Kent M. Reed

Multi-platform next-generation sequencing of the domestic Turkey (Meleagris gallopavo): Genome assembly and analysis

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome.

Intraindividual and Interspecies Variation in the 5S rDNA of Coregonid Fish

Abstract. This study was designed to characterize further the nontranscribed intergenic spacers (NTSs) of the 5S rRNA genes of fish and evaluate this marker as a tool for comparative studies. Two members of the closely related North American Great Lakes cisco species complex (Coregonus artedi and C. zenithicus) were chosen for comparison. Fluorescence in situ hybridization found the ciscoes to have a single multicopy 5S locus located in a C band-positive region of the largest submetacentric chromosome. The entire NTS was amplified from the two species by polymerase chain reaction with oligonucleotide primers anchored in the conserved 5S coding region. Complete sequences were determined for 25 clones from four individuals representing two discrete NTS length variants. Sequence analysis found the length variants to result from presence of a 130-bp direct repeat. No two sequences from a single fish were identical. Examination of sequence from the coding region revealed two types of 5S genes in addition to pseudogenes. This suggests the presence of both somatic and germline (oocyte) forms of the 5S gene in the genome of Coregonus. The amount of variation present among NTS sequences indicates that accumulation of variation (mutation) is greater in this multicopy gene than is gene conversion (homogenization). The high level of sequence variation makes the 5S NTS an inappropriate DNA sequence for comparisons of closely related taxa.

Molecular cytogenetic analysis of the double-CMA3 chromosome of lake trout, Salvelinus namaycush

The chromosome possessing two chromomycin A3 (CMA3) staining sites in lake trout (Salvelinus namaycush) was examined using FISH. All CMA3-bands in the karyotype contained ribosomal DNA (rDNA), including both sites on the double-CMA3 chromosome. One rDNA site on this chromosome was bordered by telomeric repeats, implicating a rearrangement in the origin of the second NOR.

Chromosome painting supports lack of homology among sex chromosomes in Oncorhynchus, Salmo, and Salvelinus (Salmonidae)

The sex chromosome pair has been identified previously as the largest submetacentric pair in the genome in several species of the genus Salvelinus (eastern trouts and chars) including S. namaycush (lake trout) and as a large subtelocentric/acrocentric pair in several species of the genus Oncorhynchus (Pacific trouts and salmon). Sex chromosomes have not been identified in Salmo (Atlantic salmon and brown trout). Two paint probes, one specific for the short arm (Yp) and the other for the long arm (Yq) of the sex chromosome pair in Salvelinus namaycush were hybridized to chromosomes of Oncorhynchus mykiss (rainbow trout) and O. tshawytscha (chinook salmon) and Salmo salar (Atlantic salmon) and S. trutta (brown trout). The two probes hybridized to two different autosomal pairs in each of the Oncorhynchus species, supporting lack of homology between the sex chromosomes in the two genera. The Yp probe hybridized to interstitial regions on two different chromosome pairs in S. salar and one pair in S. trutta. The Yq probe hybridized to a different pair in both species.

Application of fluorescence in situ hybridization (FISH) techniques to fish genetics: a review

Abstract The technology is now in place for major advances in the genetics and cytogenetics of fishes at the molecular level. One promising method with broad application is fluorescence in situ hybridization (FISH). Methodologies of FISH and the current and potential uses of these chromosomal techniques in fish genetics are reviewed. Highly repetitive ribosomal genes (rDNAs) and the multicopy genes for histones have been localized in several fish species and are providing new information on the evolution of salmonid genomes. Microdissection techniques such as those used to examine the Y chromosome of lake trout can produce paint probes useful in determining chromosomal arm homologies between closely related species. Repetitive sequences isolated from various fish species have been localized to centromeres, telomeres, and sex chromosomes. Some of these are currently being used as species-specific, chromosome-specific or sex-specific probes in aquaculture of fishes. Centromeric and telomeric probes have been used to examine intraspecific chromosome rearrangements such as the transposition of rDNA. Similarly, these types of probes could be used as genome markers for examining interspecific hybrids and in the study of nuclear organization (i.e. the spatial arrangement of chromosomes in interphase cells and gametes) Chromosome mapping of single copy genes, microsatellite loci and syntenic gene groups is now possible with FISH techniques and will be useful in isolating quantitative trait loci (QTL) of importance in aquaculture.

Polymorphism of the nucleolus organizer region (NOR) on the putative sex chromosomes of Arctic char (Salvelinus alpinus) is not sex related

Polymorphism of the nucleolus organizer region (NOR) on the putative sex chromosomes of Arctic char (Salvelinus alpinus) was examined using conventional cytogenetic and molecular techniques. Variation was observed in the number, size and position of rDNA loci on the sex pair. Fluorescence in situ hybridization (FISH) analyses showed that the sex chromosomes of Arctic char lack the repetitive DNA sequences (MboI/BglII family) that are a prominent feature of the sex chromosomes of lake trout (S. namaycush). Southern analyses of genomic DNAs using an rDNA fragment as probe revealed extensive restriction fragment length polymorphism (RFLP) variation among individuals. Despite the presence of variation in all aspects of this rDNA locus, no sex-specific differences were detected. Repetitive DNAs (multicopy rDNA as in Arctic char or tandem repetitive DNA as in lake trout) appear to play important but different roles in the evolution of the sex chromosomes in these species.

