Chromosomes of fish

 Genes display the mechanism of inheritance and chromosomes determine these mechnisms, thus according to W.S. Sutton (1902) the chromosomes provide the physical basis of heredity.  The chromosomes are named from the fact that they can be stained preferentially with certain dyes.  They appear thread-like at certain times and are known to be composed of linear complexes of deoxyribonucleic acid (DNA), the genetic material proper, and histone proteins which are believed to have a supporting or structural role.  The number of chromosomes per cell is characterstics of a species although in fish some variation can be observed between members of the same species and even between cells in one fish. 

The chromosome number in the cell of the body is normally made up of two sets, one of the maternal and one of paternal origin and this double state is described by the term diploid.  Within the animal kingdom the diploid number of chromosomes varies from two in some nematodes to over 200 in certain species of fish.  A single set of chromosomes of mature egg or spermatozoon is described as a haploid set.  Where more than two sets of chromosomes occur in cells, the condition is termed polyploid or more specifically, triploids for three sets, tetraploids for four sets and so on. 

The material of chromosome is called chromatin and two sorts are recognized-euchromatin which stains lightly and heterochromatin which stains darkly.  Euchromatin is believed to contain the genes in a linear array like beads on a string, while heterochromatin is regarded as genetically inert and to have a function in maintaining the structural integrity of the chromosome and perhaps regulating gene expression.  Heterochromatin is mostly made up of highly repeated simple sequences of DNA.  Two other features of chromosomes are the ends called telomeres, and a constriction called the centromere.  The telomeres are special in the sense that they are stable entities, usually heterochromatic which µround off¶ the chromosome thread.  If chromosomes are broken, the broken ends are µsticky¶ and tend to rejoin but not always in the original way. 

Telomeres are essential for maintaining the integrity of chromosome threads.  The centromere controls the movement of chromosomes during cell division.  Like the telomeres centromeres appear to be located in heterochromatin.  Of more obvious importance, however, is the position of the centromere along the length of the chromosome.  When it is located near to the centre, the chromosomes is described as metacentric.  Rod-shaped chromosomes have the centromere at or very near to one end and are called telocentric,  While acrocentric chromosomes are those with one arm very much longer than the other.  The prefix µsub¶ is sometimes used to describe intermediate cases. 

In addition to the major gross features of chromosomes sundry other constrictions may be seen following simple staining, and a feature of one or more pair of chromosome pairs in a nucleus may be a nucleolar organiser region (NOR).  Counts of chromosome arms number of arms or µnombre fondamental¶ (NF value) is extremely important in any consideration of chromosomes in evolutionary or breeding contets since this defines the genetic content of a chromosome complement.  Fish chromosomes are not easy to study as they are usually very numerous and often small and lacking individual characterstics.  The enormous taxonomic variation is obvious in fishes but some generalizations can be made.  The postulated primitive chromosome complement for teleosts is 2n = 48 with each of the 24 chromosomes acrocentric in form and of an evenly graded size. 

There are three main areas of intraspecific or intraindividual chromosomal variation 12Variation in the chromosome number (2n) or the number of chromosome arms (NF) Variation in the banding patterns of individual chromosomes. Variation in chromosomes content of the sexes.

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Chromosome number 
Chromosome number in fishes range from a low of n = 8 in the Cyprinodontid, Notobranchius rachovii and the anabantid, Sphaerichthys osphromonoides to a high of n = 84 in the Petromyzontiform lamprey, Petromyzon marinus. ‡ Acipenser ruthenus ‡ A. baceri ‡ A. naccarii 2n = 118 2n = 250 2n = 246 

About 35 to 40% of almost 500 studied fish species from 76 families and 26 orders have n = 24 chromosome. 70% of species have chromosomes numbers in the range n = 22-26 and 80% of species fall in the range 20-28. Two minor peaks are found in the ranges n = 40-52 (5-6%) and n = 82-84 (0.8%). First peak is for species from families Salmonidae, Cyprinidae, Catostomidae and Cobitidae, a few petromyzontiform lampreys, one chondrostean and a few skates of the order Rajiformes. 

The latter peak contains four species of Petromyzonti form lampreys  Salmoniform species have higher chromosome number (median = 36 haploid) than cyprinifrom species (median = 25 haploid)  They also are more variable in chromosome number. Within the salmonifarmes range of chromosome number is n = 11-51 and within Cypriniformes is n = 18-52.  Groups with chromosome numbers in the range n = 22-26 tend to be relatively invariant in chromosome number, where groups with higher or lower chromosome numbers tend to be more variable. 

There are three cytological mechanisms which may bring about changes in chromosome number 1- Polyploidization ± Duplication of chromosome number 2- Robertsonian rearrangements-where fusion of two acrocentric chromosomes or centric dissociation of a single metacentric into two acrocentrics results. 3- Aneuploidy where non-disjunction or endoreduplication results in gain or loss of individual chromosomes.

Chromosome morphology (size) 
The concept of µsymmetrical¶ versus µasymmetrical¶ karyotypes is used to indicate the apparent degree of chromosomal heterogeneity within a karyotype.  In perfectly symmetrical karyotype all chromosomes are approximately the same size and shape.  The trout karyotype is quite symmetric and by contrast minnow (cyprinidae) karyotype is highly asymmetric.  The symmetric karyotype of trout comprise the metacentrics one size group and the acrocentrics a second.  Cyprinidae karyotype is highly asymmetric. Apart from differences in chromosome size there are also obvious differences in centromere position even within groups of chromosomes of approximate size. 

The length of the average fish chromosome is between 2 and 5 m.  Very large chromosomes of 15-30 m (lung fish, Lepidosiren paradoxa or the extremely bizzare SM (Supermacro) chromosome in two forms of the family Diretmidae (Beryciformes) are rare.  Extremely small chromosomes (micro-chromosomes) have been reported in a few species.  Between 26 and 48 micro chromosomes are found in the karyotypes of three very primitive species, Hydrolagus colliei (rat fish), Scaphyrhynchus platorhynchus (sturgeon) and Lepisosteus productus (gar).

Genome size 
The amount of DNA per nucleus, also shows wide variation among fishes.  The genome size is also referred as the C-value.  The C-value can be estimated by cytophotomentry. The blood cells are at first fixed with acetic acidethanol, then hydrolysed with hydrochloric acid, stained with Feulgen stain and measured by cytophotometer to get the C-value. By µflow cytometry¶ C-value can also be estimated.  Haploid DNA contents range from 0.4 pg per nucleus in tetradontifarm puffers Fugu rubripes to 124 pg per nucleus in the lung fish, Lepidosiren paradoxa or 142 pg in Polypterus aethiopicus.  DNA contents of 195 species shows distribution around a strong mode at 1.7 pg (diploid amount).  Among teleost fishes the estimated mode (haploid amount) is 1.0 pg.  There is usually homogeneity of DNA amount within families and lower taxonomic categories and genome sizes tend to be relatively stable despite changes in morphology and / or physiology. 

However, in Cyprinidae, Cyprinodontidae and Callichthyidae, DNA content may differ by more than twofold in different species.  Decrease in DNA content is associated with increasing specialization in body form and design.  More specialised species have less DNA per cell than do more generalised forms.  Specialised families with small genome sizes tend to be less variable in genome size and almost all families with very low average DNA contents per species (0.4 to 0.6 pg) have very little variation in DNA content among species.  Cytological mechanisms leading to increases in genome size include polyploidy, lateral increases through differential polynemy, longitudinal increases through accidental DNA doubling, unequal crossing over and regional disturbances in DNA replication.  Mechanisms leading to decreases in genome size include unequal crossing over, regional disturbances in DNA replication or misrepair of chromosome breaks. 

Based on the log normal distribution of DNA content observed among fishes, it was suggested that changes in genome sizes were small, numerous and cumulative and most likely stemmed from successive duplications and / or deficiencies.  Large changes such as implied by polyploidy were exceptional.  The decreases in genome size is due to loss of unnecessary and / or redundant DNA .  For the decreases in genome size in fishes one line of suggestion is that much of the reduction in genome size occurs during chromosomal rearrangements which produce changes in chromosome number.  Among diploid teleost fishes there is a highly significant positive correlation between chromosome number and genome size.  This correlation holds when species with probable polyploidy ancestry are excluded from calculation. 

Species or species groups with higher chromosome numbers tend to have larger genome sizes.  Although exceptions exist, the clear implication is that reduction in chromosome number is accompanied by reduction in genome size, and hence may be viewed as another process correlated with increasing specialization and advancement.  The trend in fish karyotype evaluation is toward smaller genome size.  This presumably is accomplished in part by chromosomal rearrangements which reduce chromosome number.  DNA loss may in itself be adaptive by altering certain biophysical parameters related to genome size and then too, reduction in chromosome number may be adaptive tightening linkage. 

