Ralstonia Strains from Potato-Growing Regions of Kenya Reveal Two Phylotypes and Epidemic Clonality of Phylotype II Sequevar 1 Strains.

Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is the most destructive potato disease in Kenya. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with bacterial wilt of potato in Kenya, (ii) generate an RSSC distribution map for epidemiological inference, and (iii) determine whether phylotype II sequevar 1 strains exhibit epidemic clonality. Surveys were conducted in 2018 and 2019, in which tubers from wilting potato plants and stem samples of potential alternative hosts were collected for pathogen isolation. The pathogen was phylotyped by multiplex PCR and 536 RSSC strains typed at a sequevar level. Two RSSC phylotypes were identified, phylotype II (98.4%, n = 506 [sequevar 1 (n = 505) and sequevar 2 (n = 1)]) and phylotype I (1.6%, n = 30 [sequevar 13 (n = 9) and a new sequevar (n = 21)]). The phylotype II sequevar 1 strains were haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. The TRST scheme identified 51 TRST profiles within the phylotype II sequevar 1 strains with a modest diversity index (HGDI = 0.87), confirming the epidemic clonality of RSSC phylotype II sequevar 1 strains in Kenya. A minimum spanning tree and mapping of the TRST profiles revealed that TRST27 '8-5-12-7-5' is the primary founder of the clonal complex of RSSC phylotype II sequevar 1 and is widely distributed via latently infected seed tubers. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tandem repeats in precursor protein stabilize transcript levels and production levels of the fungal ribosomally synthesized and post-translationally modified peptide ustiloxin B.

Ustiloxin B is a ribosomally synthesized and post-translationally modified peptide (RiPP) first reported in Ascomycetes. Its biosynthetic pathway was recently identified in the filamentous fungus Aspergillus flavus. The precursor protein of ustiloxin B, UstA, has a signal peptide to the endoplasmic reticulum at its N-terminal and a subsequent tandemly highly repeated segment cleaved at Lys-Arg dipeptides by Kex2 protease; such proteins are called Kex2-processed repeat proteins (KEPs). RiPP biosynthetic pathways using KEPs as precursor proteins are widely distributed in the Fungi kingdom, with high diversity of precursor protein sequences. UstA in A. flavus has a 16-fold tandemly repeated segment containing the core peptide Tyr-Ala-Ile-Gly, which forms the ustiloxin B backbone structure, but it is unknown why such a costly-to-maintain highly repeated sequence is retained. Here, we replaced ustA, the gene encoding the ustiloxin B precursor protein, with synthetic genes encoding 1-, 3-, 5-, 7-, and 11-fold tandem-repeat segments in A. flavus, to investigate the relationship between the repeat number and ustiloxin B production. Ustiloxin B production increased quadratically with increasing repeat number in ustA variants, although it dropped in a previously constructed ustA variant that had a substituted synthetic gene encoding a 16-fold repeat segment probably because of the presence of the many rare codons in the sequence. We also examined the transcript levels of substituted synthetic genes in ustA variants, and surprisingly we found that the transcript levels of the synthetic genes increased linearly with increasing repeat number. This result implies that an unknown mechanism stabilizes ustA transcripts via the highly repeated structure in a feedback manner. We also constructed a transformant without the intron in native ustA, but no effect of intron removal was observed on either ustiloxin B production or the precursor gene transcript level. The costly-to-maintain highly repeated sequence in KEPs probably serves the purpose of maintaining stable transcripts and thus increasing the amount of substrate.

Molecular Diversity and Pathogenicity of Ralstonia solanacearum Species Complex Associated With Bacterial Wilt of Potato in Rwanda.

