. The results obtained in this study indicated that these bacteria from the bark beetle gut represented a novel species, for which the name Erwinia typographi sp. nov. is proposed, with the type strain DSM 22678T ( = Y1T = LMG 25347T).

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FREITAS, MAYARA L. R., ANDRÉ A. M. GOMES, ANDRÉ W. C. ROSADO, and OLINTO L. PEREIRA. "Cladosporium species from submerged decayed leaves in Brazil, including a new species and new records." Phytotaxa 482, no. 3 (February 3, 2021): 223–39. http://dx.doi.org/10.11646/phytotaxa.482.3.1.

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Fungi belonging to the genus Cladosporium are cosmopolitan and occur in various substrates and hosts. Nevertheless, this is the first time that we have described this genus colonizing leaf litter submerged in water. We surveyed Cladosporium spp. associated with submerged leaf litter, from three localities belonging to the Atlantic Forest biome in the State of Minas Gerais, Brazil. A multilocus DNA sequence typing approach employing ITS, Actin, and Translation elongation factor 1-α region/genes associated with morphological and cultural analyses were used to identify these species. The species C. angulosum and C. anthropophilum are reported for the first time in Brazil, and a new species for the genus is described here as C. puris.

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Lupien, S. L., F. M. Dugan, K. M. Ward, and K. O’Donnell. "Wilt, Crown, and Root Rot of Common Rose Mallow (Hibiscus moscheutos) Caused by a NovelFusariumsp." Plant Disease 101, no. 2 (February 2017): 354–58. http://dx.doi.org/10.1094/pdis-05-16-0717-re.

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A new crown and root rot disease of landscape plantings of the malvaceous ornamental common rose mallow (Hibiscus moscheutos) was first detected in Washington State in 2012. The main objectives of this study were to complete Koch’s postulates, document the disease symptoms photographically, and identify the causal agent using multilocus molecular phylogenetics. Results of the pathogenicity experiments demonstrated that the Fusarium sp. could induce vascular wilt and root and crown rot symptoms on H. moscheutos ‘Luna Rose’. Maximum-likelihood and maximum-parsimony phylogenetic analyses of portions of translation elongation factor 1-α and DNA-directed RNA polymerase II largest and second-largest subunit indicated that the Hibiscus pathogen represents a novel, undescribed Fusarium sp. nested within the Fusarium buharicum species complex.

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TRONCOSO-PALACIOS, JAIME, FRANCISCO FERRI-YÁÑEZ, ALEJANDRO LASPIUR, and CÉSAR AGUILAR. "An updated phylogeny and morphological study of the Phymaturus vociferator clade (Iguania: Liolaemidae)." Zootaxa 4441, no. 3 (June 28, 2018): 447. http://dx.doi.org/10.11646/zootaxa.4441.3.2.

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Species delimitation in Phymaturus has been a difficult task due to the highly conserved morphological and ecological features present in this genus. Almost all species of Phymaturus have been described without DNA data or lacking statistical analyses which makes even more difficult to compare species. Although two molecular phylogenetic studies have been recently published, here we provide the first multilocus phylogenetic reconstruction including all Chilean species, with samples from all type localities and some previously unsampled populations. We also estimate pairwise distances among the Chilean species of Phymaturus (P. vociferator and P. mallimaccii clades) and compare our results with the P. payuniae clade, where previous studies have used multiple lines of evidence. Additionally, we performed univariate and multivariate morphological analyses and skeletal comparisons (clavicle) for the species of the P. vociferator clade. As a result of this integrative approach, we describe a new species.

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Kerkhove, Thomas R. H., Jens Boyen, Annelies De Backer, Jan H. Mol, Filip A. M. Volckaert, Frederik Leliaert, and Marleen De Troch. "Multilocus data reveal cryptic species in the Atlantic seabob shrimp Xiphopenaeus kroyeri (Crustacea: Decapoda)." Biological Journal of the Linnean Society 127, no. 4 (June 1, 2019): 847–62. http://dx.doi.org/10.1093/biolinnean/blz065.