It has been suggested that reduction in chromosome number (and also in genome size) in fishes may be associated with increasing habitat stability and effectiveness of food resource utilization.  One may further speculate that once a species or species group reaches a small genome size, chromosome structural changes which result in further DNA loss should no longer be easily tolerated.  If this is true highly specialized taxa should be relatively invariant in genome size and in karyotype. In general this is the case.  In the highly specialized order perciformes, the average species excluding those from the families (Scaridae and Gobiidae) has ca. 0.97 pg of DNA per haploid nucleus.  This estimate is relatively low when compared with most other teleostean orders. 

The perciformes are also relatively homogenous in genome size and chromosome number.  In contrast, species from the less specialized order Cypriniformes (excluding two cyprinids, Carassius auratus and Cyprinus carpio, and the family catostomidae, which are apparently polyploid) have on the average ca. 1.33 pg of DNA per haploid nucleus and also are more variable in genome size and chromosome number.  The same trend holds for comparisons within orders. Many species in the families Gobiidae (Perciformes), Callichthyidae and Loricarriidae (Siluriformes) each have average DNA contents higher than the average species in their respective orders.  They also appear to be more heterogenous in genome size and chromosome number.  This indication is that taxa with high DNA contents may have greater flexibility in terms of chromosomal rearrangement. 

There is no correlation between the genome size (the C-value) and organisimal or genetic complexity (Cavalier-Smith, 1978, 1985). This is called as C-value paradox.  A glaring example is the genome size difference between man (3.5 pg) and lung-fish (124 pg) or Polypteus (142 pg). Although man is the most advance organism from evolutionary point of view, its Cvalue is almost 35 times smaller than that of the lung-fish.

Units of measurement of genome size
1- Nuclear genome size of eukaryotes is usually measured in picograms (pg) of DNA (1 pg = 10 ± 12g). 2- 1 dalton (unit of relative atomic or molecular mass) = 1.66×10± 12pg. 3- 1 pg = 6.02X1011 dalton = 0.98 X 106 kb. 4- 1 kb = 1000 base pairs of double stranded DNA.

Chromosome numbers are numbers and DNA contents of a range of fish species

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Chromosomal basis of sex determination in fishes 
Majority of fishes reproduce bisexually and have separate sexes.  In nature generally sex ratio is approx 1:1 in most cases.  It is frequently assumed that sex determination depends to a large extent on genes which reside on a single pair of ³sex´ chromosomes or heterosomes.  Chromosomal basis of sex determination in fishes is quite variable.

1- Male heterogamety - I 
In this system males possess a pair of genetically nonhomologous heterosomes (X & Y) and produce both X- and Y- bearing sperm (heterogamety) while females possess two X chromosomes and produce only X- bearing ova (homogamety).  The two heterosomes, X and Y get segregated in different sperm (heterogamety) producing two different types of gametes.

2- Male heterogamety - II 
Evidence of XX : XO type of male heterogamety has also been found in a few fishes.  This form of heterogamety usually arises either from loss of the Y chromosome, or from fusion of the Y chromosome with an autosome or with the X.  Spermatogonial metaphases of salmoniform, Sternoptyx diaphana contained 2n = 35 chromosomes, the X being the largest among five acro-centric chromosomes. In meiotic II metaphases, two morphotypes were observed, one with n=18 and one with n=17.

3- Female heterogamety 
Female heterogamety of the WZ : ZZ type (WZ = has been found cytologically in fishes. , ZZ ) also 

Mosquitofish, Gambusia affinis and the stickleback, Apeltes quadracus have been observed to be female heterogametic.  The platyfish, Xiphophorus maculatus is both male (XX : XY) and female (WZ : ZZ) heterogametic.

4- Muliple Sex chromosomes 
Multiple sex chromosomes (X1X2Y) have been reported in a cyprinodontid killifish.  Mitotic karyotypes revealed that females (2n = 48) possessed five pairs of acrocentric chromosomes whereas males (2n = 47) possessed only 4 pairs of acrocentrics plus a single outsized metacentric.

5- Sex determining genes on autosomes 
In Poecilia reticulata is heterogametic (XX : XY ). Its exceptional individuals were heterosomally of one sex, but phenotypically and functionally of other sex.  These exceptional individuals were fertile in crosses to normal individuals of same sex chromosome constitution but of the opposite sex.  Exceptional females. (XX) crossed with normal (XX) produced all 

Exceptional (XY) crossed to normal (XY) produced male (2 XY, 1 YY) and female 1 XX offspring in a 3:1 ratio.  It was interpreted that male and female determining genes are situated throughout the genome.  Normally these autosomal genes are hypostatic to the hetersomal sex determining genes, the sex of an individual is function of its sex- chromosome constitution.  Through chance genetic or chromosomal recombinations, the sum of the autosomal male ± or female potency could override the usually epistatic sex chromosome genes, and thus produce the exceptional individuals.

6- Polygenic Sex determination 
In several species sex determination appears to be completely polygenic, there being no genetic or cytological evidence of sex chromosomes heterogamety.  Most thoroughly studied fish is Xiphophorus helleri in which polygenes in their manifold recombinations decide about the sex of a specimen.

Hermaphroditism and Sex determination in fishes 
Hermaphroditism is the normal and functional coexistence of both maleness and femaleness in an individual.  Two basic types of hermaphroditism (synchronous or balanced hermaphroditism and asynchronous or consecutive hermaphroditism) are observed in fishes.  Asynchronous or consecutive protandrous or protogynous. hermaphrodites are either 

It is suggested that sexuality in a hermaphrodtitic species is a process of sex differentiation rather than sex determination.  Evidence of genetically or morphologically differentiated sex chromosomes (heterosomes) in either synchronous or asynchronous hermaphrodites is not expected.

Qualitative and quantitative inheritance 
Phenotype- observable expression Genotype is its genetic composition. of a character and 

Different forms of a character are called as its traits tallness or dwarfness are the phenotypic traits of the character, height.  A gene may also occur in different forms called alleles. If a gene controls a character, the alleles control the trait of that character. In eukaryotes, each gene occurs in paired form.  A lacus is the point of location of a gene in the chromosome. Two identical alleles- homozygous, dissimilar allele heterozygous.

A comparison of qualitative and quantitative characters
Qualitative
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Quantitative

Traits fall into discrete 1- Traits show continuous phenotypic classes, e.g. variation, e.g. height of round vs. wrinkled seed students in a classroom. shape in a pea plant. Generally one gene 2- A number of genes control a controls a character and character and the effects of the effects of its alleles can individual genes can be perceived easily. not be observed. The environment apparently has no effect on the phenotype. 3- Environment affects the ultimate expression of the phenotype greatly.

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Mechanism of inheritance 4- Mechanism of inheritance investigated by counting investigated by measuring and comparing the ratios in the traits in a population the offspring. using statistical method.

Mendelian Inheritance 
Medaka albino arose from selected matings of pale coloured but normal eyed fish and exhibited the familiar features of absence of pigment on the body and in the eyes. 

Two matings between albino fish generated a total of 800 embryos all detectable as albinos.  This illustrates as useful characterstics of the albino trait that the albino trait clearly µbred true¶.  When normal wild type medaka were cross mated to albino, whichever parent was the albino, the offspring were all normal in appearance.  When these F1, individuals were mated together, their offspring comprised normal and albino embryos in the ratio 3:1 (actually 423:141, a good result from the statistical point of view). 

The albino parent in the Po genaration has the genotype aa (homozygous) comprising an albino allele received from each of its parents.  The wild type genotype is also homozygous, designated AA, these alleles being dominant to the recessive a.  In the F, generation, the offspring are all Aa heterozygotes and normal in appearance but produce eggs and spermatozoa which carry either A or a in roughly equal proportions.  Therefore in the F2, random union of eggs and spermatozoa leads to the creation of AA, Aa and aa offspring in the ratio of 1:2:1 which because of the normal appearance of Aa, leads to a ratio of 3 normal to 1 albino embryo.

Complex effects of alleles 
Two complicating issues associated with the above expositioned simplistic representation of mendelian inheritance are.

1- An effect of the albino allele on viability. 2  An effect on the interaction of the albino allele with other colour determining genes. The viability complication is reasonably straightforward from the 800 albino embryos only 29 reached adulthood. Among the F2 offspring of other cross-matings the ratio of normal to albino was not 3:1 as in embryos but 20 or more to 1. The albino homozygote aa was much less fit than the AA or Aa genotypes, both of which exhibit the normal wild type of phenotype.  

This finding was exactly in line with earlier studies of albinism in the Guppy, Poecilia reticulata, Swordtail, Xiphophorus helleri and threespine sticklebacks Gasterosteus aculeatus and the condition was described as a semilethal mutation. This secondary consequence of an allele on fitness is called Pleiotropy and is exceedingly common.  The second complication involved genes which control other aspects of pigmentation.  These other genes in Guppy (Poecilia reticulata) were first named as Gold and Blond.  Wild-type background colour of Guppy is usual greenish grey and the two genes represented by recessive alleles in the homozygous condition, gg or bb, both produced a xanthic body colour but by different mechanism.  Gold recessive phenotype±slightly reduced numbers of melanophores restricted to the edges of the scales this giving a more pronounced reticulated pigment pattern against an overall gold coloration. 

