DNA Loci Research Articles

A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes

ObjectivesInvestigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF.ResultsWe developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes.

Molecular and acoustic evidence support the species status of Anthus rubescens rubescens and Anthus [rubescens] japonicus (Passeriformes: Motacillidae).

The Buff-bellied Pipit Anthus rubescens comprises two allopatric subspecies groups: A. r. rubescens and A. r. alticola in North America and A. [r.] japonicus in north-east Asia. Despite their great morphological resemblance in breeding plumage, most individuals can be assigned to one or the other subspecies group in non-breeding plumage. Allopatric distributions, morphological differentiation and previously reported molecular divergence suggested the need for additional taxonomic study to assess the rank of these two populations. To resolve the taxonomy of the Buff-bellied Pipit species complex we analysed i) two mitochondrial DNA (mtDNA) loci and ii) nine bioacoustic parameters across 69 sound recordings (338 flight calls) recovered from public databases using principal component analysis and Euclidean distance measures. By comparing our mtDNA and call divergence measures with similar values measured between long-recognised species pairs of the genus, we show that the level of mitochondrial and acoustic divergence between the two Buff-bellied Pipit subspecies groups is typical of species-level divergence in the genus Anthus. Therefore, we recommend splitting the Buff-bellied Pipit species complex into two species: Anthus rubescens (American Pipit) and Anthus japonicus (Siberian Pipit). Our results also suggest that the Water Pipit A. spinoletta deserves taxonomic reassessment as its lineages are highly divergent in acoustics and mtDNA, while mtDNA relationships suggest paraphyly relative to the Rock Pipit A. petrosus. Our work highlights the crucial importance of integrative approaches in taxonomy and the usefulness of bioacoustics in studying cryptic diversity.

The Self-incompatibility Locus and Chloroplast DNA Regions of Prunus domestica Reflect the Origin and Genetic Diversity of Traditional Cultivars

A traditional cultivar, Besztercei Bt.2, and a clone of an autochthonous landrace (Nemtudom P3) of the hexaploid European plum (Prunus domestica) were studied to highlight their breeding perspectives. Five self-incompatibility ribonuclease (S-RNase) alleles were detected in both cultivars, with one allele shared. DNA sequence analysis confirmed it as a new, previously unidentified allele in P. domestica, which we labeled as S18. This allele was found to share ∼99% identity with the Prunus spinosa SB-RNase allele. Because Prunus species are readily hybridizing, sequence variations in 10 chloroplast DNA regions and nuclear internal transcribed spacers were studied to check if ‘Nemtudom P3’ and ‘Besztercei Bt.2’ are indeed P. domestica. The majority-rule consensus tree of maximum likelihood and Bayesian inferences confirmed it, and also indicated genetic differentiation with ‘Nemtudom P3’ and ‘Besztercei Bt.2’ forming a statistically supported subclade within the P. domestica germplasm. Our results pointed to some regions of the P. domestica chloroplast genome (trnS-trnG-trnG, trnC-ycf6, and trnD-trnT) that can be used to detect intraspecific variations. The proportion of parsimony informative characters compared with the total length of amplified regions was the highest in the case of nrITS with 12.1%. The S-genotyping of 68 wild-growing Nemtudom trees showed the genetic consequences of long-term vegetative propagation and occasional crossing between Besztercei and Nemtudom accessions. Controlled pollinations confirmed the self-compatibility of ‘Nemtudom P3’. By clarifying their phylogenetic position, and characterizing the S-locus, our results will help breeding P. domestica cultivars and pave the way to understanding how the S-locus works in a hexaploid Prunus species.

Molecular Identification, Effector genes and Virulence of Isolates of <i>Parastagonospora nodorum</i> from Altai Krai (Russia)