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Abstract The recognition of cryptic biodiversity provides valuable insights for the management of exploited species. The Atlantic seabob shrimp (Xiphopenaeus kroyeri) is a commercially important fishery resource in the Guianan ecoregion, South America. Previous research in Brazil suggested the presence of cryptic species within the genus. Here, we confirm this presence and delimit the species by applying a multilocus approach based on two mitochondrial (COI and cytb) and two nuclear (PEPCK and NaK) genes. Species boundaries were tested using BPP, GMYC and bPTP delimitation algorithms. These analyses provided strong support for three clades within the genus Xiphopenaeus, including one undescribed clade, which occurs sympatrically with X. kroyeri in the Western Atlantic. Unexpectedly, this undescribed clade is more closely related to the Pacific Xiphopenaeus riveti than to their Atlantic congener. Our DNA-based species delimitation was further supported by new ecological information on habitat and morphology (colour). We also expand the known distribution range of the cryptic species, currently restricted to Brazil, to include French Guiana, Suriname and Colombia. Our findings have important consequences for the management of the species, in terms of both biodiversity management and fisheries management.

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Johnson-Mackinnon, Crosbie, Karlsbakk, Marcos-Lopez, Paley, Nowak, and Bridle. "Multilocus Sequence Typing (MLST) and Random Polymorphic DNA (RAPD) Comparisons of Geographic Isolates of Neoparamoeba perurans, the Causative Agent of Amoebic Gill Disease." Pathogens 8, no. 4 (November 19, 2019): 244. http://dx.doi.org/10.3390/pathogens8040244.

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Neoparamoba perurans, is the aetiological agent of amoebic gill disease (AGD), a disease that affects farmed Atlantic salmon worldwide. Multilocus sequence typing (MLST) and Random Amplified Polymorphic DNA (RAPD) are PCR-based typing methods that allow for the highly reproducible genetic analysis of population structure within microbial species. To the best of our knowledge, this study represents the first use of these typing methods applied to N. perurans with the objective of distinguishing geographical isolates. These analyses were applied to a total of 16 isolates from Australia, Canada, Ireland, Scotland, Norway, and the USA. All the samples from Australia came from farm sites on the island state of Tasmania. Genetic polymorphism among isolates was more evident from the RAPD analysis compared to the MLST that used conserved housekeeping genes. Both techniques consistently identified that isolates of N. perurans from Tasmania, Australia were more similar to each other than to the isolates from other countries. While genetic differences were identified between geographical isolates, a BURST analysis provided no evidence of a founder genotype. This suggests that emerging outbreaks of AGD are not due to rapid translocation of this important salmonid pathogen from the same area.

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Zamora, J. C., and S. Ekman. "Phylogeny and character evolution in the Dacrymycetes, and systematics of Unilacrymaceae and Dacryonaemataceae fam. nov." Persoonia - Molecular Phylogeny and Evolution of Fungi 44, no. 1 (June 29, 2020): 161–205. http://dx.doi.org/10.3767/persoonia.2020.44.07.

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We present a multilocus phylogeny of the class Dacrymycetes, based on data from the 18S, ITS, 28S, RPB1, RPB2, TEF-1α, 12S, and ATP6 DNA regions, with c. 90 species including the types of most currently accepted genera. A variety of methodological approaches was used to infer phylogenetic relationships among the Dacrymycetes, from a supermatrix strategy using maximum likelihood and Bayesian inference on a concatenated dataset, to coalescence-based calculations, such as quartet-based summary methods of independent single-locus trees, and Bayesian integration of single-locus trees into a species tree under the multispecies coalescent. We evaluate for the first time the taxonomic usefulness of some cytological phenotypic characters, i.e., vacuolar contents (vacuolar bodies and lipid bodies), number of nuclei of recently discharged basidiospores, and pigments, with especial emphasis on carotenoids. These characters, along with several others traditionally used for the taxonomy of this group (basidium shape, presence and morphology of clamp connections, morphology of the terminal cells of cortical/marginal hyphae, presence and degree of ramification of the hyphidia), are mapped on the resulting phylogenies and their evolution through the class Dacrymycetes discussed. Our analyses reveal five lineages that putatively represent five different families, four of which are accepted and named. Three out of these four lineages correspond to previously circumscribed and published families (Cerinomycetaceae, Dacrymycetaceae, and Unilacrymaceae), and one is proposed as the new family Dacryonaemataceae. Provisionally, only a single order, Dacrymycetales, is accepted with in the class. Furthermore, the systematics of the two smallest families, Dacryonaemataceae and Unilacrymaceae, are investigated to the species level, using coalescence-based species delimitation on multilocus DNA data, and a detailed morphological study including morphometric analyses of the basidiospores. Three species are accepted in Dacryonaema, the type, Da. rufum, the newly combined Da. macnabbii (basionym Dacrymyces macnabbii), and a new species named Da. macrosporum. Two species are accepted in Unilacryma, the new U. bispora, and the type, U. unispora, the latter treated in a broad sense pending improved sampling across the Holarctic.