Blond recessive phenotype on the other hand comprised greatly reduced numbers of melanophores leading to a soft yellow body colour.  Both phenotypes included normal eye pigmentation and thus contrasted with the albino phenotype.  When homozygous albino fish crossed with homozygous Blond or homozygous Gold individuals all the offspring were normal.  Since these alleles are recessive it follows that the normal F1 offspring are heterozygote for both the albino allele and either the Gold or the Blond.  In other words the albino allele represents a different gene from the Blond or Gold alleles. 

Similarly Gold and Blond had been shown to be different genes i.e. non-allelic, so we have three genes all different, generating a reduction in or absence of melanin.  Finally in the F2 offspring the different genes segregated independently and some offspring were homozygous for the albino gene and either Blond or Gold recessive genes-these were not intermediate in colour between the albino and Gold or Blond type but indistinguishable from albino.  The albino phenotype over rode that of either of the other double recessives, this is termed epistasis and is a very common phenomenon in genetics.

Colour Genetics of Oryzias (Medaka)
In its natural habitat the fish of both sexes of medaka are a dull greenish grey colour with flecks of iridescence. Several colour varieties were developed by Japanese aquarists, which in contrast to the greenish colour of normal, wild type fish, were either solid red, white or blue and variegated red or white. Two different genes have been shown to be involved. One gene was responsible for the red or variegated red patterns. The red phenotype arises when the melanophore cells lack pigment and normally obscured erythrophores shows through. The variegated pattern arises when the melanophores are segregated into clumps instead of being distributed fairly evenly over the body. 

When a red fish is crossed with a wild type, the offspring were all wild type.  But when these offspring were crossed, the approximately three greenish offspring to one red. result was 

The gene for normal development of melanin ± B dominant over the recessive form of the gene (allele) for no pigment, symbolized b.  The variegated pattern was inherited in a rather different manner.  In crosses between red and variegated red the offspring were all variegated, the allele for variegated was therefore dominant over the allele b and was given the symbol B1.  In crosses between homozygous wild type (BB) and homozygous variegated (B1B1), the heterozygous offspring were also all variegated but to a lesser extent than in homozygous B1B1 individuals.  Thus B, B1 and b represented an allele series in which b was recessive to the other two which themselves interacted in a more or less additive fashion and are described as co-dominant.

Colour Polymorphism in Poecilia 
Brilliant coloration of the males was taken as an example of sexual dimorphism. The colouration of this stock of males comprised red and black spots and patches of metallic lustre which was constant from generation to generation, however contrasted in some populations which led to the examination of the geneties of the different colour forms.  Females have uniform grey colour.  The male colouration has two patterns which differered in many respects. Particularly the presence of a black spot on the dorsal fin.  The B has a spot on dorsal fin but not in A.  A cross between a male of type B and a female from stock A produced males of type B only. 

Similarly when type A males were used, male offspring always showed the type A pattern.  This father to son inheritance led to the conclusion that sex was determined chromosomally in Poecilia, male was XY and that the genetic control of male colour pattern was located in the Y chromosome  X- linked criss-cross type inheritance in which genes on the X chromosome are transmitted to offspring of both sexes from the mother but to female offspring only from father.  These genes were named sulphureus (Su) and Elongatus (El).  Their inheritance passes from the father to his daughters where they are not actually expressed because they are sex limited.  The F1 daughters pass the X-linked gene to their daughters or sons and in the latter they are expressed along with whatever genes the Y chromosome contains.

The important points.
12  3Y chromosome always has some colour pattern gene, Yo never observed. Although females carry colour pattern genes they do not show the appropriate phenotype These genes are therefore sex-linked and at the same time sex-limited in that they are expressed in males only. The Y are, in fact, expressed if females are masculinized with male sex hormones. In the male the genes are always expressed, whether on X or Y or on both, in other words they are dominant genes or alleles.

Colour in Platyfish Xiphophorus maculatus : most variable colouration 
The genetic control of its various colour patterns differs from that of Poecilia in that most of the genes are not sex-limited and although many of them are sex-linked, they do not form an integral part of sexual dimorphism in X. maculatus.  Thus although the Y chromosome in Poecilia always carries colour genes, the equivalent chromosome in the platy need not.  Colour is not a secondary sexual character in X. maculatus.  Its black strain is known as µNigra¶ inherited as a dominant sex-linked allele but female was the possessor of the different or contrasting pair of sex chromosomes.  Thus a cross between a Nigra female and a lightly pigmented maledescribed as µwhite¶ produced Nigra sons and white daughters.  Which in F2 produced approximately equal frequencies of Nigra and white individuals of each sex. 

The results of above crosses were interpreted that the differential sex chromosome in the female had a null allele O.  This type of sex determination, with a heterogametic female is conventionally described as WZ ( ) and ZZ ( ) system.  It is the normal mode of sex determination in birds and some moths and is not uncommon in fish.  Colour variations in X. maculatus involved three types of pigment cell; micromelanophore, and macromelanophore which produce black spotting of various degrees and erythrophores which produce a red colouration.  This red colouration is partly sex limited, showing more vividly in the male than in the female.  The mlanophore patterns are expressed equally strongly in males and females. 

A fourth type of pigment cell, the xanthophore, produces a yellow background colouration and is always present in wild fish.  Gold lacked both types of melonophores and also erythrophores, thus giving expression to the remaining yellow xanthophore pigments.  The presence or absence of micromelanophores was inherited independently of the macro-melanophore and erythrophore patterns which themselves were linked to the W chromosome.  The presence or absence of micro-melanophores determined by a gene on an autosome. was 

In a cross between a Gold female and a Red male, all the offspring's were Red .  Containing all four types of chromatophore, demonstrating the dominance of genes for the presence of chromatophores over those for their absence.  In the F2 offspring of the cross between these Red males and females, however, the patterns segregated into Red with micromelanophores (RSt), Red without micromelanophores (Rst), stippled (St) and Gold (st).  The numbers of offspring of each type approximated to the 9:3:3:1 ratio expected in a dihybrid cross (i.e. 3:1 × 3:1). 

It is clear that colour in fish can be determined by the action of genes with alternate alleles and generally it is not a simple dominance / recessive relationship.  Where complex pleiotropisms or epistatic interactions occur, further complicated by multiple genes and multiple alleles plus the complications of sex chromosome inheritance, the situation may be enormously variable.

Scale patterns of carp 
On the basis of scale patterns four basic types of carp are recognized. 1- Scale carp ± frequently described as wild carp 2- Mirror carp ± with a few large scales scattered about the body 3- Linear carp, in which the normal overlapping tiled effect of the scales is transformed into linear arrays running the length of the body. 4- The leather carp, with very few scales at all.  There are two µscaly¶ alleles at locus designated S and s and the mirror carp is homozygous for s; i.e. the genotype is ss (ss : nn). The allele s is fully recessive, so that genotypes Ss and SS are indistinguishable and fully scaled. 

The second gene also has two alleles but the one producing the linear form of carp is dominant over the recessive normal alleles.  The heterozygote Nn is a linear (Ss : Nn or SS : Nn) carp, the recessive homozygote nn is normally scaled and the dominant homozygote NN is not viable.  The fourth of four forms, the leather carp, is the combination of two phenotypes, mirror and linear, and such fish have the single genotype ss : Nn .

Scale pattern phenotypes in the common carp Cyprinus carpio. 

Because of the heterozygous nature of the linear genotype, linear and leather carp do not breed true.  Linear carp crossed together (Nn × Nn) produce offspring of which one third are homozygous for the normal allele (nn) and are therefore scaled or wild-type in appearance. The other two thirds are heterozygotes (Nn), and hence show the linear scale patterns and NN individuals do not survive.  The same segregation at the N gene occurs in the leather × leather cross, but interaction with the s gene, where only one allele is represented, generates one third mirror carp and two third leather carp.  Crossing of linear × leather produces either linear plus scaled fish or linear scaled and mirror carp, depending on whether or not the linear fish were heterozygotes.

Body shape 
The genetic control of body shape in fact deals with skeletal abnormalities, particularly those involving the spine.  Two different abnormalities of the spine in Oryzias latipes (medaka) are due to non-alletic genes and the recessive alleles were called wavy and fused, respectively.  Wavy in the homozygous condition caused a curvature of the spinal column in the vertical plane and in consequence reduced the length of the body relative to its depth.  The allele fused produced a similar body shortening reducing the length of the spinal column. by 

Studies of the inheritance of the two genes suggest that they are on different chromosomes. 