Septoria blotches represent some of the most harmful wheat diseases that encompass all grain-producing countries worldwide. They are caused mainly by fungi of the genera Parastagonospora and Zymoseptoria. The aims of the research were to analyze the nucleotide sequences of two phylogenetically informative DNA loci, ITS and tub2, of Parastagonospora nodorum isolates from the Altai Krai, to detect the presence of Tox1, Tox3, ToxA effector genes and to study the virulence of the isolates. Microscopic analysis of fragments of affected plant tissue was used for primary identification of the Septoria blotch. The causative agent from the studied plant samples was identified as P. nodorum. Fungal colonies cultivated on potato-glucose agar displayed a well-developed light brown velvety mycelium with dark periphery (mixed type) and numerous pycnidia exhibiting high sporulation capacity. Microbiological diagnostics were complemented by molecular genetic studies. Sequencing of ITS and tub2 loci isolates revealed complete genetic identity of all eight studied monoconidial isolates obtained from different samples. PCR-based detection of tree Tox-genes demonstrated that the only Tox3 was present in the geno-types of 80 tested P. nodorum monoconidial isolates. The ToxA and Tox1 genes were not found in the studied isolates. The virulence of P. nodorum isolates was evaluated in laboratory conditions using an isolated wheat leaves assay. Mixture of four isolates from spring wheat and one isolate from winter wheat, were characterized as virulent. When spring and winter wheat cultivars were infected with two isolates obtained from oats, pathogenic but avirulent properties were detected. The isolate from triticale was avirulent to winter wheat cultivars and virulent to spring wheat cultivars.

Preservation of genetic diversity in a highly fragmented population of the gray-sided vole Myodes rufocanus in an intensive farming region.

Individual dispersal plays an important role in preserving genetic diversity in density-fluctuating populations of arvicoline rodents. When habitats are fragmented and dispersal between habitats is severely constrained, genetic diversity can be lost. Here, I investigated whether genetic diversity in the gray-sided vole Myodes rufocanus was preserved in an intensive farming region in Japan, where voles inhabited isolated windbreak forests along the borders of plowed lands. Genetic structure was examined in 673 vole samples (330 in spring and 343 in fall) collected at 34 windbreak forests located 0.35-20 km apart. A part of the control region (425 bp) of mitochondrial DNA (mtDNA) was sequenced in 673 voles, yielding 76 haplotypes. Genetic differentiation of maternally inherited mtDNA among trapping sites was markedly lower in males than in females in both seasons, indicating strong male-biased dispersal. Genotypes at six microsatellite DNA loci were determined in 494 voles (245 in spring and 249 in fall) from 18 trapping sites, and loci harbored 16-24 alleles. The mean number of alleles per locus (allelic diversity) at trapping sites was positively correlated with the number of examined individuals (density) in both seasons, and the relationship was very similar to that of a previous study performed in much less fragmented populations. The genetic differentiation of microsatellite DNA among trapping sites decreased considerably from spring to fall. In a STRUCTURE analysis with a most probable cluster number of two, closer trapping sites showed more similar mean values of cluster admixture proportions. The present findings indicate that gene flow among isolated windbreak forests, which occurred mainly by dispersal of males, was not restrained in this intensive farming region. Furthermore, the results suggest that genetic diversity in the study population was preserved as well as in less fragmented populations.

New extralimital breeding records of saltmarsh sparrows (Ammospiza caudacuta) and Nelson's sparrows (Ammospiza nelsoni) and their implications.

Saltmarsh (Ammospiza caudacuta) and Nelson's (A. nelsoni) sparrows are sister taxa that breed in tidal marshes along the coast of the Northeastern United States and Canada. The Saltmarsh Sparrow breeds from mid-coast Maine south to Virginia, while the Acadian Nelson's Sparrow breeds from the Canadian maritime provinces south to northern Massachusetts. Here, we present three extralimital observations of breeding Saltmarsh (n = 2) and Nelson's (n = 1) sparrows. In 2021 and 2022, we observed Saltmarsh Sparrow females attending nests at Mendall Marsh, ME, and Milbridge, ME, respectively, approximately 60 and 110 km beyond the documented northern extent of the Saltmarsh Sparrow breeding range. In 2022, we observed a breeding-condition male Nelson's sparrow singing in the upriver portion of a marsh on Cape Cod, Massachusetts, approximately 115 km beyond the previously documented southern extent of the Nelson's Sparrow breeding range. We confirmed morphological species identification using a panel of microsatellite DNA loci. Due to both the well-documented population declines of these species in the region and the intensity of sampling effort undertaken in recent years, we suggest that these observations likely are not indicative of range expansion. However, they do indicate that these 2 taxa have the capacity to use and successfully reproduce in marshes well beyond their established breeding limits. Our findings provide novel insight into the potential for these taxa to occur and successfully breed outside their documented breeding ranges. Given increased interest in their conservation, these results support the idea that management actions aimed at creating or maintaining nesting habitat across both species ranges could benefit both taxa.