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Bart, Aldert, Ilse G. A. Schuurman, Mark Achtman, Dominique A. Caugant, Jacob Dankert, and Arie van der Ende. "Randomly Amplified Polymorphic DNA Genotyping of Serogroup A Meningococci Yields Results Similar to Those Obtained by Multilocus Enzyme Electrophoresis and Reveals New Genotypes." Journal of Clinical Microbiology 36, no. 6 (1998): 1746–49. http://dx.doi.org/10.1128/jcm.36.6.1746-1749.1998.

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Randomly amplified polymorphic DNA (RAPD) genotyping was applied to one representative strain of each of the 84 electrophoretic types (ETs) of Neisseria meningitidis serogroup A previously defined by multilocus enzyme electrophoresis (MEE) (J.-F. Wang et al., Infect. Immun. 60:5267–5282, 1992). Twenty-seven additional isolates comprising six ETs were also tested. MEE and RAPD genotyping yielded similar dendrograms at the subgroup level. Similar results were obtained by both methods for 18 serogroup A meningococci isolated in The Netherlands between 1989 and 1993. Ten of these isolates defined a new subgroup, designated subgroup IX. One isolate belonged to the ET-5 complex, normally associated with serogroup B strains (D. A. Caugant et al., Proc. Natl. Acad. Sci. USA 83:4927–4931, 1986). By RAPD genotyping, meningococci can be linked to previously characterized genotypes by using a computerized database, and dendrograms based on cluster analyses can easily be generated. RAPD analysis offers advantages over MEE since intermediate numbers of isolates of serogroup A meningococci can quickly be assigned to known subgroups and new subgroups can be defined.

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Brady, Carrie L., Ilse Cleenwerck, Stephanus N. Venter, Katrien Engelbeen, Paul De Vos, and Teresa A. Coutinho. "Emended description of the genus Pantoea, description of four species from human clinical samples, Pantoea septica sp. nov., Pantoea eucrina sp. nov., Pantoea brenneri sp. nov. and Pantoea conspicua sp. nov., and transfer of Pectobacterium cypripedii (Hori 1911) Brenner et al. 1973 emend. Hauben et al. 1998 to the genus as Pantoea cypripedii comb. nov." International Journal of Systematic and Evolutionary Microbiology 60, no. 10 (October 1, 2010): 2430–40. http://dx.doi.org/10.1099/ijs.0.017301-0.

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Bacterial strains belonging to DNA hybridization groups (HG) II, IV and V, in the Erwinia herbicola–Enterobacter agglomerans complex, of Brenner et al. [Int J Syst Bacteriol 34 (1984), 45–55] were suggested previously to belong to the genus Pantoea, but have never been formally described and classified. Additionally, it has been shown in several studies that Pectobacterium cypripedii is more closely related to species of Pantoea than to those of Pectobacterium. In this study, the phylogenetic positions of Brenner's DNA HG II, IV and V and Pectobacterium cypripedii were re-examined by both 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) based on the gyrB, rpoB, atpD and infB genes. The analyses revealed that DNA HG II, IV and V and Pectobacterium cypripedii form five separate branches within the genus Pantoea (strains from HG V were split into two branches). DNA–DNA hybridization data further confirmed that DNA HG II, IV and V constitute four separate species. Pectobacterium cypripedii was shown to be a close phylogenetic relative of Pantoea dispersa and DNA HG IV by both 16S rRNA gene sequence and MLSA analyses. Biochemical analyses performed on strains from DNA HG II, IV and V and Pectobacterium cypripedii confirmed their taxonomic position within the genus Pantoea and revealed phenotypic characteristics that allow the differentiation of these species from each other and from their closest phylogenetic neighbours. It is proposed to emend the description of the genus Pantoea and to describe Pantoea septica sp. nov. for DNA HG II (type strain LMG 5345T =BD 874T =CDC 3123-70T), Pantoea eucrina sp. nov. for DNA HG IV (type strain LMG 2781T =BD 872T =CDC 1741-71T =LMG 5346T), Pantoea brenneri sp. nov. for strains of DNA HG V excluding LMG 24534 (type strain LMG 5343T =BD 873T =CDC 3482-71T) and Pantoea conspicua sp. nov. for the remaining strain of DNA HG V (type strain LMG 24534T =BD 805T =CDC 3527-71T) and to transfer Pectobacterium cypripedii to the genus as Pantoea cypripedii comb. nov. (type strain LMG 2657T =ATCC 29267T =DSM 3873T =LMG 2655T).