A mutant form in the guppy, similar to wavy in the Medaka determined by a recessive allele has also been reported.  The second of the spinal deformities in the Guppy was shown not to be inherited.  The cause for such deformities can be attributed environmental accidents, particularly around hatching time. to

Quantitative Genetics (I. Metrical) 
Quantitative genetics includes the study of two quite different branches of heredity.  The first, the genetic control of metrical traits- things one can measure such as size, growth rate, survival or number of scales or vertebrae ± is of very great importance in breeding of fish.  The second, on the structure of populations, deals in allele frequencies and is also of value in breeding practice, but its greatest impact is on the study of evolutionary mechanisms in the broader sense i.e. the dynamics of changes in the genetic structure of populations under selection, whether that selection is natural or artificial.  The genetics of metrical traits attempts to follow the fate of a multiplicity of genes, each of which has only a small effect and is indistinguishable from all of others. 

These minor genes are not definable like major genes of Mendelian hereditary pathways.  Polygenic inheritance is not measured in individuals but in populations, and it is the numbers of polygenes transmitted from one individual to the next which is of importance.  The second branch of quantitative genetics also concerns populations but with respect to the behavior of populations themselves.  The distribution statistics of the alleles within populations are of importance, not the nature of traits themselves, except in those cases the allele in question has an impact on fitness.  Any sort of genetic attribute can be used in population genetics but the most noteworthy advances have come from studies with electrophoretically detected variations which have simple genetic determination.

1- Polygenic inheritance 
The term polygene was proposed by K. Mather (1941) to idenitify genes which each had only a small effect on the magnitude of a measurable character such as height or length, numbers of vertebrae or number of bristles or wing veins in insects.  As the mendelian model of inheritance could not explain the continuous nature of the variation between individuals for such characters but the postulation of multifactorial gene control provided the means.  It is surprising how few polygenes are needed to provide for a more or less continuously variable inherited trait.  The inheritance of platemorphs in Gasterosteus aculeatus is controlled by two gene loci each with two alleles. If the range of plate numbers is 6 to 30 and each dominant allele contributes a fixed number of plates to the series of any fish. 

Then a fish with four recessives would have 6 plates one with one dominant 12 plates, one with two dominants 18 plates, with three dominants 24 plates and 4 dominants 30 plates.  If three loci comprising six alleles were involved the steps would be even smaller at 4 scales per dominant allele on top of the basic 6, such as 6, 10, 14, 18, 22, 26, 30.  Thus by increasing the number of possible genes involved it is possible to move towards a continuous or in the case of scale quasi- continuous variation.  Thus with only a few gene loci with small additive effects it is possible not only to construct continuosly variable characters but also to explain the frequency distributions which occur in natural and in reared populations of fish.  It is believed that polygenes are much more numerous than in the hypothetical example used here. 

The effect of a large number of genes plus the smoothing effect of a environ mental factors which have plus and minus effects on all measurable characterstics, leads to smooth variations and distributions.  Metrical traits are therefore assumed to be determined by polygenic and environmental agencies working together.  The task of quantitative genetics in this context is chiefly to estimate the extent of the genetic component of the variability.

2- Heritability 
Another aspect of the quantitative genetics is the assessment of the genetic and environmental control of measurable characterstics, such as performance traits defined by the concept of heritability  Most measurable characterstics in living things have an inverse bell shaped frequency distribution which approximates to the mathematically defined µnormal distribution¶ in which the key parameters are the mean and the standard deviation, which is the square root of the variance (the variance being computed as the mean of the summed squared deviations of individual observatioins from the mean. 

In statistics the standard deviation is the normal parameter used in calculations, in genetics the variance is usually used to describe populations.  The value of the variance (V) can be apportioned to an environmental component (VE) and a genetic component (Vg) and the latter can be further divided into additive (VA) and interactive (Vi) components. Thus V = VE + VI + VA  The additive variance contributes most to the similarity between relatives and provides for a successful selection programme for genetic improvement.  Heritability (h2) is that part of total variance which is additive and genetic in nature.  Heritability quantifies the percentage of phenotypic variance that is inherited in a predictable and reliable manner.

Thus V= VE +Vi + VA h2 = VA / Vp VA ± additive genetic variance Vp ± Phenotypic variance Heritability is expressed as a percentage (0-100% or 0.0-1.0) Total phenotypic variation (VP) with respect to a particular phenotypic character has three components : genotype (G), environmental (E) and genotype ± environmental (GE) interaction. This may be represented as VP = VG + VE + VGE  The genetic component (VG) can be further divided into three subcomponents. i) Additive effects (A), ii) dominance effect (D) and iii) epistatic effect (I). Thus VG = VA + VD + VI VA- Additive genetic variance is the sum of the effect of all alleles that helps to produce the phenotype. Additive effects are transmitted from the parents to the offspring without being disrupted during meiosis. VD and VI ± are disrupted during meiosis hence are not transmitted from parents but created afresh in every generation.

Broad Sense heritability
VG (total genetic variance) h2 = VP (total phenotypic variance) 

Since dominance and epistatic interactions are difficult components to estimate, only additive genetic component is generally considered for heritability estimation. VA (additive genetic variance) h2 = VP (total phenotypic variance)  This is called as Narrow Sense heritability. For a selection programme for improvement R = h2 S 

S- the selection pressure / selection differential is measured as the difference between the mean of the whole population and the mean of that group of organisms chosen to be parents of the next generation.  R is the selection response expressed in similar terms i.e. the difference between the mean of the original population and that of the next.  The estimation of h2 is done either by measuring correlations between relatives such as offspring and parents, or by recording progress under a selection programme- where h2 is assessed as the slope of the improvement curve, and sometimes called the realized heritability.  For fish it is imperative to stress that environmental factors, including maternal effects are of extreme importance. However, control of environmental variance in fish farming is at best very difficult.  Heritabilities = 0.25 or above±selection will produce good gains 0.15- selection will be ineffective

3- Growth rate 
This more than any other commercial trait has dominated the discussion on genetic improvement within fish farming.  However, most fancy fish are small and have a determinate growth pattern with a fixed final size whereas food fish on the other hand seem to have an indeterminate growth pattern with growth continuing albeit at an ever decreasing rate during the lifetime of the fish.  The fish of a determinate growth pattern generate eggs and spermatozoa by direct metabolism of food whereas those of indeterminate growth rate use food for increasing body size and eventually convent body materials into sex products due to which their continuous growth is possible.  It is considered that even slight improvement in body weight would result in millions of kilograms of additional production. 

Most fish farm outputs at the gross level are limited by food, space, oxygen and temperature and almost never by genetic potential.  The main barrier to a production application of quantitative genetics to growth rate, however, is the lack of strict control on the life cycle of the species used in fish farming.  The selection programme in trout culture after 10 years had attained maturity at 3yrs age and 3 kg weight or even 3 kg weight in 2 years with modern technology of feeding compared to attainment of maturity after attaining 600 g weight after 4 yrs.  The natural and hatchery productions are incomparable and difficulty in data interpretation was experienced as an unselected control line was not maintained.  The Washington strain of rainbow trout does not appear to have had much impact on commercial production in U.K. 

The most systematic study of the inheritance of growth rate in fish is that conducted by Moav and Wohlfarth, Israeli group.  The overall result of five generations of selection of carp for improved growth rate was disappointingly negative.  They had performed mass selection coupled with unselected controls.  It was concluded that failure to improve growth rate was because the additive variance for this trait had reached a plateau due to natural selection and past, unconscious, artificial selection.  Growth is dependent upon environmental factor and maturity.

4- Stock and strains 
There are no generally accepted definition of the terms stock, strain, line or variety.  They each represent genetic derivations from µspecies¶ with some natural and some domestication implications .  The stocks within a species, e.g. geographic µraces¶ might be expected to show variation for growth rate and other characterstics ± developed over tens of thousands of years or longer.  Recent domestication may have added to this by the natural choice of better µstrains¶ studies. With simple genetic scale pattern types suggested that the scaly form grew best followed in order of performance by mirror carp, leather and line. 

A similar analysis of strains of rainbow trout from different regions has also demonstrated very large differences in growth rate.  The growth rate differences between these rainbow trout strains were genetic in that successive generations exhibited continuity of the trait and more specifically, hybrids between slow and fast of rowers grew at an intermediate rate.

Spawning time 
Spawning can be controlled by environmental manipulations and hormonal control but methods are expensive and not always reliable.  The use of individual strains with natural inherent tendency of spawning at different times of the year is the best solution. 

This seems practical only for rainbow trout at present.  The domesticated strains of this species in use world- wide have a range of spawning times which spans the entire year.  The various strains collected for use in U.K. had the spawning times covering the autumn, winter and spring periods.  The spawning time in rainbow trout is genetically controlled and hybrids between the extremes were intermediate between two parents.  Anecdotal evidence suggests that the wide range of spawning times has been developed by selection and that a shift of about 1 month can be achieved in four or five generations of selection.