Evaluation of Effectiveness of a New Kit of Reagents for Detection of Mutations Associated with Drug Resistance of Tuberculous Mycobacteria to Rifampicin and Isoniazid, by PCR according to Evidences of Clinical Trials

The objective: to evalute effectiveness in clinical trials of the new kit of reagents of AmpliTest® MBT-Resist-I based on real-time PCR (RT-PCR) versus similar kits of reagent registered in Russia, to identify genetic markers of M. tuberculosis complex (MTB) resistance to rifampicin (RIF) and isoniazid (INH) in human biological samples and MTB cultures.Subjects and Methods. 200 DNA samples were studied, they had been obtained from biological specimens (sputum (N=100), bronchoalveolar lavage (N=50), biopsy (surgical material; N=50)) of pulmonary tuberculosis patients and containing MTB DNA at a concentration of at least 1 x 103 GE/ml, and from 100 DNA samples of MTB cultures. Efficiency of detecting mutations associated with MTB resistance to RIF and INH using the new kit of reagent of AmpliTest® MBT-Resist-I was evaluated in comparison with HP Amplitube-MDR-RV (OOO Sintol, Russia). In the case of discordant results, the target MTB DNA loci were sequenced by Sanger.Results. We observed complete agreement of the results and consequently high efficiency rates (positive and negative concordance of results - 100%, respectively) of the new kit of reagents of AmpliTest® MBT-Resist-I in comparison with the kit of reagent of Amplitube-MDR-RV in detection of mutations associated with MTB resistance to both RIF and INH, both for human samples and for MTB cultures. In two DNA samples of MTB cultures, an additional mutation (I572L) in the rpoB gene was detected using the new kit of reagents which was not detected by the comparison kit and was confirmed by Sanger sequencing.Conclusion. Inhalers vary significantly by the patients' ability to use them correctly. Asthma patients master the correct inhalation technique better versus COPD patients. Liquid inhalers were more difficult to be used correctly, and the best results were observed with multi-dose powder inhalers. The patient's training by a healthcare professional is critical to assure the correct use of inhalers.

Brevibacterium sp. strain CS2: A potential candidate for arsenic bioremediation from industrial wastewater

A multiple metal-resistant Brevibacterium sp. strain CS2, isolated from an industrial wastewater, resisted arsenate and arsenate upto 280 and 40 mM. The order of resistance against multiple metals was Arsenate > Arsenite > Selenium = Cobalt > Lead = Nickel > Cadmium = Chromium = Mercury. The bacterium was characterized as per morphological and biochemical characteristics at optimum conditions (37 ℃ and 7 pH). The appearance of brownish color precipitation was due to the interaction of silver nitrate confirming its oxidizing ability against arsenic. The strain showed arsenic processing ability at different temperatures, pH, and initial arsenic concentration which was 37% after 72 h and 48% after 96 h of incubation at optimum conditions with arsenite 250 mM/L (initial arsenic concentration). The maximum arsenic removal ability of strain CS2 was determined for 8 days, which was 32 and 46% in wastewater and distilled water, respectively. The heat-inactivated cells of the isolated strain showed a bioremediation efficiency (E) of 96% after 10 h. Genes cluster (9.6 kb) related to arsenite oxidation was found in Brevibacterium sp. strain CS2 after the genome analysis of isolated bacteria through illumine and nanopore sequencing technology. The arsenite oxidizing gene smaller subunit (aioB) on chromosomal DNA locus (Prokka_01508) was identified which plays a role in arsenite oxidation for energy metabolism. The presence of arsenic oxidizing genes and an efficient arsenic oxidizing potential of Brevibacterium sp. strain CS2 make it a potential candidate for green chemistry to eradicate arsenic from arsenic-contaminated wastewater.

DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The dark side of circular RNA, a new driver of genome instability

Circular RNAs (circRNAs) are covalently closed RNA molecules whose functions are still largely uncharacterized. In the July issue of Cancer Cell, Conn etal.1 demonstrate that circRNA can bind cognate DNA loci, forming circRNA-DNA hybrids (circR loops), driving genetic rearrangements of MLL/KMT2A, which are associated with the most aggressive acute leukemias.

First report of anthracnose on spotted laurel caused by Colletotrichum fructicola in South Korea.