Bacterial wilt (BW), caused by Ralstonia solanacearum species complex (RSSC), leads to substantial potato yield losses in Rwanda. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with BW of potato, (ii) generate an RSSC distribution map for epidemiological inferences, and (iii) test the pathogenicity of predominant RSSC phylotypes on six commercial potato cultivars. In surveys conducted in 2018 and 2019, tubers from wilting potato plants were collected for pathogen isolation. DNA was extracted from 95 presumptive RSSC strain colonies. The pathogen was phylotyped by multiplex PCR and typed at sequevar level. Phylotype II sequevar 1 strains were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. Pathogenicity of one phylotype II strain and two phylotype III strains were tested on cultivars Kinigi, Kirundo, Victoria, Kazeneza, Twihaze, and Cruza. Two RSSC phylotypes were identified, phylotype II (95.79%, n = 91) and phylotype III (4.21%, n = 4). This is the first report of phylotype III strains from Rwanda. Phylotype II strains were identified as sequevar 1 and distributed across potato growing regions in the country. The TRST scheme identified 14 TRST haplotypes within the phylotype II sequevar 1 strains with moderate diversity index (HGDI = 0.55). Mapping of TRST haplotypes revealed that a single TRST '8-5-12-7-5' haplotype plays an important epidemiological role in BW of potato in Rwanda. None of the cultivars had complete resistance to the tested phylotypes; the level of susceptibility varied among cultivars. Cultivar Cruza, which is less susceptible to phylotype II and III strains, is recommended when planting potatoes in the fields with history of BW.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tandem repeat sequence of duck circovirus serves as downstream sequence element to regulate viral gene expression.

Duck circovirus (DuCV) has a small, single-stranded circular DNA genome of approximately 1.99 kb. Through a genome sequence analysis using the dottup program, we found that a quadruple tandem repeat sequence (QTR) in the intergenic region between the rep and cap genes of the DuCV genome, but not in other circoviruses. The QTR was also substantially different and evolutionarily conserved in the genotype 1 and 2 DuCV strains. Furthermore, a luciferase reporter assay demonstrated that QTR functioned as a downstream sequence element (DSE) of polyadenylation signals to enhance mRNA stability, which was dependent on four copies but not the QTR direction. Cap and Rep expression derived by subgenomic constructs also revealed a critical role of QTR in regulating viral gene expression. Finally, a reverse genetic study of a DuCV-based minicircle DNA technique found that a deletion of QTR induced a significant deficiency in viral genes transcription and replication. Our findings were the first to report that QTR only exists in the DuCV genome and serves as a novel molecular marker of DuCV genotyping, and has revealed its crucial biological function in regulating viral gene expression.

An extra insertion of tandem repeat sequence in African swine fever virus, China, 2019.

On 7 March 2019, African swine fever in a domestic pig farm was detected in Guangxi Province of China. The phylogenetic analysis showed that its causative strain contained two tandem repeat sequence insertions in the intergenic region between the I73R and the I329L genes, and was different from previously reported strains in China and other countries.

Structural characteristics of a mitochondrial control region from M yotis bat (Vespertilionidae) mitogenomes based on sequence datasets.

The datasets included sequences of a control region from Myotis bat mitogenomes. The control region (1706-2005 bp) of the Myotis mitogenomes was divided into three domains similar to that of other mammals, which included the common conserved blocks (ETAS domain, Central domain, and CSB domain). Several long tandem repeat sequences were present between the upstream of control regions and ETAS1. The size, base composition, and copy number of the long tandem repeat sequences differed between the Myotis species. Short tandem repeat sequences were also found between CSB1 and CSB2 in the CSB domain.

Infection of African swine fever in wild boar, China, 2018.

On 16 November 2018, a wild boar infected with African swine fever was reported in China. The phylogenetic analysis showed that its causative strain belonged to the p72 genotype II, CD2v serogroup 8 and contained no additional tandem repeat sequences between the I73R and the I329L protein genes, which was different from previously reported strains in China.

PowerPlex® fusion 6C system: internal validation study.