Food Conversion efficiency 
Above the level of maintenance ration, increasing the rations produces an increased weight increment up to a maximum beyond which further addition of the diet has no effect.  The FCE starts below zero, rises with increasing rations to a maximum then declines.  There are several points on these curves where selection might be applied, but in practice the parameters are so dependent on uncontrollable environmental variation than quantitative genetic management seems impractical.

Disease resistance 
Fish immune response systems are weakly developed.  Two basic systems are humoral (i.e. antibody mediated) and cellular (i.e. under the control of major histocompatibility systems).  Antibody formation is only very weak in fish and the cellular types of defence mechanisms seem to be the more important components of immunity.  Attempts to improve by selection the resistance of fish to specific diseases do not appear to have been highly successful.  Selection of brooktrout and browntrout for resistance to furunculosis for ten years did show some improvement but was impossible to quantify.

Population Studies by DNA analysis

Mitochondrial DNA (mt DNA)
‡ It is a circular molecule of double stranded DNA comprising about 1800 nucleotide bases. ‡ Segregation and crossing over do not occur in mtDNA. Nevertheless, some evolution can arise by base substitution. ‡ Presence of more than one type of mtDNA molecule in an individual does happen (heteroplasmy).

Nuclear DNA
‡ Nuclear DNA , nuclear deoxyribonucleic acid (nDNA), is DNA contained within a nucleus of eukaryotic organisms. In most cases it encodes more of the genome than the mitochondrial DNA and is passed sexually rather than matriclinally. Nuclear DNA is the most common DNA used in forensic examinations.

Principle of DNA isolation
‡ For the isolation of nuclear DNA the cells are disrupted with gentle homogenization and the nuclei are pelleted by low-speed centrifugation. ‡ The nuclear membrane is then lysed by sodium-dodecyl-sulphate (SDS) treatment that releases chromatin mass into the solution.

‡ The proteins present in the chromatin are removed by the treatment of protease enzyme followed by extraction with phenolchloroform. ‡ DNA in the solution is precipitated out by adding alcohol and salt. ‡ Isolated DNA in Tris-EDTA solution can be stored in the refrigerator at 4°C for years.

Satellites, Minisatellites & Microsatellites
‡ Differences occur in the number of repeated copies of a segment of DNA. These sequences can be classified on the basis of decreasing size into satellites, minisatellites and microsatellites. ‡ Satellite: ‡ (i) DNA consisting of long repeat units (100-1000 base pairs) i.e. Units of upto several thousand base pairs repeated thousands or millions of times. ‡ (ii) Due to abundance of repeats and slightly different base pair composition compared to bulk genomic DNA called ´Satelliteµ.

‡ Minisatellite: ‡ (i) DNA sequence of 10-64 base pairs with repetition of two to several hundred times at a locus. ‡ (ii) Second class of tandemly repeated DNA, consisting of shorter repeat units. ‡ Microsatellite: It has unit length of 1-4 base pairs repeated upto hundred times at a locus. It is also called simple sequence or short tandem repeat (STR) DNA.

‡ Length variation in tandemly arrayed repititive DNA particularly in the later two classes, is usually due to increases or decreases in repeat unit copy number. ‡ These differences in repeat numbers represent the basis for most DNA profiling technologies used today.

Random Amplified Polymorphic DNA (RAPD)
‡ RAPD techniques relies on the differential amplification of small DNA fragments using PCR with arbitrarily chosen oligonucleotide primers. ‡ This allows the detection of polymorphism without prior knowledge of nucleotide sequence. Polymorphism results either from chromosomal changes in the amplified region or base changes that alter the primer binding site. ‡ The procedure is rapid, which requires only small amount of DNA and involves no radioactivity. ‡ The RAPD-PCR primers are not designed to amplify a specific target sequence, so the amplified loci are scattered presumably throughout the genome. RAPD markers are dominant since polymorphism is detected by the presence or absence of bands.

Restriction Fragment Length Polymorphism (RFLP)
‡ It arises by loss or gain of restriction sites in the DNA sequence. ‡ Insertion or deletion of a DNA sequence also creates RFLP.

Molecular Genetics
‡ Combination of recombinant DNA technology with genetics. ‡ Molecular genetics is the field of biology which studies the structure and function of genes at a molecular level. The field studies show that the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology.

Restriction Endonucleases
‡ Restriction endonuclease enzymes or Restriction enzymes (RE) occur widely in bacteria. ‡ These enzymes protect them from the invasion of viral DNAs by cleaving them into pieces. ‡ A restriction enzyme cleaves both the strands of DNA duplex at paliondromic sites, i.e. the sequence with two fold axis of symmetry.

DNA Fingerprinting
‡ Microsatellite or minisatellite probe is used for DNA fingerprinting. ‡ DNA fingerprinting analysis uses multi-locus or singlelocus minisatellite probes. DNA fingerprinting with multilocus probe generates a large number of bands. It uses the whole minisatellite DNA probe. Single-locus DNA fingerprinting produces less number of bands. Singlelocus minisatellite probes are isolated from the genomic library by the use of oligonucleotide designed from the core sequence of the repeat present in intron region of the gene. This is done after computer analysis of the published nucleotide sequence. The probes used for fingerprinting are more polymorphic than RFLP probe.

‡ Multi-locus probes are universal while single locus probes are largely species or genus specific. ‡ The profiles of bands are referred as ³Fingerprint´.

Recombinant DNA Technology 
It is also called gene cloning or molecular cloning and is an umbrella term that encompasses a number of experimental protocols leading to the transfer of genetic information (DNA) from one organism to another.  No single set of methods used, but a recombinant DNA experiment often follows the following format. 1- The DNA (cloned DNA, insert DNA, target DNA, foreign DNA) from a donor organism is extracted, enzymatically cleaved (cut, digested) and joined to another DNA entity (cloning vector) to form a new recombined DNA molecular (cloning vector-insert DNA construct, DNA construct. 2- This cloning vector-insert DNA construct is transferred into and maintained within a host cell. The introduction of DNA into a bacterial host cell is called transformation. 3- These host cells that take up the DNA construct (transformed cells) are identified and selected (separates, isolated) from those that do not.

Plasmid Cloning Vectors 
Plasmids are self-replicating, double stranded, circular DNA molecules that are maintained in bacteria as independent extrachromosomal entities.  Virtually all bacterial genera have plasmids.  F plasmids- Carry information for their own transfer from one cell to another.  R plasmids- Encode resistance to antibiotics.  Degradative plasmids- Carry specific set of genes for the utilization of unusual metabolites.  Cryptic plasmids- Have no apparent functional coding genes.  Size- Less than 1 to more than 500 kb. 

Each plasmid has a sequence that functions as an origin of DNA replication; without this site it can not replicate in a host cell.  Some have 10 to 100 copies per host cell (high- copy numbered plasmids).  Others 1 to 4 copies per cell (low copy number plasmids).  Population of plasmids in a bacterium approx to 0.1 to 5% of the total DNA.  As autonomous, self replicating genetic elements, plasmids have the basic attributes to make them potential vectors for carrying cloned DNA.  Some plasmids, because of the specificity of their origin of replication, can only replicate in one specific species of host cell-narrow-host-range plasmid. 

Other plasmids have less specific origins of replication and can replicate in a number of bacterial species-broad-host-range plasmids.  When two or more types of plasmids can not coexist in the same host cell, they are said to belong to a single incompatibility group.  But plasmids from different incompatibility groups can be maintained together in the same cell.  Some microorganisms have been found to contain as many as 8 to 10 different plasmids.  Naturally occurring (unmodified, unengineered) plasmids often lack several important features that are required for a high quality cloning vector.

Important qualities of a quality Cloning vector are1- Small size which is necessary because the efficiency of transfer of exogenous (foreign) DNA into E. coli decreases significantly with plasmids that are greater than 15 kb. 2- Unique (single) restriction endonuclease recognition sites into which the insert DNA can be cloned. 3- One or more selectable markers (genetic) for identifying recipient cells that carry the cloning vector-insert DNA construct.  Consequently plasmid cloning vectors have to be genetically engineered.

Plasmid Cloning Vector pBR322 
pBR322 in one of the best studied and most often used µgeneral purpose¶ plasmid cloning vector.  In general, plasmid cloning vectors are designated by a lower case p, which stands for plasmids, and some abbreviation that may be descriptive, or as is the case with pBR322, anecdotal.  The BR of pBR322 recognizes the work of the researchers F. Bolivar and R. Rodriguez, who created the plasmid, and 322 is a numerical designation that has relevance to those workers.