Spotted laurel (Aucuba japonica) is a popular ornamental bush (it has two-colored leaves and red berries) and is used outdoors and indoors for decoration in South Korea. Anthracnose reduces the aesthetic value of spotted laurel leaves. In August 2022, anthracnose symptoms were observed on leaves in a park at Jeju Island, South Korea. Approximately 55% of bushes were infected by this disease. Symptoms consisted of round or irregular lesions that initially appeared as black spots and coalesced into larger, black lesions covering whole leaves and twigs. Entire leaves wither and finally die. To identify the putative causal agent, 12 affected leaves were collected, placed in a plastic box containing moist tissue, and incubated at 25 ºC in the dark to obtain conidial mass. Conidial masses were produced on leaf lesions after 2 days, and then 12 morphologically similar fungal isolates were recovered following single the spore isolation technique on solid potato dextrose agar (PDA) (Cai et al. 2009). Ten-day-old colonies were olivaceous gray with immersed perithecia on the upper side and black at the center on the reverse side. Conidia were aseptate, cylindrical with round ends and measured 14.9 - 22.7 × 5.5 - 9.4 μm (n = 80). Appressoria were brown, irregular in shape, and 7.0 - 16.1 × 5.00 - 9.9 μm (n = 50). Asci were eight-spored, banana-shaped, and measuring 60.8 - 123.1 × 13.00 - 18.9 μm (n = 30). Hyaline ascospores were single-celled, curved or straight with round ends, and ranged in size was 15.5 - 23.3 × 5.1 - 11.8 μm (n = 50). The morphological characteristics of the isolates overlapped with those of Colletotrichum species within the C. gloeosporioides complex, including Colletotrichum fructicola (Weir et al. 2012). Five genomic DNA loci of the isolates, including the partial ITS rDNA region, ACT, GAPDH, TUB, and ApMat genes, were amplified and sequenced using ITSF1/ITS4, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting consensus sequences were deposited in the GenBank and the accession numbers (ITS = LC739331- LC739334, TUB = LC739335- LC739338, GAPDH = LC739339- LC739342, ACT = LC739343 -LC739346, ApMat = LC742925 - LC742928) were obtained. A maximum phylogenetic tree was constructed based on the combined data sets of ITS, ACT, GAPDH, TUB, ApMat sequences. The isolates were clustered with reference isolates of C. fructicola (isolates ICMP18581). The pathogenicity test was performed on uninfected, healthy spotted laurel cuttings in the pot. Five leaves per seedling were selected, surface sterilized with 70% ethanol, and rinsed with sterile distilled water (SDW). A sterile pin was used to make 3 to 4 wounds on each side of the leaf from the midrib. 10 μl of spore suspension per wound spot (1 × 106 spores/ml) was applied on the wounds of one site from midrib, and SDW was placed on the wounds of other site as a control. The treated seedlings were covered with sterile plastic bag and kept in a 12-h fluorescent light/dark cycle under greenhouse conditions at 25 ± 2°C and 80% relative humidity. Two seedlings were inoculated with a single isolate, and this experiment was repeated twice. Circular or irregular lesions appeared after 5 days of inoculation, while the control remained asymptotic. Koch's postulates were fulfilled by reisolating and reidentifying the causal agent from the lesions of inoculated leaves. Colletotrichum fructicola has been reported as the causal agent of anthracnose on mango (Joa et al. 2016), apple (Kim et al. 2018), grapes (Lim et al. 2019), peaches (Lee et al. 2020), and hybrid pear (Choi et al. 2021) in South Korea. To the best of our knowledge, it is the first report of C. fructicola causing anthracnose on spotted laurel. This study will be helpful to develop effective management strategies to minimize leaf lesions.

Differentially methylated DNA loci between Eastern and Western Finns associate with CHD risk factors

Differentially methylated DNA loci between Eastern and Western Finns associate with CHD risk factors

Diversity of Colletotrichum species associated with torch ginger anthracnose

ABSTRACT Anthracnose caused by Colletotrichum species is one of the most important diseases of torch ginger. The disease leads to loss of aesthetic and commercial value of torch ginger stems. This study aimed to characterize Colletotrichum species associated with torch ginger anthracnose in the production areas of Pernambuco and Ceará. A total of 48 Colletotrichum isolates were identified using molecular techniques. Pathogenicity tests were performed on torch ginger with representative isolates. Phylogenetic analyses based on seven loci—DNA lyase (APN2), intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), calmodulin (CAL), intergenic spacer between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a hypothetical protein (GAP2-IGS), glutamine synthetase (GS), and β-tubulin (TUB2)—revealed that they belong to five known Colletotrichum species, namely, C. chrysophilum, C. fructicola, C. siamense, C. theobromicola, and C. tropicale, and three newly discovered species, described here as C. atlanticum, C. floscerae, and C. zingibericola. Of these, C. atlanticum was the most dominant. Pathogenicity assays showed that all isolates were pathogenic to torch ginger bracts. All species are reported for the first time associated with torch ginger in Brazil. The present study contributes to the current understanding of the diversity of Colletotrichum species associated with anthracnose on torch ginger and demonstrates the importance of accurate species identification for effective disease management strategies.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