Defining the turkey MHC: sequence and genes of the B locus.

The MHC, the most polymorphic and gene dense region in the vertebrate genome, contains many loci essential to immunity. In mammals, this region spans ∼4 Mb. Studies of avian species have found the MHC to be greatly reduced in size and gene content with an overall locus organization differing from that of mammals. The chicken MHC has been mapped to two distinct regions (MHC-B and -Y) of a single chromosome. MHC-B haplotypes possess tightly linked genes encoding the classical MHC molecules and few other disease resistance genes. Furthermore, chicken haplotypes possess a dominantly expressed class I and class II B locus that have a significant effect on the progression or regression of pathogenic disease. In this study, we present the MHC-B region of the turkey (Meleagris gallopavo) as a similarly constricted locus, with 34 genes identified within a 0.2-Mb region in near-perfect synteny with that of the chicken MHC-B. Notable differences between the two species are three BG and class II B loci in the turkey compared with one BG and two class II B loci in the chicken MHC-B. The relative size and high level of similarity of the turkey MHC in relation to that of the chicken suggest that similar associations with disease susceptibility and resistance may also be found in turkey.

Localization of repetitive DNAs to zebrafish (Danio rerio) chromosomes by fluorescence in situ hybridization (FISH)

The genome of the zebrafish, Danio rerio, contains two major classes of tandem repetitive elements (AT-rich and GC-rich). The AT-rich repeats can be further subdivided into two subgroups which differ by about 10% of 185 bp in the repeating unit. The chromosomal location of these sequences and the moderately repetitive 5S rDNA sequences was determined in two diploid zebrafish cell lines using in-situ hybridization with fluorochrome-labeled probes. The AT-rich sequences were found at the centromeres of all chromosome pairs and the GC-rich sequences were found in paracentromeric location on over half of the chromosomal pairs. Different patterns of hybridization were found for the two subgroups of the AT-rich family. One type hybridized primarily to centromeres of one half to two thirds of the chromosomal pairs and the other type to centromeres of about three fourths of the chromosomal pairs. The pattern of hybridization with the GC-rich sequences varied somewhat between the cell lines consistent with interindividual variation in the location of paracentromeric heterochromatin. The 5S rRNA genes are found on the long arm of chromosome 3. Most of this chromosome arm is late replicating, but apparently does not contain either the AT-rich or GC-rich repetitive sequences.

Molecular characterization and cytogenetic analysis of highly repeated DNAs of lake trout, Salvelinus namaycush

The chromosomes of lake trout (Salvelinus namaycush) contain a considerable amount of heterochromatin located at the centromeres and/or telomeres of several chromosomes, including a sex-specific block located distally on the X chromosome. In order to investigate further the repetitive DNAs of lake trout, genomic DNA from a female was size fractionated (<600 bp) with the restriction endonuclease AluI and fragments were cloned into the bacteriophage M13. A total of 42 clones were isolated. Relative copy number of individual inserts within the lake trout genome was estimated by Southern analysis. Twelve clones were determined to be highly repetitive and were chosen for further investigation. Inserts of these clones contained sequences similar to the AluI/RsaI, EcoRI/DraI, DraI/BstEII, and MboI/BglII families reported from Arctic char (Salvelinus alpinus). The chromosomal location of several of these fragments was determined in lake trout by fluorescence in situ hybridization (FISH). Two related AluI/RsaI sequences (Type A, ∼ 140 bp, and Type B, ∼ 120 bp) showed differential hybridization. Type A hybridized to the centromeres of all metacentric as well as several acrocentric chromosomes. Type B hybridized to the centromeres of most acrocentric chromosomes. A sequence with homology to the EcoRI/DraI family hybridized to the centromeres of several acrocentric chromosomes. Sequences with partial similarity to the DraI/BstEII family hybridized to the major rDNA sites (nucleolar organizer regions, NORs) and several minor telomeric sites. The interstitial and telomeric heterochromatin of lake trout, including that of the X chromosome, appears to comprise sequences belonging to the MboI/BglII family.

Characterization of charr chromosomes using fluorescence in situ hybridization

The chromosomal locations of several families of tandem repetitive DNA sequences and the 5S rDNA were determined using fluorescence in situ hybridization (FISH) in the five North American charr species: Salvelinus namaycush, S. fontinalis, S. alpinus, S. malma, and S. confluentus. The pattern of hybridization of three centromeric repetitive sequences previously isolated from S. namaycush and S. alpinus was unique in each species. Dual-color FISH experiments showed that in several species many of the centromeres had the EcoRI-DraI family in addition to either the AluI-RsaI type A or type B families. The EcoRI-DraI family which was found only at the centromeres of acrocentric chromosomes in S. namaycush, S. fontinalis and S. malma was also found at centromeres of selected metacentrics in S. alpinus (one pair) and S. confluentus (four pairs) whose chromosomes have undergone additional centric fusions compared to the other species. The locations of 5S rDNA sequences were different in each species except for the two most closely related (S. alpinus and S. malma). Two whole-arm chromosome paint probes, one specific for the short and the other for the long arm of the lake charr sex chromosomes, hybridize to the same chromosome pair in all species. Results with other paint probes suggest that independent centric fusions have occurred in S. alpinus and S. confluentus which is consistent with the phylogenetic tree obtained previously for Salvelinus with cytogenetic and DNA data.