Fig-

Genetic map of the plasmid cloning vector pBR322. Unique HindIII, salI BamHI, and PstI recognition sites are present within the genes for tetracycline resistance (Tetr) and ampicillin resistance (Ampr). The unique EcoRI site is just outside the tetracycline resistance gene. The origin of replication functions in bacterium E. coli. The complete DNA sequence of pBR322 consists of 4,361 bp. 

pBR322 carries two antibiotic resistance genes: one confers resistance to ampicillin (Ampr) and the other to tetracycline (Tetr). This plasmid also has unique BamHI, HindIII, and SalI recognition sites within the Tetr gene; a unique PstI site in the Ampr gene; a unique EcoRI site that is not within any coding DNA; and an origin or replication that functions only in E.coli. It is maintained at a high copy number in E. coli and cannot be readily transferred to other bacteria.  Purified, closed-circular pBR322 molecules are cut with a restriction enzyme that lies within either of the antibiotic resistance genes, which cleaves the plasmid DNA only once to create single, linear, sticky-ended DNA molecules.  These linear molecules are combined with prepared target DNA from a source organism. 

This DNA has been cut with the same restriction enzyme, which generates the same sticky ends as those on the plasmid DNA.  The DNA mixture is then treated with T4 DNA ligase in the presence of ATP.  Under these conditions, a number of different ligated combinations are produced, including the original closedcircular plasmid DNA.  To reduce the amount of this particular unwanted ligation product, the cleaved plasmid DNA preparation is treated with the enzyme alkaline phosphatase to remove the 5'-phosphate groups from the linearized plasmid DNA.  As a consequence, T4 DNA ligase cannot join the ends of the dephosphorylated linear plasmid DNA.

Fig- Cloning foreign DNA into a plasmid vector. After restriction endonuclease cleavage alkaline phosphatase treatment the plasmid DNA is ligated to the restriction endonuclease-digested target DNA and two of the four nicks are sealed This molecular configuration is stable and the two DNA molecules are covalently joined. After introduction into a host cell, ensuing replication cycle produce new complete circular DNA molecules with no nicks. 

However, the two phosphodiester bonds that are formed by T4 DNA ligase after the ligation and circularization of alkaline phosphatase-treated plasmid DNA with restriction endonuclease-digested source DNA (which provides the phosphate groups) are sufficient to hold both molecules together, despite the presence of two nicks.  After transformation, these nicks are sealed by host cell DNA ligase system.  In addition, although unwanted, fragments from the source DNA are also joined to each other by T4 DNA ligase.

Other Plasmid Cloning Vectors 
The plasmid pBR322 was a well-conceived cloning vector. It only has a few unique cloning sites, however, and its use requires a timeconsuming selection procedure. Thus, it was inevitable that other systems would be developed.  For example, the plasmid pUCI9 has 2,686 base pairs and contains an ampicillin-resistance gene; a regulatable segment of the ß galactosidase gene (lacZ') of the lactose operon of E. coli; a lacI gene that produces a repressor protein that regulates the expression of the lacZ' gene; a short sequence with multiple unique cloning sites (i.e., EcoRI, SacI, Kpnl, BamHI, XbaI, HincII, AccI, BspMI, PstI, SphI, HindIII) and the origin of replication from pBR322

Fig 2.12- Genetic map of the plasmid cloning vector pUCI9. The multiple cloning sequence contains unique sites for the restriction endonucleases EcoRI, SacI, KpnI, XmaI, SmaI, BamHI, XbaI, SalI, HincIII, AccI, PstI, BspMI, SphI, and HindIII and is used for the insertion or cloned DNA. The plasmid contains an ampicillin resistance gene, an origin of replication that function in E. coli and the lacI gene, which produces a repressor that blocks the transcription of the lacZ' gene in the absence or the inducer IPTG. The complete DNA sequence of pUC19 is 2,686 base pair. 

The pUC19 selection procedure has the following rationale.  When cells carrying unmodified pUC19 are grown in the presence of isopropylthiogalactoside (IPTG), which is an inducer of the lac operon, the product of the lacI gene cannot bind to the promoteroperator region of the lacZ' gene, so the lacZ' gene encoded in the plasmid is transcribed and translated.  The lacZ' protein combines with protein that is encoded by chromosomal DNA to form an active hybrid ß -galactosidase.  In pUC19 the multiple cloning sequence is incorporated into the lacZ' gene in the plasmid without interfering with the production of the functional hybrid ß-galactosidase.  Finally, if the substrate 5-bromo-4-chloroindo-lyl- ß -galactoside (X-gal) is present in the medium, it is hydrolyzed by this hybrid ßgalactosidase to a blue product.  Under these conditions, colonies containing unmodified pUCI9 appear blue.

Enzymes Commonly used in recombinant DNA Technology
Restriction endonucleases 
For molecular cloning, both the source DNA that contains the target sequence and the cloning vector must be consistently cut into discrete and reproducible fragments.  Subjecting isolated chromosomal DNA either to passage through a small-bore needle or to sonication produces doublestranded pieces of DNA that may range from 0.3 to 5 kilobase pairs (kb) in length. Unfortunately, these simple procedures induces breaks randomly, so each time a DNA sample is treated, a different set of fragments is generated.  It was only after bacterial enzymes that cut DNA molecules internally at specific base-pair sequences were discovered that molecular cloning decame feasible. These enzymes are called type II restriction endonucleases. 

Restriction endonuclease enzymes or Restriction Enzymes (RE) occur widely among bacteria.  These enzymes protect them from the invasion of viral DNAs by cleaving them into pieces.  A restriction enzyme cleaves both the strands of the DNA duplex at paliondromic site, i.e. the sequence with two-fold axis of symmetry.  The length of the paliondromic sequence varies between 4-8 nucleotides.  Several hundreds of restriction enzymes have been discovered so far but few are used commonly in genetic engineering work.  The naming protocol for these enzyme is the genus is the capitalized letter and the first two letters of the species name are in lowercase letters.  The strain designation is often omitted from the name and Roman numerals are used to designate the order of characterization of different restriction endonucleases from the same organism. 

Reaction between the RE and DNA is called restriction digestion, which is done by incubating certain amount of DNA and RE in appropriate buffer at 370 C for few hours.  Restriction enzymes produce two types of ends in the DNA fragments after digestion: i) cohesive or sticky ends with protruding or overhanging unpaired single stranded sequence of 2-5 nucleotides e.g. EcoRI, HindIII and ii) blunt ends, with no such unpaired nucleotides e.g. HpaI, Alul

Restriction enzyme digestion of DNA leads to formation of fragments with; a) staggered ends and b) blunt ends. 

Restriction enzymes are called molecular scissors in genetic engineering because each enzyme recognises the same specific base sequence irrespective of the DNA source.  This helps in cutting the large DNA molecule into fragments, which can be separated by gel electrophoresis.  These enzymes also help to establish some fixed landmarks along a large DNA molecule.

DNA polymerases 
i) DNA polymerases synthesize complementary nucleotide sequence on a template nucleotide strand. DNA Polymerase I, isolated from E. coli, synthesizes a complementary strand on a template DNA in 5' 3' direction. It also possesses low level of exonuclease activity in both 5' 3' and 3' 5' directions. This enzyme is used in labelling of DNA to prepare probe. Klenow enzyme is the large fragment of the DNA Polymerase I of E. coli. It possesses polymerase activity but lacks the exonuclease activity in 5' 3' direction. Bacteriophage T4 and T7 polymerases are other two polymerase enzymes used for specific purpose.

ii)

iii) Taq DNA Polymerase is isolated from a bacterium, Thermus aquaticus, living in hot springs. This enzyme is highly thermostable for which it is used for DNA amplification during Polymerase Chain Reaction (PCR). iv) Reverse transcriptase, isolated from retroviruses, is an enzyme that is used for synthesizing complementary DNA (cDNA) from mRNA in a process called reverse transcription.

DNA ligase 
Two DNA fragments from the same or different sources can be joined in a test tube to produce a recombinant DNA. DNA ligase performs this function by catalysing the formation of phosphodiester bond between adjacent 3' hydroxyl and 5' phosphate termini of the two different DNA fragments. DNA ligase produced by T4 bacteriphage is used, which needs ATP for its activity.

DNA cloning 
DNA cloning or molecular cloning is the technique of producing identical copies of DNA in a larger amount with the help of a microbial host.  For this, the desired DNA fragment is inserted into a DNA carrier molecule known as vector by ligation. 

Vectors are capable of self replication inside a bacterial cell leading to the multiplication of the insert DNA also.  Plasmid and phage vectors are commonly used for molecular cloning purpose.  A plasmid is a small, circular, double-stranded, selfreplicating DNA molecule present in the bacteria. Phages are viruses which infect bacteria.  The genetic material of phages replicates within the bacterial host. The DNA viruses like M 13 and phage lambda are useful vector for molecular cloning.

The basic principle of cloning a DNA fragment by shot-gun approach 
Firstly, both the vector and the genomic DNA are cut by the restriction enzyme digestion.  These two DNA fragments are joined by ligation to produce recombinant DNA molecule.  The recombinant DNA molecules are introduced into E. coli by the process, called transformation.  Once inside the bacterial cell the plasmid carrying the DNA insert multiplies.  The recombinant DNA clones are selected in the presence of antibiotic resistance gene in the plasmid vector or by X-gal test and confirmed using DNA probe.

