Physical mapping of the Nile tilapia ( Oreochromis niloticus) genome by fluorescent in situ hybridization of repetitive DNAs to metaphase chromosomes—a review

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  Physical mapping of the Nile tilapia ( Oreochromis niloticus) genome by fluorescent in situ hybridization of repetitive DNAs to metaphase chromosomes—a review
  Physical mapping of the Nile tilapia( Oreochromis niloticus ) genome by fluorescent in situ hybridization of repetitive DNAsto metaphase chromosomes—a review Cesar Martins a, *, Claudio Oliveira a  ,Adriane P. Wasko a  , Jonathan M. Wright   b a   Departamento de Morfologia, Instituto de Biocieˆncias, Universidade Estadual Paulista,CEP 18618-000, Botucatu, SP, Brazil   b  Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1 Received 30 May 2003; received in revised form 14 July 2003; accepted 6 August 2003 Abstract The Nile tilapia ( Oreochromis niloticus ) has received increasing scientific interest over the past few decades for two reasons: first, tilapia is an enormously important species in aquacultureworldwide, especially in regions where there is a chronic shortage of animal protein; and second, thisteleost fish belongs to the fascinating group of cichlid fishes that have undergone a rapid andextensive radiation of much interest to evolutionary biologists. Currently, studies based on physicaland genetic mapping of the Nile tilapia genome offer the best opportunities for applying genomics tosuch diverse questions and issues as phylogeography, isolation of quantitative trait loci involved in behaviour, morphology, and disease, and overall improvement of aquacultural stocks. In this review,we have integrated molecular cytogenetic data for the Nile tilapia describing the chromosomallocation of the repetitive DNA sequences, satellite DNAs, telomeres, 45S and 5S rDNAs, and theshort and long interspersed nucleotide elements [short interspersed nuclear elements (SINEs) andlong interspersed nuclear elements (LINEs)], and provide the beginnings of a physical genome mapfor this important teleost fish. D  2004 Elsevier B.V. All rights reserved.  Keywords:  Physical mapping; Chromosomes; Repetitive sequences; Nile tilapia; Genome;  Oreochromis niloticus 0044-8486/$ - see front matter   D  2004 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2003.08.017* Corresponding author. Tel.: +55-14-68026322; fax: +55-14-68213744.  E-mail address: (C. Martins) 231 (2004) 37–49  1. Introduction Cichlids are the predominant fishes of the Afr ica’s Great Lakes t hat are now distributedworldwide in tropical and subtropical regions (Trewavas, 1983). Owing to their rapid adaptive radiation, which has led to an extensive ecological diversity, and the great importance of some species to tropical and subtropical aquaculture, the cichlid fishes havereceived increasing scientific interest  (Pullin, 1991). The diversity of the species—more than 1500 species just in Africa—is of primary scientific interest. The African cichlids can be divided into three major grou ps: (1) Pelmatochromine cichlids, (2) Haplochrominecichlids and (3) Tilapiine cichlids (Lowe-McConnell, 1991). The Haplochromine cichlids of East and Central Africa, especially those of the Rift Valley Lakes, have undergone aremarkable speciation event and are now considered as a classic example of adaptiveradiation (Liem, 1991). It is believed that there ar e 500–1000 diff erent cichlid species adapted to specific niches solely in Lake Malawi (Stiassny, 1991). The Tilapiine tribe, commonly named tilapia, includes the genera  Sarotherodon ,  Oreochromis  and  Tilapia , andthe fourth genus,  Danakilia , which comprises a single species.Although approximately 70 species of cichlids are referred to as ‘‘tilapia’’, only Oreochromis niloticus ,  Oreochromis mossambicus  and  Oreochromis aureus , and their hybrids, have great importance in world fisheries. Nowadays, the Nile tilapia,  O. niloticus ,represents one of the most widely farmed freshwater fish in the world. Aquaculture practices may inadvertently decrease the genetic variability present in farmed stocks by theselection and breeding of related individuals, or by the use of a small number of parents as broodstock. Unless careful genetic records are maintained, there is a likelihood for increased inbreeding. Many of the domesticated strains of the Nile tilapia suffer frominbreeding depression and the best-performing strains are those most recently isolatedfrom nature. In genetic terms, knowledge of the Nile tilapia genome is rather preliminary,and far behind pufferfish and the zebrafish. Owing to its scientific importance tofundamental and applied biology, genomic studies and genome maps for the Nile tilapiaare timely and surely warranted. 