Mapping by Sequencing the Pneumocystis Genome Using the Ordering DNA Sequences V3 Tool

Abstract A bioinformatics tool called ODS3 has been created for mapping by sequencing. The tool allows the creation of integrated genomic maps from genetic, physical mapping, and sequencing data and permits an integrated genome map to be stored, retrieved, viewed, and queried in a stand-alone capacity, in a client/server relationship with the Fungal Genome Database (FGDB), and as a web-browsing tool for the FGDB. In that ODS3 is programmed in Java, the tool promotes platform independence and supports export of integrated genome-mapping data in the extensible markup language (XML) for data interchange with other genome information systems. The tool ODS3 is used to create an initial integrated genome map of the AIDS-related fungal pathogen, Pneumocystis carinii. Contig dynamics would indicate that this physical map is ∼50% complete with ∼200 contigs. A total of 10 putative multigene families were found. Two of these putative families were previously characterized in P. carinii, namely the major surface glycoproteins (MSGs) and HSP70 proteins; three of these putative families (not previously characterized in P. carinii) were found to be similar to families encoding the HSP60 in Schizosaccharomyces pombe, the heat-shock Ψ protein in S. pombe, and the RNA synthetase family (i.e., MES1) in Saccharomyces cerevisiae. Physical mapping data are consistent with the 16S, 5.8S, and 26S rDNA genes being single copy in P. carinii. No other fungus outside this genus is known to have the rDNA genes in single copy.

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Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Genome ◽

10.1139/g94-102 ◽

1994 ◽

Vol 37 (5) ◽

pp. 717-725 ◽

Cited By ~ 179

Author(s):

Jiming Jiang ◽

Bikram S. Gill

Keyword(s):

In Situ Hybridization ◽

Physical Mapping ◽

Dna Sequences ◽

Genome Mapping ◽

Molecular Cytogenetics ◽

Gene Families ◽

Single Copy ◽

Plant Genome ◽

Metaphase Chromosomes

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.Key words: in situ hybridization, physical mapping, genome mapping, molecular cytogenetics.

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Physical mapping of repetitive oligonucleotides facilitates the establishment of a genome map-based karyotype to identify chromosomal variations in peanut

BMC Plant Biology ◽

10.1186/s12870-021-02875-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Liuyang Fu ◽

Qian Wang ◽

Lina Li ◽

Tao Lang ◽

Junjia Guo ◽

...

Keyword(s):

In Situ Hybridization ◽

Physical Mapping ◽

Tandem Repeats ◽

Genetic Research ◽

Diploid Species ◽

Reference Sequence ◽

A Genome ◽

Genome Map ◽

Chromosomal Variants

Abstract Background Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge. Results A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligo probes were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3 and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1 and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of the peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of the chromosomes. Conclusions The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.

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A Linkage-Based Genome Assembly for the Mosquito Aedes albopictus and Identification of Chromosomal Regions Affecting Diapause

Insects ◽

10.3390/insects12020167 ◽

2021 ◽

Vol 12 (2) ◽

pp. 167

Author(s):

John H. Boyle ◽

Pasi M. A. Rastas ◽

Xin Huang ◽

Austin G. Garner ◽

Indra Vythilingam ◽

...

Keyword(s):

Linkage Map ◽

Aedes Albopictus ◽

Dna Sequences ◽

Genome Assembly ◽

Single Copy ◽

Health Concern ◽

Candidate Snps ◽

Single Location ◽

Asian Tiger ◽

Photoperiodic Diapause

The Asian tiger mosquito, Aedes albopictus, is an invasive vector mosquito of substantial public health concern. The large genome size (~1.19–1.28 Gb by cytofluorometric estimates), comprised of ~68% repetitive DNA sequences, has made it difficult to produce a high-quality genome assembly for this species. We constructed a high-density linkage map for Ae. albopictus based on 111,328 informative SNPs obtained by RNAseq. We then performed a linkage-map anchored reassembly of AalbF2, the genome assembly produced by Palatini et al. (2020). Our reassembled genome sequence, AalbF3, represents several improvements relative to AalbF2. First, the size of the AalbF3 assembly is 1.45 Gb, almost half the size of AalbF2. Furthermore, relative to AalbF2, AalbF3 contains a higher proportion of complete and single-copy BUSCO genes (84.3%) and a higher proportion of aligned RNAseq reads that map concordantly to a single location of the genome (46%). We demonstrate the utility of AalbF3 by using it as a reference for a bulk-segregant-based comparative genomics analysis that identifies chromosomal regions with clusters of candidate SNPs putatively associated with photoperiodic diapause, a crucial ecological adaptation underpinning the rapid range expansion and climatic adaptation of A. albopictus.

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Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

Plant Cell Reports ◽

10.1007/s00299-011-1086-y ◽

2011 ◽

Vol 30 (9) ◽

pp. 1779-1786 ◽

Cited By ~ 5

Author(s):

Kun Yang ◽

Hecui Zhang ◽

Richard Converse ◽

Yong Wang ◽

Xiaoying Rong ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Repetitive Dna ◽

Dna Sequences ◽

Single Copy ◽

Repetitive Dna Sequences

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High frequency repeat-induced point mutation (RIP) is not associated with efficient recombination in Neurospora.

Genetics ◽

10.1093/genetics/138.4.1093 ◽

1994 ◽

Vol 138 (4) ◽

pp. 1093-1103 ◽

Cited By ~ 3

Author(s):

J T Irelan ◽

A T Hagemann ◽

E U Selker

Keyword(s):

Point Mutation ◽

Dna Sequences ◽

High Frequency ◽

Recombination Frequency ◽

Single Copy ◽

Sexual Cycle ◽

Base Pairs ◽

Copy Gene ◽

The Relationship ◽

Repeat Induced Point Mutation

Abstract Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.