Polymerase chain reaction (PCR) 
Polymerase chain reaction (PCR) is a chemical process of amplifying a specific DNA sequence in vitro (lnnis et at., 1990). The components amplification are: A target DNA Two synthetic oligonucleotide primers Four deoxyribonucleotides (dATP, dCTP, dTTP, dGTP) and a divalent metal ions (preferably Mg 2+) A heat stable DNA polymerase enzyme, usually Taq polymerase. that are necessary for PCR 

1. 2. 3. 4. 

The DNA amplification occurs in vitro in three steps namely, denaturation, primer annealing and primer extension. The target DNA should be denatured by raising the temperature above 900C. Then the oligonucleotide primers are allowed to anneal on the target DNA by lowering the temperature to the range of 35-55 0C; The DNA is synthesized next by the extension of the oligonucleotide primers with the help of the polymerase enzyme at a temperature around 700C.

1. 2.

3. 

These steps are repeated for 30 to 40 cycles using an automatic, programmable bath called thermal cycler.  Thus, by PCR, repeated duplication of DNA takes place in a chain reaction.  The amount of target DNA increases exponentially to a million folds within 3-5 hours.  Thus, amplification of one nanogram of genomic DNA would produce microgram amount of target DNA.

Production of transgenic with GH- hormone gene Molecular cloning of fish GH cDNA 
µClone¶ is a population of identical cells derived from a single parental cell.  For study large amount of cDNA or a gene is needed which is achieved by gene cloning.  Desired DNA fragment by itself can not be identified by the bacterial system for replication, instead it will be degraded by host endonucleases.  The piece of interested DNA is linked (ligation) to a carrier DNA (plasmid vector) and it is transferred (transformation) into bacterial cells.  The DNA fragment attached to the vector DNA is easily identified by the bacterial DNA polymerase and repidally amplified to several hundred copies.

Ligation of GH cDNA to Vector 
Purified, closed-circular pBR322 molecules are cut with a restriction enzyme that lies within either of the antibiotic resistance genes.  It cleaves the plasmid DNA only once to create single, linear, sticky ended DNA molecules.  These linear molecules are combined with prepared target DNA. This DNA has been cut with the same restriction enzyme, which generates the same sticky ends as those on the plasmid DNA.  The DNA mixture is then treated with T4 DNA ligase in the presence of ATP.  Under these conditions a number of different ligated combinations are produced, including the original closedcircular plasmid DNA. 

To reduce unwanted ligation product the cleaved plasmid DNA preparation is treated with the enzyme alkaline phosphatase to remove the 5¶- phosphate groups from the linearized plasmid DNA.  As a consequence, T4 DNA ligase can not join the ends of the dephosphorylated linear plasmid DNA.  Two phosphodiester bonds that are formed by T4 DNA ligase after the ligation and circularization of alkaline phosphatase ± treated plasmid DNA with restriction endonuclease digested source DNA (which provide the phosphate groups) are sufficient to hold both molecules together.  After transformation the nicks are scaled by host cell DNA ligase system.  In addition, although unwanted fragments from the source DNA are also joined to each other by T4 DNA ligase.

Fig- Cloning foreign DNA into a plasmid vector. After restriction endonuclease cleavage alkaline phosphatase treatment the plasmid DNA is ligated to the restriction endonuclease-digested target DNA and two of the four nicks are sealed This molecular configuration is stable and the two DNA molecules are covalently joined. After introduction into a host cell, ensuing replication cycle produce new complete circular DNA molecules with no nicks.

Screening & selection of recombinant clones E. coli cells are plated on an ampicillin containing LB (Labine Bile) agar plate.  Only those E. coli cells which contain drug (ampicillin) resistance plasmid alone can grow otherwise others will be killed by the antibiotic.  Cells (colnies) having recombinant plasmid (vector with insert) can be distinguished from the non-recombinant (without insert vector) harboring cells by µblue white selection¶.  The pBluescript plasmid codes for a small portion of ß-gal enzyme, which in combination with the other portion of the host ß-gal enzyme acts on X-gal (a galactose analog) which turns blue upon cleavage and gives a characterstics blue colour to the colonies. 

The recombinant plasmids, which have been disturbed by inserts, can not produce the enzyme and cells harboring recombinant cells remain white on x-gal.  So the white colonies are the real recombinant clones.  Such white recombinant colonies are selected out and grown separately in LB medium, plasmid isolated and analysed for presence of the insert.

Electroporation of fish gametes & embryo 
The induction of changes to augment the permeability of a cell by an electric field is called electroporation. Electroporation is a commonly used technique for the transfer of macromolecules such as DNA and protein into cells of animals, plants and bacteria. Most important electrical parameters in an electroporation experiment are i) field strength & ii) pulse length.

Electroporation of sperm:
1. 2. 3. 4. 5. 6. 7. 8. 9. Collect milt by gentle abdominal stripping of a one day starved male Rinse the milt with Fish physiological saline (FPS); centrifuge at 300 rpm Add appropriate amount of foreign DNA to the milt and place it in the transfection chamber Place the transfection chamber between two electrodes of electroporatic apparatus Set the desired voltage, capacitance and resistance in the electroporatic apparatus. Press the pulse deliver keys till you hear a beep sound Collect the electroporated sperm by gentile centrifugation Fertilize the eggs with the electroporated sperm Allow the fertilized eggs to develop in water

Electroporation of fertilized eggs:
1. 2. Collect the eggs by gentle abdominal stripping of one day female and carryout artificial fertilization Place the fertilized eggs in the transfection chamber and add appropriate amount of foreign DNA Follow the steps 4, 5 & 6 in the above mentioned protocol Collect the electroporated eggs, rinse it in water incubate until hatching

3. 4.

Microinjection of GH gene 
GH encoding cDNA of rohu and other fish are cloned and sequenced. GH cDNA is cloned into different vectors.  The plasmid is commonly used to transform E. coli from which plasmid DNA is isolated.  Plasmid microinjection is carried out in a sterile medium  Tilapia egg is highly opaque and the chorion is very tough. It can not be pierced through even with a very fine microneedle. Therefore, foreign DNA is injected into the germinal disc through the micropyle before the first cell division under an inverted microscope fitted with a micromanipulator.

PROTOCOL
1. 2. 3. 4. 5. Collect egg and milt by gentle abdominal stripping of adult female and male fish and carry out artificial fertilization Fill the injection pipette with the foreign DNA solution by suction Transfer egg to a sterile petridish containing sterile water (230C) and place it under the microscope Turn the fertilized egg until the micropyle is visible at the tip of the animal pole Fix the egg by suction to a holding pipette formed like an egg cup to prevent lateral movement during manipulation

6-

Hit the centre of the micropyle with the injection pipette to pass the membrane. Several trials may be needed to hit the centre. Inject foreign DNA by air pressure (use the control panel). Solution could be seen flowing into the egg. Gently withdraw the needle using the control panel Repeat microinjection using embryos at 2, 4 and 8 cell stages

789-

10 Incubate the injected eggs and monitor survival 12 hrs till they hatch. Fry hatched out from the injected eggs have to be used for identifying integration through dot and southern blot analyses

Fig. MICROMANIPULATION IN TILAPIA EGG

Fig. GENE MANIPULATION IN ZEBRA EFF

Sexuality in Fishes 
The sexuality in fishes is greatly diversified, including various types of sex chromosomal mechanisms, gynogenetic reproduction, differentiated and undifferentiated gonochorism, synchronous, protandrous and protogynous hermaphroditism, species specific external and diversified ethological sexes.  These diversified sexualities can be considered with respect to the control mechanisms, physiological and genetic sex.

Classification of physiological sex in fishes
Uindifferentiated Gonochorism Differentiated Gondal sex

Synchronous

Physiological sex (Biochemical)

Hermaphroditism

Protogynous

Sex accessories External sex Secondary Sex characterstics Ethological sex Sexal behaviour

Protandrous 

Biggest group of hermaphrodites Serranidae of order- Perciformes.

is

Family- 

Most of the hermaphrodites belong to the most advanced order-Percifromes. Sparid family of the order Perciformes exhibit varying forms of hermaph roditism and also gonochorism.  It represents diverse forms such as protogynous, protandrous and synchronous hermaphrodites to straight forward gonochorists.

Gonochorism 
It is the existence of either testes or ovaries in one individual fish. Most cultured fish species have this type of sexuality.  In the undifferentiated gonochorism, the undifferentiated gonad first develops into an ovary-like gonad, then about one half of the individuals become males and the other half become the females.  In the differentiated species, the undifferentiated gonad directly differentiates into either a testis or an ovary.  Labile phase ± duration required for sex determination

Sex determination 
Determinate type ± Short labile phase  Indeterminate type ± Long labile phase

Sex control in fishes 
  

Sex specific growth rate Population control Sexual maturity Ornamental value

Manipulation of physiological sex 
The physiological sex can be manipulated by using sex steroids and selective breeding and chromosomal manipulation.