2. Genome mapping In the past decade, DNA-based genetic markers have been developed for use inaquaculture primarily with the goal of improving fish stocks and strains for important traitssuch as growth enhancement and resistance to viral and bacterial disease. Various DNAmarkers have been used for the construction of genetic maps that will also be of particular  benefit in aquaculture, specifically for stock identification, selective breeding analysis of quantitative traits, and assessing the genetic variability of populations. Genome maps willalso find applications in behavioural, morphological, phylogeographic and other evolu-tionary studies.Currently, genome maps are constructed in one of three ways; (1) physical maps that localize DNA segments onto the karyotype of a species by cytogenetic methodologies; (2)genetic linkage maps that assign the linear order of DNA markers along a chromosome based on recombination frequencies between loci; and (3) the ultimate map, the complete C. Martins et al. / Aquaculture 231 (2004) 37–49 38  nucleotide sequence of a species’ genome. The construction and integration of physicaland genetic maps for the Nile tilapia currently represents the best strategy for theunderstanding of the structure and evolution of the cichlid genome.Cytogenetic st udies have shown that the haploid genome of the Nile tila pia consists of 22 chromosomes (Kornfield et al., 1979; Majumdar and McAndrew, 1986) with a genome size of 1.2 pg, or about 10 9  bp (Hinegardner and Rosen, 1972). To date, polymorphic DNA markers have been ap plied in the construction of a genetic linkage map for the Niletilapia (Kocher et al., 1998) with the goal of identifying single genes or quantitative trait loci which might be useful for the improvement of Nile tilapia production and other applications. Physical mapping of the chromosomes of   O. niloticus  also represents a powerful way to trace useful genetic markers for the improvement of the Nile tilapia production. Molecular cytogenetic information described for the Nile tilapia is related tothe structure and the distribution of repetitive DNA sequences in the chromosomes of thisspecies. Except for the first chromosome pair, the remaining chromosomes in the Niletilapia karyotype are nearly identical in morphology and size, which makes identificationof particular karyotypic elements difficult. The integration of a physical and a genetic maphas been hindered due to the absence of specific chromosome markers for this species and,until recently, limited information on the structure of the Nile tilapia chromosomes andtheir identification was available. 3. Chromosome markers in the Nile tilapia A substantial fraction of the eukaryotic genome, in some instances greater than 90%,consists of repetitive DNA sequences. These include satellite, minisatellite and micro-satellite sequences, transposable elements, and multigene families such as the ribosomalRNA gene clusters. Although studied extensively for the past four decades, the molecular forces that propagate and maintain repetitive DNAs in the genome are still not wellunderstood. However, the role of these DNAs in genome organization and evolution, andtheir likely impact on speciation is increasingly appreciated (Charlesworth et al., 1994).In the course of identification of chromosome markers for the Nile tilapia, severalrepetitive DNA families have been isolated and their organization investigated in thegenome of this species. Here we review the molecular cytogenetic data concerning satelliteDNAs, telomeric sequences, 45S rDNA, 5S rDNA, SINEs and LINEs in the Nile tilapia. 3.1. Satellite DNAs C-banding of metaphase chromosomes of   Oreochromis  provided evidence that heterochromatin is localized along and around the centromeres of the chromosomes of all species of the  Oreochromis  genus so far studied (Majumdar and McAndrew, 1986;Oliveira and Wright, 1998). As C-band positive segments are mostly composed of satellite DNAs, investigations have been directed towards the isolation and character-ization of such sequences in the genome of the Nile tilapia. Two highly repeatedtandemly arrayed sequences have been cloned, sequenced and their genomic organiza-tion determined. The first isolated satellite, SATA (Table 1), is approximately 237-bp C. Martins et al. / Aquaculture 231 (2004) 37–49  39  length (size and sequence variation has been observed in related tilapiine species)(Wright, 1989; Franck et al., 1992) and is present in the genomes of other Africancichlids with copy numbers ranging from 0.1 to 5.41  10 5 depending on the speciesexamined (Franck et al., 1994). The second satellite sequence, SATB (Table 1), although not as abundant as SATA, is a highly reiterated sequence (1–10  10 3 copies) of 1900 bp present in the Nile tilapia genome and also in the closely related cichlid tribe,Haplochrominae, but not present in the genomes of Asian or New World cichlids(Franck and Wright, 1993).Fluorescent