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A systematic comparison of eight new plastome sequences from Ipomoea L

PeerJ ◽

10.7717/peerj.6563 ◽

2019 ◽

Vol 7 ◽

pp. e6563

Author(s):

Jianying Sun ◽

Xiaofeng Dong ◽

Qinghe Cao ◽

Tao Xu ◽

Mingku Zhu ◽

...

Keyword(s):

Comparative Analysis ◽

Next Generation Sequencing ◽

Chloroplast Genome ◽

Dna Sequences ◽

Repetitive Sequences ◽

Single Copy ◽

Next Generation ◽

Variable Regions ◽

Chloroplast Genomes ◽

Generation Sequencing

Background Ipomoea is the largest genus in the family Convolvulaceae. The species in this genus have been widely used in many fields, such as agriculture, nutrition, and medicine. With the development of next-generation sequencing, more than 50 chloroplast genomes of Ipomoea species have been sequenced. However, the repeats and divergence regions in Ipomoea have not been well investigated. In the present study, we sequenced and assembled eight chloroplast genomes from sweet potato’s close wild relatives. By combining these with 32 published chloroplast genomes, we conducted a detailed comparative analysis of a broad range of Ipomoea species. Methods Eight chloroplast genomes were assembled using short DNA sequences generated by next-generation sequencing technology. By combining these chloroplast genomes with 32 other published Ipomoea chloroplast genomes downloaded from GenBank and the Oxford Research Archive, we conducted a comparative analysis of the repeat sequences and divergence regions across the Ipomoea genus. In addition, separate analyses of the Batatas group and Quamoclit group were also performed. Results The eight newly sequenced chloroplast genomes ranged from 161,225 to 161,721 bp in length and displayed the typical circular quadripartite structure, consisting of a pair of inverted repeat (IR) regions (30,798–30,910 bp each) separated by a large single copy (LSC) region (87,575–88,004 bp) and a small single copy (SSC) region (12,018–12,051 bp). The average guanine-cytosine (GC) content was approximately 40.5% in the IR region, 36.1% in the LSC region, 32.2% in the SSC regions, and 37.5% in complete sequence for all the generated plastomes. The eight chloroplast genome sequences from this study included 80 protein-coding genes, four rRNAs (rrn23, rrn16, rrn5, and rrn4.5), and 37 tRNAs. The boundaries of single copy regions and IR regions were highly conserved in the eight chloroplast genomes. In Ipomoea, 57–89 pairs of repetitive sequences and 39–64 simple sequence repeats were found. By conducting a sliding window analysis, we found six relatively high variable regions (ndhA intron, ndhH-ndhF, ndhF-rpl32, rpl32-trnL, rps16-trnQ, and ndhF) in the Ipomoea genus, eight (trnG, rpl32-trnL, ndhA intron, ndhF-rpl32, ndhH-ndhF, ccsA-ndhD, trnG-trnR, and pasA-ycf3) in the Batatas group, and eight (ndhA intron, petN-psbM, rpl32-trnL, trnG-trnR, trnK-rps16, ndhC-trnV, rps16-trnQ, and trnG) in the Quamoclit group. Our maximum-likelihood tree based on whole chloroplast genomes confirmed the phylogenetic topology reported in previous studies. Conclusions The chloroplast genome sequence and structure were highly conserved in the eight newly-sequenced Ipomoea species. Our comparative analysis included a broad range of Ipomoea chloroplast genomes, providing valuable information for Ipomoea species identification and enhancing the understanding of Ipomoea genetic resources.

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Electron microscopy of Achlya deoxyribonucleic acid sequence organization

Molecular and Cellular Biology ◽

10.1128/mcb.1.2.136-143.1981 ◽

1981 ◽

Vol 1 (2) ◽

pp. 136-143

Author(s):

M Pellegrini ◽

W E Timberlake ◽

R B Goldberg

Keyword(s):

Dna Sequences ◽

Deoxyribonucleic Acid ◽

Microscopic Analysis ◽

Single Copy ◽

Electron Microscopic ◽

Sequence Organization ◽

Double Stranded Dna ◽

Copy Dna ◽

Gene 32 ◽

Single Copy Dna

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.

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LOCALIZATION OF SINGLE COPY DNA SEQUENCES ON G-BANDED HUMAN CHROMOSOMES BY IN SITU HYBRIDIZATION

Mobilization and Reassembly of Genetic Information ◽

10.1016/b978-0-12-633360-2.50076-0 ◽

1980 ◽

pp. 443

Author(s):

Mary E. Chandler ◽

Michael B. Mann ◽

Grady F. Saunders

Keyword(s):

In Situ Hybridization ◽

Dna Sequences ◽

Single Copy ◽

Human Chromosomes ◽

Copy Dna ◽

Single Copy Dna

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Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

Phytopathology ◽

10.1094/phyto.1997.87.8.853 ◽

1997 ◽

Vol 87 (8) ◽

pp. 853-861 ◽

Cited By ~ 33

Author(s):

Dallice Mills ◽

Brian W. Russell ◽

Janet Williams Hanus

Keyword(s):

Dna Sequences ◽

Bacterial Species ◽

Single Copy ◽

Cross Reactivity ◽

Clavibacter Michiganensis ◽

Subtraction Hybridization ◽

Detection Systems ◽

Clavibacter Michiganensis Subsp ◽

Polymerase Chain ◽

Primer Sets

Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

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