RECENT ADVANCES OF CYTOGENETICS IN FISHERIES

Kiran Rasal; M. Khan; Makwana Nayan; Murali S.;

Avinash Rasal; Archana Durgude; Rashmi Ambulkar

Central Institute of Fisheries Education; Mumbai

Corresponding Author - kirancife@gmail.com

 

INTRODUCTION

Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes . Barbara McClintock and Harriet Creighton were the ones who worked on maize in early years popularizing cytogenetics.

              Fish make up half of the extant vertebrate species, they exhibit a huge level of biological diversity and, as food resource, they play major roles in the culture and economy of human populations. For most of the biologists, cytogenetics begins to be recognized as an essential tool to approach questions in both basic and applied ichthyology.

 

CYTOGENETICS-A BREIF HISTORY

Modern cytogenetics is generally said to have begun in 1956 with the discovery that normal human cells contain 46 chromosomes by Tjio and Levan. This discovery was aided by a new technique of slide preparation utilizing a hypotonic solution discovered by TC Hsu in 1952. With that advent of harvest procedures which allowed easy enumeration of chromosomes, discoveries were quickly made in abnormalities arising from nondysjunction events which cause cells with aneusomy.

      In the late 1960's Caspersson developed banding techniques which differentially stain chromosomes. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Deletions within one chromosome could also now be more specifically named and understood. Diagrams indentifying the chromosomes based on the banding patterns are known as cytogenetic maps. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations.. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid, and elongation techniques for all culture types that allow for higher resolution banding

 

Cytogenetics Ð RECENT ADVANCES, THE Beginning of molecular cytogenetics

FISH

In the 1980s advances were made in molecular cytogenetics. FISH (fluorescence in situ hybridization) is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

   This change significantly increased the usage of probing techniques as fluorescently labeled probes are safer and can be used almost indefinitely.

 Chromosome micro dissection

 Chromosome micro dissection is a technique that physically removes a large section of DNA from a complete chromosome. The smallest portion of DNA that can be isolated using this method comprises 10 million base pairs - hundreds or thousands of individual genes. To prepare cells for chromosome micro dissection, a scientist first treats them with a chemical that forces them into metaphase: a phase of the cell's life-cycle where the chromosomes are tightly coiled and highly visible. Next, the cells are dropped onto a microscope slide so that the nucleus, which holds all of the genetic material together, breaks apart and releases the chromosomes onto the slide. Then, under a microscope, the scientist locates the specific band of interest, and, using a very fine needle, tears that band away from the rest of the chromosome. The researcher next produces multiple copies of the isolated DNA using a procedure called PCR (polymerase chain reaction). The scientist uses these copies to study the DNA from the unusual region of the chromosome in question.

 

Comparative genomic hybridization

Comparative genomic hybridization (CGH) or Chromosomal Microarray Analysis (CMA) is a molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA and often in tumor cells.

CGH will detect only unbalanced chromosomal changes. Structural chromosome aberrations such as balanced reciprocal translocations or inversions can not be detected, as they do not change the copy number.

Virtual Karyotype

Recently, platforms for generating high-resolution karyotypes in silico from disrupted DNA have emerged, such as array comparative genomic hybridization (arrayCGH) and SNP arrays. Conceptually, the arrays are composed of hundreds to millions of probes which are complementary to a region of interest in the genome. The disrupted DNA from the test sample is fragmented, labeled, and hybridized to the array. Knowing the address of each probe on the array and the address of each probe in the genome, the software lines up the probes in chromosomal order and reconstructs the genome in silico .

Virtual karyotypes have dramatically higher resolution than conventional cytogenetics. The actual resolution will depend on the density of probes on the array. Currently, the Affymetrix SNP6.0 is the highest density commercially available array for virtual karyotyping applications. It contains 1.8 million polymorphic and non-polymorphic markers for a practical resolution of 10-20kbÑabout the size of a gene. This is approximately 1000-fold greater resolution than karyotypes obtained from conventional cytogenetics.

 

Fish cytogenetics

         Only limited knowledge was available in fisheries about assessing biodiversity in a river basin, to control the reproduction in a cultured species, from studying the ancestry of a given lineage to tracking down DNA sequences in a fish genome, etc. These new molecular techniques when introduced into fisheries created a revolution as there were a lot of things still to be learnt in this field. Summarized below are some of the recent findings in fish cytogenetics.

 

 

Advances

1)    A  comprehensive compendium of chaotic killifish karyotypes

    Martin Vlker , Petr Rb , Harald Kullmann karyotyped killifish.  Fish karyotypes are generally quite well conserved in evolution, with more than 50% of species having 2n=48 or 2n=50 chromosomes. However, some groups of killifishes (Cyprinodontiformes) show an enormous karyotypic variability which is most distinct in the African family Nothobranchiidae. Using a combination of cytogenetic and molecular phylogenetic methods, our studies of the nothobranchiid genus Chromaphyosemion revealed chromosome numbers ranging from 2n=20 to 2n=40, several phylogenetically independent reductions of 2n, a high diversity of NOR phenotypes, variability in the number of chromosome arms due to inversions and heterochromatin additions and possibly also independent evolutions of XX/XY sex chromosome systems.