Genome-wide analysis reveals distinct global populations of pink bollworm (Pectinophora gossypiella)

The pink bollworm (Pectinophora gossypiella) is one of the world’s most destructive pests of cotton. This invasive lepidopteran occurs in nearly all cotton-growing countries. Its presence in the Ord Valley of North West Australia poses a potential threat to the expanding cotton industry there. To assess this threat and better understand population structure of pink bollworm, we analysed genomic data from individuals collected in the field from North West Australia, India, and Pakistan, as well as from four laboratory colonies that originated in the United States. We identified single nucleotide polymorphisms (SNPs) using a reduced-representation, genotyping-by-sequencing technique (DArTseq). The final filtered dataset included 6355 SNPs and 88 individual genomes that clustered into five groups: Australia, India-Pakistan, and three groups from the United States. We also analysed sequences from Genbank for mitochondrial DNA (mtDNA) locus cytochrome c oxidase I (COI) for pink bollworm from six countries. We found low genetic diversity within populations and high differentiation between populations from different continents. The high genetic differentiation between Australia and the other populations and colonies sampled in this study reduces concerns about gene flow to North West Australia, particularly from populations in India and Pakistan that have evolved resistance to transgenic insecticidal cotton. We attribute the observed population structure to pink bollworm’s narrow host plant range and limited dispersal between continents.

Parsing variance by marker type: Testing biogeographic hypotheses and differential contribution of historical processes to population structure in a desert lizard.

A fundamental goal of population genetic studies is to identify historical biogeographic patterns and understand the processes that generate them. However, localized demographic events can skew population genetic inference. Assessing populations with multiple types of genetic markers, each with unique mutation rates and responses to changes in population size, can help to identify potentially confounding population-specific demographic processes. Here, we compared population structure and connectivity inferred from microsatellites and restriction site-associated DNA loci among 17 populations of an arid-specialist lizard, the desert night lizard, Xantusia vigilis, in central California to test among historical processes structuring population genetic diversity. We found that both marker types yielded generally concordant insights into population genetic structure including a major phylogenetic break maintained between two populations separated by less than 10 km, suggesting that either marker type could be used to understand generalized demographic patterns across the region for management purposes. However, we also found that the effects of demography on marker discordance could be used to elucidate population histories and distinguish among competing biogeographic hypotheses. Our results suggest that comparisons of within-population diversity across marker types provide powerful opportunities for leveraging marker discordance, particularly for understanding the creation and maintenance of contact zones among clades.

Genetic methods in honey bee breeding.

The honey bee Apis mellifera is a rather difficult object for selection due to the peculiarities of its biology. Breeding activities in beekeeping are aimed at obtaining bee colonies with high rates of economically useful traits, such as productivity, resistance to low temperatures and diseases, hygienic behavior, oviposition of the queen, etc. With two apiaries specializing in the breeding of A. m. mellifera and A. m. carnica as examples, the application of genetic methods in the selection of honey bees is considered. The first stage of the work was subspecies identification based on the analysis of the polymorphism of the intergenic mtDNA locus tRNAleu-COII (or COI-COII) and microsatellite nuclear DNA loci Ap243, 4a110, A24, A8, A43, A113, A88, Ap049, A28. This analysis confirmed that the studied colonies correspond to the declared subspecies. In the apiary with A. m. mellifera, hybrid colonies have been identified. A method based on the analysis of polymorphisms of the tRNAleu-COII locus and microsatellite nuclear DNA loci has been developed to identify the dark forest bee A. m. mellifera and does not allow one to differentiate subspecies from C (A. m. carnica and A. m. ligustica) and O (A. m. caucasica) evolutionary lineages from each other. The second stage was the assessment of the allelic diversity of the csd gene. In the apiary containing colonies of A. m. mellifera (N = 15), 20 csd alleles were identified. In the apiary containing colonies of A. m. carnica (N = 44), 41 alleles were identified. Six alleles are shared by both apiaries. DNA diagnostics of bee diseases showed that the studied colonies are healthy. Based on the data obtained, a scheme was developed for obtaining primary material for honey bee breeding, which can subsequently be subjected to selection according to economically useful traits. In addition, the annual assessment of the allelic diversity of the csd gene will shed light on the frequency of formation of new allelic variants and other issues related to the evolution of this gene.