This work is aimed at describing the proceedings and parameters used to validate PowerPlex® Fusion 6C System, the polymerase chain reaction (PCR) amplification kit by Promega, for posterior implementation in the laboratorial routine of the Forensic Genetic Service. The PowerPlex® Fusion 6C System allows multiplex PCR, through simultaneous amplification and posterior detection by fluorescence of 27 loci. Characterization of the kit was made according to the laboratory's internal validation procedure based on validation guidelines from Scientific Working Group on DNA Analysis Methods. Some parameters were evaluated, such as specificity, analytical thresholds, sensitivity, precision, mixture studies, DNA control samples, a proficiency test and changes in the PCR-based procedures: final reaction volume and cycle number, changes in the reaction mixture for direct amplification. This kit proved to be very robust and the results are in concordance with previous developmental validation by the manufacturer. In some parameters, the results were better than expected.

Observation and analysis of STR loci mutation among 1 786 cases of paternity test in Guangxi area / 重庆医学

Objective To observe and analyze the mutation characteristics of 17 STR loci among the paternity test cases in Guangxi area .Methods Among 1 786 cases of non—exclusion parentage ,1 430 cases were parental triplet and 356 cases were uniparental diad ,1 001 persons were Han people ,2 102 persons were Zhuang people and 113 persons were other ethnic group in the parents .The genome DNA was extracted by Chelex-100 method .17 short tandem repeat (STR) loci were detected by Power Plex ? 18D System Kit .The paternity testing containing mutant STR loci were screened out from 1786 cases .The locus-specific ,specificity of paternal and maternal ,and allele-specific mutation rates were observed and analyzed ,respectively .The characteristics of the muta-tions were studied .Results In total ,75 mutations events were observed at 16 of the 17 loci .Among them ,73 (97 .34% ) times were one step mutation ,onece(1 .33% ) was two—step mutation ,and once(1 .33% ) was three—step mutation ,no mutation was found at the TPOX locus .The mutation rates ranged 0 .031 1% —0 .404 2% ,and the mean mutation rate was 0 .145 8% .The proportion of the paternal mutations and the maternal mutations was 5 .4:1 .0 ,the difference had statistical significance(P0 .05) .Conclusion STR loci mutation is common phenomenon in paternity test .The data of STR loci mutations should be constantly accumulated for selecting the genetic characteristics in line with the Guangxi population and the genetic markers of STR loci with high identification ability to ensure ac-curate and reliable identification results .

Novel multiplex format of an extended multilocus variable-number-tandem-repeat analysis of Clostridium difficile correlates with tandem repeat sequence typing.

Subtyping of Clostridium difficile is crucial for outbreak investigations. An extended multilocus variable-number tandem-repeat analysis (eMLVA) of 14 variable number tandem repeat (VNTR) loci was validated in multiplex format compatible with a routine typing laboratory and showed excellent concordance with tandem repeat sequence typing (TRST) and high discriminatory power.

Unusual features of control region and a novel NADH 6 genes in mitochondrial genome of the finespot goby, Chaeturichthys stigmatias (Perciformes, Gobiidae).

In this article, we determined the complete mitogenome of finespot goby Chaeturichthys stigmatias with emphasis on the arranged gene order and gene feature with published Gobiidae species. The C. stigmatias mtDNA was 18,562 bp in length (56.94% AT), and comprised 37 genes (13 protein genes, 2 rRNAs and 22 tRNAs) that was typical for mitochondrial genome of Gobiidae species. Unusually, the NADH 6 gene was very large in length compared with other Gobiidae species. Mitogenome of C. stigmatias had a long putative control region with high AT content (71.28%). Within this sequence, we determined repeat regions, the termination-associated sequence and the conserved sequence block for this region. The origin of L-strand replication in C. stigmatias was located in a cluster of five tRNA genes (WANCY). The conserved motif (5'-GCCGG-3') was also determined at the base of the stem in the tRNA-Cys gene. This study will provide a better understanding of Gobiidae mitogenomes and offer useful information for future studies concerning Gobiidae mitogenome evolution.