in situ hybridization (FISH) of SATA (Oliveira and Wright, 1998) showedthat this abundant satellite DNA is localized primarily, if not exclusively, in thecentromeric region of all the Nile tilapia chromosomes, and is also present in the short arms of two chromosome pairs (Fig. 1).The presence of abundant copies of the SATA satellite at the centromeres of allchromosomes of the Nile tilapia, as detected under high stringency conditions, suggeststhat some form of amplification and intragenomic turnover must occur in this species. Insitu hybridization data from mouse (Hamilton et al., 1990), in which satellite DNAs are distributed in all chromosomes, suggest that in some species, there is a tremendousintragenomic movement of these repetitive elements among non-homologous chromo-somes and that mechanisms of genomic turnover are capable of distributing and homog-enizing repeat units of a given satellite DNA family throughout the genome. Consideringthat SATA is also present in the genome of other tilapiine and haplochromine species(Franck et al., 1994), a possible alternative hypothesis is that this satellite sequenceoriginated and spread in an ancestor of this group, and has been maintained in thecentromeres of all chromosomes owing to the functionality of this repetitive sequence.Most copies of the satellite SATB sequence are located in the short arm of only onechromosome pair (chromosome 4— Fig. 1) of the Nile tilapia, as demonstrated by FISH experiments. However, copies of this repetitive DNA are probably also present in other chromosomes of the species (Oliveira and Wright, 1998) as weak fluorescent signals were Table 1Characteristics of repetitive sequences mapped in the chromosomes of the Nile tilapia ( O. niloticus )Repetitive sequence Repeat type Monomer size (bp) Chromosome locationSATA Satellite 237 Centromeric region of all chromosomesSATB Satellite 1900 Short arm of chromosome 4(TTAGGG) n  Telomeric All telomeres and two interstitial loci inthe long arm of chromosome 1CiLINE2 LINE 1165 a  Dispersed over all chromosomes andenriched in the long arm of chromosome 1ROn-1 SINE 345 Small clusters dispersed in all chromosomesand a large cluster in the long armof chromosome 1ROn-2 SINE 359 Small clusters dispersed in all chromosomes18S rDNA 45S rDNA Chromosomes 8, 10 and 155S rDNA type I 5S rDNA 1417 Chromosome 35S rDNA type II 5S rDNA 475 Chromosomes 9 and 13 a  Length of cloned element. Other CiLine2 elements in the Nile tilapia genome may be considerably larger. C. Martins et al. / Aquaculture 231 (2004) 37–49 40  observed in many chromosomes. Alternatively, these weak signals may be related to the presence of sequences exhibiting partial nucleotide identity to SATB sequences. Thedistribution of satellite DNA sequences in just one or in few chromosomes, as observed for SATB, seems to be a common pattern found in several fish species (Garrido-Ramos et al.,1994; Canapa et al., 2002).Restriction digestion experiments with  Eco RI and  Hae III, enzymes that have recogni-tion sites within the SATA and SATB sequences (Franck et al., 1992; Franck and Wright,1993), were also performed on the chromosomes of the Nile tilapia (Oliveira and Wright,1998) and supported the chromosomal location of SATA and SATB sequences determined by FISH. Most C-bands of the chromosomes of the Nile tilapia seem to be composed of the satellite DNA sequences, SATA and SATB. However, since some heterochromatinsegments do not correspond to SATA or SATB, as for example the short arm of chromosome pair 1 (Oliveira and Wright, 1998), it is more likely that other repetitive sequences exist in the chromosomes of   O. niloticus  and their further isolation andcharacterization could contribute to the identification of other chromosome markers for the Nile tilapia. 3.2. Telomeres Telomeres are composed of DNA and proteins found at the ends of linear eukaryoticchromosomes (Shippen, 1993). The basic structure and function of the telomere have been conserved throughout evolution, reflecting its important role in stabilizing chromosomesand blocking chromosome end-to-end fusion and degradation (Blackburn and Szostak,1984). Telomeric DNA is composed of tandem arrays of a species-specific 5–8-bp GT-rich sequence, termed telomeric repeat  (Shippen, 1993). Vertebrates from fish to humans share a common telomeric repeat sequence, (TTAGGG) n . The length of these arrays variesfrom species to species. Fig. 1. Ideogram representing the physical mapping of repetitive sequences in the chromosomes of the Niletilapia ( O. niloticus ). The chromosome position of the different sequences is distinguished by colour: red,SATA; speckled in black, SATB; yellow, 45S rDNA; blue, telomere; faint green, 5S rDNA type I; dark green,5S rDNA type II. C. Martins et al. / Aquaculture 231 (2004) 37–49  41
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