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MLADINEO, I., N. J. BOTT, B. F. NOWAK, and B. A. BLOCK. "Multilocus phylogenetic analyses reveal that habitat selection drives the speciation of Didymozoidae (Digenea) parasitizing Pacific and Atlantic bluefin tunas." Parasitology 137, no. 6 (December 23, 2009): 1013–25. http://dx.doi.org/10.1017/s0031182009991703.

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SUMMARYParasite communities of wild and reared bluefin tuna display remarkable diversity. Among these, the most prevalent and abundant are the Didymozoidae (Monticelli, 1888) (Trematoda, Digenea), considered one of the most taxonomically complex digenean families. The aim of this study was to evaluate phylogenetic structure of Didymozoidae occurring in Pacific (Thunnus orientalis) and Atlantic bluefin tuna (T. thynnus) in order to increase our knowledge of didymozoid zoogeography and identify species that could successfully be employed as biological tags for stock assessment studies. For the present analyses we used 2 nuclear ribosomal DNA loci, part of the 28S gene and the second internal transcribed spacer (ITS-2) as well as a portion of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). In most parasitic groups, morphology is the primary factor in the structuring of phylogenetic relationships. In rare examples, however, habitat has been suggested as a primary factor affecting parasite evolution. During their evolution, didymozoids have spread and inhabited a remarkable number of different sites in their hosts, colonizing exterior as well as strictly interior niches. Our data suggest that habitat selection has been the leading force in shaping didymozoid phylogenetic relationships. For 2 didymozoid species (D. wedli and D. palati), cox1 sequences indicate intraspecific differences between Mexican and Adriatic populations.

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Chen, Ruey-Shyang, and Bruce A. McDonald. "Sexual Reproduction Plays a Major Role in the Genetic Structure of Populations of the Fungus Mycosphaerella graminicola." Genetics 142, no. 4 (April 1, 1996): 1119–27. http://dx.doi.org/10.1093/genetics/142.4.1119.

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Abstract The relative contributions of sexual and asexual reproduction to the genetic structure of populations can be difficult to determine for fungi that use a mixture of both types of propagation. Nuclear RFLPs and DNA fingerprints were used to make indirect and direct measures of departures from random mating in a population of the plant pathogenic fungus Mycosphaerella graminicola during the course of an epidemic cycle. DNA fingerprints resolved 617 different genotypes among 673 isolates sampled from a single field over a 3-month period. Only 7% of the isolates represented asexual clones that were found more than once in the sample. The most common clone was found four times. Genotypic diversity averaged 85% of its maximum possible value during the course of the epidemic. Analyses of multilocus structure showed that allelic distributions among RFLP loci were independent. Pairwise comparisons between individual RFLP loci showed that the majority of alleles at these loci were in gametic equilibrium. Though this fungus has the capacity for a significant level of asexual reproduction, each analysis suggested that M. graminicola populations maintain a genetic structure more consistent with random-mating over the course of an epidemic cycle.