Complete mitochondrial genome of the two-spotted stag beetle, Metopodontus blanchardi (Coleoptera: Lucanidae).

We have completely sequenced the mitochondrial genome (mitogeome) of the two-spotted stag beetle, Metopodontus blanchardi, which is listed as a first-degree endangered species in Korea. The complete mitogenome of M. blanchardi was determined to be 21,628 bp, indicating at least 5 kb larger in size than typical animal mitogenomes. Such a long M. blanchardi genome stems from a 3100-bp long A + T-rich region and a 4051-bp long, large non-coding sequence located between tRNA(Ile) and tRNA(Gln). The A + T-rich region is composed of duplicated repeat sequences (each 965 bp and 969 bp), and three non-repeat sequences encompassing the repeat sequences. The 4051-bp long non-coding sequence is composed of ∼ 17 tandem repeat sequences, each of which is composed of two subunits (113-bp and 104-bp long subunits) and this is encompassed by non-repeat sequences. The start codon for COI gene of M. blanchardi was designated as unconventional AAG (Lysine) by following a previous study.

Genotipificación de Mycobacterium leprae colombiano para la determinación de patrones de transmisión de la enfermedad / Genotyping colombian Mycobacterium leprae for determining disease transmission patterns

Objetivo Evaluar la variabilidad de VNTR (variable-number tandem repeat) de Mycobacterium leprae de pacientes colombianos con y sin tratamiento previo para identificar posibles fuentes de infección y entender los patrones de transmisión de la enfermedad. Metodología Estudio transversal descriptivo, en donde mediante un muestreo electivo a conveniencia se tomaron 161 biopsias de pacientes multibacilares de lepra, que habían sido solicitadas para diagnóstico y seguimiento de la enfermedad, de las cuales se realizó extracción de ADN de M. leprae y usando la técnica de PCR para VNTRs de M. leprae estandarizada, se establecieron los genotipos y los diferentes clusters mediante el agrupamiento apareado UPGMA. Resultados En las 161 muestras totales se hallaron 22 genotipos VNTRs diferentes, de las cuales 100 muestras (62,1 por ciento) pertenecían al genotipo único VNTRU, y de los genotipos restantes, los mayoritarios, es decir los que dieron lugar a formación de grupos o clusters fueron VNTR17 (5,6 por ciento), VNTR20 (4,3 por ciento), VNTR18 (4,3 por ciento), VNTR14 (4,3 por ciento) y VNTR13 (3,7 por ciento). Conclusión En este estudio se evidencia por análisis de agrupamiento que se pueden detectar clones con diferente grado de virulencia/agresividad, lo cual implica la necesidad de incrementar varias de las actividades del programa de control que darán como resultado la verdadera disminución de la transmisión del microorganismo.

Objective Assessing VNTR (variable-number tandem repeat) variability of Mycobacterium leprae from Colombian patients with and without prior treatment to identify potential sources of infection and to understand the patterns of disease transmission. Methodology This was a descriptive cross-sectional study where a convenience sample of biopsies was taken from 161 multibacillary leprosy patients; diagnosis and monitoring of the disease had been requested for these patients. DNA was extracted from M. leprae and standardised using the PCR technique for M. leprae VNTR, ge­notypes were established and different clusters grouped by unweighted pair group method with arithmetic mean (UPGMA). Results 22 different VNTR genotypes were found from 161 samples, of which 100 samples (62.1 percent) had a single u-VNTR genotype and the remaining genotypes were VNTR 17 (5.6 percent), VNTR 20 (4.3 percent), VNTR 18 (4.3 percent), VNTR 14 (4.3 percent) and VNTR 13 (3.7 percent), namely those forming groups or clusters. Conclusion This study showed that clones can be detected with varying degrees of virulence / aggressiveness by cluster analysis, implying the need for more monitoring programme activities which will result in a real decline in microorganism transmission.