Blood meal analysis and molecular detection of mammalian Leishmania DNA in wild-caught Sergentomyia spp. from Tunisia and Saudi Arabia.

Phlebotomine sand flies (Diptera: Phlebotominae) belonging to the genus Phlebotomus are vectors of pathogens such as arboviruses, bacteria, and parasites (Leishmania). Species of the genus Sergentomyia (Se.) transmit Sauroleishmania (Reptile Leishmania) and feed on cold-blooded vertebrates; recently, they have been incriminated in mammalian Leishmania transmission. In addition, they have been reported to feed on warm-blooded vertebrates. This study aimed to (i) screen wild-caught Sergentomyia species for the detection of mammalian Leishmania and (ii) identify the blood meal origin of engorged females. The sand flies were collected using centers for disease control and prevention (CDC) traps, mounted and identified morphologically. Only females of the genus Sergentomyia were screened for Leishmania infection using PCR targeting the 18S ribosomal DNA locus. For positive specimens, Leishmania parasites were typed using nested PCR targeting ribosomal internal transcribed spacer 1 followed by digestion with HaeIII. The PCR-RFLP results were confirmed through sequencing. Blood meal identification was performed through PCR amplification of the vertebrate cytochrome b gene using degenerate primers followed by sequencing. In total, 6026 sand fly specimens were collected between 2009 and 2018. Among these, 511 belonged to five species of Sergentomyia genus: Se. minuta (58.51%), Se. fallax (18.01%), Se. clydei (14.68%), Se. dreyfussi (6.26%), and Se. antennata (2.54%). A total of 256 female Sergentomyia sp. specimens were screened for Leishmania infection. Seventeen (17) were positive (6.64%). Two Leishmania species were identified. Leishmania major DNA was detected in five specimens; this included three Se. fallax, one Se. minuta, and one Se. dreyfussi collected from Tunisia. Leishmania infantum/L. donovani complex was detected in four Se. minuta and three Se. dreyfussi specimens collected from Tunisia. In addition, we identified the blood meal origin of five engorged Se. minuta specimens collected from Tunisia. Sequencing results revealed two blood sources: humans (n = 4) and reptiles (n = 1) indicating possible role of Sergentomyia species in the transmission of human Leishmania. In addition, these species could be involved in the life cycle of L. infantum/L. donovani complex and L. major. The results of the blood meal origin showed that Sergentomyia fed on both cold- and warm-blooded vertebrates. These findings enable a better understanding of the behavior of this sand fly genus. Further studies should focus on the role of Sergentomyia in human Leishmania transmission and possible control of this disease.

Conservation Genetics of Clinch Dace Chrosomus sp. cf. saylori

Clinch Dace (Chrosomus sp. cf. saylori) is a newly recognized and yet-undescribed species of minnow with a restricted and fragmented distribution in the upper Tennessee River basin in southwestern Virginia, USA. We collected Clinch Dace from seven streams and observed variations at nine selectively neutral microsatellite DNA loci to infer population genetic processes and identify units for conservation management. Bayesian cluster analysis showed that three of the seven surveyed populations were genetically distinct, while the other four populations showed signs of recent admixture. Estimated effective population sizes and m-ratios were low within most populations, suggesting loss of alleles due to recent genetic drift. Positive FIS values, high average individual inbreeding coefficients, and high degrees of inferred relatedness among individuals suggested that inbreeding is taking place in some populations. FST values were high, and analysis of molecular variance indicated genetic divergence among populations. These indicators suggest that Clinch Dace populations are subject to the genetic processes that are characteristic of small and isolated populations.

Stochastic motion and transcriptional dynamics of pairs of distal DNA loci on a compacted chromosome.

Chromosomes in the eukaryotic nucleus are highly compacted. However, for many functional processes, including transcription initiation, the pairwise motion of distal chromosomal elements such as enhancers and promoters is essential and necessitates dynamic fluidity. Here, we used a live-imaging assay to simultaneously measure the positions of pairs of enhancers and promoters and their transcriptional output while systematically varying the genomic separation between these two DNA loci. Our analysis reveals the coexistence of a compact globular organization and fast subdiffusive dynamics. These combined features cause an anomalous scaling of polymer relaxation times with genomic separation leading to long-ranged correlations. Thus, encounter times of DNA loci are much less dependent on genomic distance than predicted by existing polymer models, with potential consequences for eukaryotic gene expression.