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Bourdin, Thibault, Emilie Bedard, Marie-Ève Benoit, Michèle Prévost, Etienne Robert, Caroline Quach, Eric Déziel, and Philippe Constant. "Development of a New High-Throughput Multilocus Sequence Typing Method to Monitor Causative Agents of Nosocomial Infections." Infection Control & Hospital Epidemiology 41, S1 (October 2020): s187. http://dx.doi.org/10.1017/ice.2020.726.

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Background: Nosocomial infections cause 4%–56% mortality in newborns. Several epidemiological studies have shown that transmission of opportunistic pathogens from the sink to the patient, including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Serratia marcescens are associated with nosocomial infections in neonatal intensive care units (NICUs). In this project, we aimed to develop fast, accurate, and high-throughput multilocus sequence typing assays (HiMLST-Illumina) to detect opportunistic pathogens to assess their distribution in the sink environment of NICUs and their transfer to patients. Methods: Genome sequences of P. aeruginosa (n = 45), S. maltophilia (n = 23) and S. marcescens (n = 34) strains were retrieved from public genome databases to build their pangenomes, using the open-source PGAdb-builder server. The core genome was identified for each opportunistic pathogen and was searched for genes displaying the highest polymorphism. The minimal number of loci to include in a HiMLST-Illumina assay was determined by comparing topology of phylogenetic trees of concatenated loci based on genome similarity, computed as the average nucleotide identity (ANI) score. The primers used for HiMLST-Illumina schemes were designed in silico on a conserved domain and were tested on reference strains of each species. Results: Bioinformatics analyses showed that 3–4 loci (<300 base pairs per locus) distinguished strains with the same performances than ANI scores. The assays were tested using opportunistic pathogen isolates and environmental DNA originating from NICU sinks. The HiMLST-Illumina analysis of environmental DNA revealed the presence of at least 1 of the 3 studied opportunistic pathogens in 50% of sampled drains (n = 20). In a previous sampling, P. aeruginosa was isolated on selective culture media before and 48 hours after disinfection of a sink drain with chlorine. S. marcescens was also isolated from another sink 2 weeks after disinfection. Identification of the isolates was confirmed by HiMLST-Illumina analyses and will be typed to compare with clinical isolates. Conclusions: Initial in silico tests predict a high discriminating power of the HiMLST-Illumina method, suggesting that it would be possible to quickly identify strains of interest in a large number of samples. The power of this method is also in the possibility for molecular typing without a need for cultivation. Preliminary results suggest that sinks are readily colonized by opportunistic pathogens. This HiMLST-Illumina scheme will be applied in a 2-year intensive survey of NICUs in 3 hospitals in Montreal to evaluate the performance of new sink designs in limiting bioaerosol production and transmission of opportunistic pathogens to patients.Funding: NoneDisclosures: None

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Guo, Mengyue, Yanqin Xu, Li Ren, Shunzhi He, and and Xiaohui Pang. "A Systematic Study on DNA Barcoding of Medicinally Important Genus Epimedium L. (Berberidaceae)." Genes 9, no. 12 (December 17, 2018): 637. http://dx.doi.org/10.3390/genes9120637.

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Genus Epimedium consists of approximately 50 species in China, and more than half of them possess medicinal properties. The high similarity of species’ morphological characteristics complicates the identification accuracy, leading to potential risks in herbal efficacy and medical safety. In this study, we tested the applicability of four single loci, namely, rbcL, psbA-trnH, internal transcribed spacer (ITS), and ITS2, and their combinations as DNA barcodes to identify 37 Epimedium species on the basis of the analyses, including the success rates of PCR amplifications and sequencing, specific genetic divergence, distance-based method, and character-based method. Among them, character-based method showed the best applicability for identifying Epimedium species. As for the DNA barcodes, psbA-trnH showed the best performance among the four single loci with nine species being correctly differentiated. Moreover, psbA-trnH + ITS and psbA-trnH + ITS + rbcL exhibited the highest identification ability among all the multilocus combinations, and 17 species, of which 12 are medicinally used, could be efficiently discriminated. The DNA barcode data set developed in our study contributes valuable information to Chinese resources of Epimedium. It provides a new means for discrimination of the species within this medicinally important genus, thus guaranteeing correct and safe usage of Herba Epimedii.

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Journal articles on the topic 'Multilocus DNA analyses'

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