2)    Cytogenetic studies of Atlantic salmon, Salmo salar L., in Scotland.

Chromosome numbers for Atlantic salmon, Salmo salar L., from ten populations in Scotland were ascertained. The majority of fish had 2n = 58, NF = 74 karyotypes, and no polymorphisms between populations were found. The findings suggest that Atlantic salmon in Scotland are cytogenetically homogeneous.

3)    FISH and DAPI staining of the synaptonemal complex of the Nile Tilapia

K. Ocalewicz , 2, J. C. Mota-Velasco, R. Campos-Ramosand , D. J.Penman of Institute of Aquaculture, University of Stirling used FISH to stain synaptonemal complex of Nile fish. Bivalent 1 of the synaptonemal complex (SC) in XY male Oreochromis niloticus shows an unpaired terminal region in early pachytene. This appears to be related to recombination suppression around a sex determination locus. To allow more detailed analysis of this, and unpaired regions in the karyotype of other Oreochromis species, they developed techniques for FISH on SC preparations, combined with DAPI staining. DAPI staining identified presumptive centromeres in SC bivalents, which appeared to correspond to the positions observed in the mitotic karyotype.

4) Cytogenetic characterization of the dwarf oyster (Ostreola  stentina )

 The chromosomes of O. stentina were studied using conventional Giemsa staining, chromosome measurements, C-and restriction endonuclease banding, and fluorescent in situ hybridization (FISH). Comparative analysis of the different karyotype patterns obtained for this species to those of other flat oyster for which data has been previously published was performed and provided new insights into oyster evolution and systematics within this family.

 

5) gene mapping of 28S rDNA sites in allotriploid Cobitis females

Among many natural Cobitis populations some polyploid hybrid forms were detected. There is a little information about genome rearrangement regarding diplo- and polyploid individuals.). Chromosomes were examined by Fluorescence in situ hybridization method (FISH) with 28S rDNA as a probe. The rDNA sites were identified in the terminal telomeric position of three submetacentric and four subtelo-acrocentric chromosomes. Moreover some of observed signals were stronger then others. One small sm/st chromosome possessing rDNA sites on both p and q arms seemed to be a marker of the karyotype of allotriploid females. This study brings useful information about polymorphism of 28S rDNA sites regarding their number, location and size in triploid specimens.

6) Mugilidae: towards a cytogenomic approach        

Approximately 25% among the over 70 species of Mugilidae (Teleostei) have been cytogenetically analysed. Most of them show the conservative 48 uni-armed karyotype, with small differences concerning the absence or the presence of short arms on a single subtelocentric chromosome pair. The Mugil curema species complex constitutes an exception, with karyotypes mainly or exclusively composed of bi-armed chromosomes and a conserved NF=48. Moreover, preliminary data from some species with the conservative karyotype have suggested the existence of different types of satellite DNA.

7) Chromosome studies of European leuciscine fishes

Leuciscine cyprinids possess 2n = 50 and extremely uniform karyotype with 8 pairs of m, 13 -15 pairs of sm and 2-4 pairs of st/a chromosomes. The largest pair is characteristically st/a element - "leuciscine" cytotaxonomic marker. However, the interspecific homology of this chromosome pair could not be assessed due to inability to produce serial banding patterns in fish chromosomes. In a  study, laser was used to dissect (10 - 15 copies of marker chromosome) whole chromosome probe (WCP) from karyotype of roach, Rutilus rutilus to ascertain the interspecific homology of marker chromosome using cross-species in situ hybridization. WCP was hybridized to chromosomes of widely distributed species,  consistently hybridized to the various proportions of distal part of longer arm indicating either sequence homology here and/or problem with production of WCP.

 

 

Reference:

1) http://en.wikipedia.org/wiki/Cytogenetics

2) http://en.wikipedia.org/wiki/DNA_microarray

3) http://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridization

4) Gene mapping of 28S rDNA sites in allotriploid Cobitis females (Pisces, Cobitidae) from a diploid-polyploid population

5) Cytogenetic studies of Atlantic salmon, Salmo salar L., in Scotland

    S. E. Hartley

    Department of Biological Science, University of Stirling, Stirling FK9 4LA, U.K.

6) Cytogenetic characterization of the dwarf oyster

 J Pereira (1), A Leit‹o (1,2), R Chaves (1), F M Batista (1,3), H Guedes-Pinto (1)

1. Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology (CGB-UTAD/IBB), 5001-801 Vila Real (Portugal)
2. INRB/L-IPIMAR. Avenida 5 de Outubro, 8700-305 Olh‹o (Portugal)
3. School of Ocean Sciences, Bangor University, Menai Bridge, Gwynedd, LL59 5AB, (UK).