Use of sex steroid hormones 
In teleosts, sex steroids are capable of influencing the course of sex differentiation.  Artificial sex reversal can be carried out by using heterologous sex steroids, androgen acting as male inducer and estrogen as female inducer.  In differentiated species the artificially sex reversed sex is probably permanent because the action of sex genes is believed to be restricted to a relatively short period during the early development of gonads, then latent or inactive after the onset of gonadal sex differentiation.  In hermaphroditic or undifferentiated species, sex reversal is more complicated because the sex genes can be active until later stages of gonadal development.

Methods of Hormone treatment 
  

Dietary supplementation Immersion Silastic implantation Injection

Male sex inducer 
The synthetic androgen, 19- norethynyltestosterone, is the most potent sex inducer from female to male in medaka, having AD50 value of 1mg/kg diet.  AD50 is represented as dose of androgen at which 50% of genetic female are induced into males. 

The 19- norethynyltestosterone is about 110 times more potent than 11keto-testosterone which is most potent of naturally occuring androgens.  AD50 of widely used methyltestosterone is estimated to be 15mg / kg diet.  Methyltestosterone is easily available, fairly stable and effective when administered orally.  The dose levels of naturally occuring androgens (testosterone & 11-keto testosterone) required to induce sex reversal are roughly two to three times higher than those to induce secondary sex characterstics.  100% sex reversal is achieved in all fish species by using methyltestosterone.  Effective dosage level of methyltestosterone is species specific.  The oral administration of methyltestosterone is effective in any species starting from first feeding.

Medaka Gold fish Tilapia mossambica & Tilapia nilotica

100 % 100 % 100 %

20-25 mg / kg diet 25 mg / kg diet 30 mg / kg diet

Tilapia aura Salmonids

100 % 100 %

30-60 mg / kg diet 0.5 to 3 mg / kg diet

Female inducer 
Estrogens used to induce feminization are 17ß-estradiol, estrone, estriol, diethylstilbestrol, ethynylestradiol, estradiol, butyryl acetate etc.  Most commonly and effectively used estrogens are 17ßestradiol and estrone.  The effective dose of 17ß- estradiol is 20 mg / kg diet for producing 100% female in Salvelinus fontinalis 10 mg / kg diet combined with immersion treatment for coho salmon.  An immersion dose of 0.5 to 5 mg/l of water at the alevin stage in masu salmon for 18 days gave 100% sex reversal  The effective dose of estrone to induce sex several in medaka and goldfish was found to be 125 mg / kg diet or 100 mg / kg diet. 

In Tilapia nilotica, doses of 100 mg /kg or 200 mg / kg diet are not optimal giving 62-78% of females.  In salmon, this steroid increases the percentage of females but does not induce 100% sex several with doses ranging from 10 mg / kg to 100 mg / kg diet.  Amongst the synthetic estrogens 100% feminization is induced by ethynylestradiol at a dose of 50 mg / kg diet in Tilapia mossambica by feeding for 19 days starting 6 days after hatching and by estradiol butyrylacetate at a concentration of 0.25 mg/l aqueous solution in Hemihaplochromis multicolor by immersion for 4-5 days.

Time and duration of treatment 
To induce sex reversal in fish, the treatment with exogenous sex steroid should be started at the optimum time and should continue for the proper duration.  The time of treatment depends primarily on the species specific time of phenotypic sex differentiation when the natural sex inducer is secreted endogenously.  Experimental data on sex reversal indicate that sex differentiation commences after hatching, either before or after the iniation of feeding.  But in viviparous fish such as the guppy, Poecilia reticulata, the sex is differentiated prior to parturition, hence the administration of exogenous steroid should be started at the embryonic stage in the ovary of gravid mothers.

Fig. Schedule for all-female production is an XX female, XY male sex control system

Fig. Schedule for the creation of YY stock for an all-male production system wheresex control is of the XX female, XY male type

Control of Sex in fish through chromosomal manipulation
Three broad classes of treatments are-

1- Induced Gynogenesis All maternal inheritance, the paternal chromosomes in the sperms are inactivated before fertilization.

2- Induced androgenesis All paternal inheritance, the maternal chromosomes in the eggs are inactivated before or shortly after fertilization.

3- Induced Polyploidy Early cell divisions in the fertilized egg are blocked to produce triploid or tetraploid individuals. 

Treatments inactivating chromosomes of sperm without destroying its ability to fertilize the egg and trigger development are needed. First such example was described by Hertwig (1911).  Phenomenon known as µHertwig effect¶ is attributed to total destruction of the chromosome at high levels of radiation resulting in production of haploid embryos which survive longer than diploid embryos expressing dominant lethal mulations induced by the radiation.  Variety of radiation and chemical treatments are available to inactivate sperm chromosomes.  rays from
60Co

or 137Cs. 

X- ray and ultraviolet light. 

& x-ray have good penetrating ability hence large quantities can be treated. They act principally by inducing chromosome breaks. 

Radiation treated sperm have lower viability and fertilizability and similar results are also obtained for chemical treatments.  Ultraviolet light in easier to work with and less dangerous. It damages chromosomes mainly by inducing thimine dimers. Possibility of their repair by photoreactivation process is to be taken care. Ultraviolet source is inexpensive and eggs are to be kept in thin layer. 

Chemicals- Toluidine blue, ethylene urea, dimethyl sulphate.

Gynogenesis Mitotic gynogenesis 
Milt stripped from the adult fish is diluted three to four times with cold Hank¶s balanced salt solution without bicarbonate. It can be stored for 1 to 6 hrs at 4oC.  Irradiate stored or freshly collected milt with UV rays (254 nm, power 1.0 mw/cm at the surface of the milt) under a germicidal lamp for 15-20 min.  Cobalt 60 (60Co) - irradiation is to be used with a dose of 50150 Krad at the rate of 100-250 rad/sec.  Eggs stripped as soon as ovulation commences.

Fig. Schematic representation of gynogenesis 

Freshly stripped eggs are fertilized with normal and inactivated sperm separately.  Fertilized eggs are subjected to heat, cold or hydrostatic pressure shock to inhibit 1st cleavage (I mitotic division of the zygote).  Incubate the fertilized eggs artificially.  Hatched larvae are reared and analysed for chromosome number.

Meiotic gynogenesis 
All other steps are same except shock treatment. Instead of arresting 1st cleavage the extrusion of 2nd polar body is inhibited.

Androgenesis 
Adult mature male is stripped for milt. If stripping not feasible fish is sacrificed and testis macerated in Ringers solution or cold Hank¶s balanced salt solution.  Diluted milt is stored at 4-50C for 1-6 hrs.  Mature eggs are stripped after ovulation and kept in Ringer¶s solution or cold Hank¶s balanced salt solution.  Eggs should be exposed to tap water.  In case of sticky eggs, milt and distilled water in the ratio of 1:9 is added just before fertilization to prevent clumping of eggs.  A group of eggs are kept as control without irradiation.

Fig. Schematic representation of androgenesis 

Single layer of eggs are irradiated with UV rays (254 nm, power 1.0 mw/cm at the surface of egg under a germicidal lamp for 2-3 min. To facilitate uniform exposure and irradiation eggs are rotated at 25 rpm.  Cobalt 60 (60Co) -irradiation is used at a dose of 3.6×104 F dose rate of 100-200 rad/sec.  The irradiated eggs photoreactivation. must be kept in dark to prevent 

Irradiated and unirradiated eggs are fertilized with normal sperm.  Fertilized eggs are subjected to heat, cold or hydrostatic pressure shock to inhibit the first cleavage (I mitotic division of the zygote) or II polar body extrusion. This is done to restore ploidy.  Fertilized eggs are incubated in dark condition for about 4-5 hrs, then under normal light.  Comparison of androgenetic fish is done with normal diploid fish.

Methods of inducing triploidy or tetraploidy 
Freshly stripped eggs are fertilized with stored diluted or freshly collected milt.  Fertilized eggs are subjected to cold, heat, hydrostatic pressure or chemical shock shortly after fertilization before II polar body is released.

Fig. Schematic representation of induced polyploidy 

Percentage of triploidy induction is estimated.  For producing tetraploids fertilized eggs are subjected to cold, heat, hydorstatic pressure or chemical shock after fertilization and release of II polar body and prior to first cleavage to suppress I mitotic division.  Triploids can also be produced by crossing diploids with tetraploids if fertile specimens available.  In ploidy manipulation physical/chemical inductors cause retention of II polar body. Shock causes depolymerisation of polymers (tubulin) that form microtubules, which are essential for the formation of spindle apparatus.  Thus shock results in the inhibition of spindle formation and subsequent extrusion of the II polar body. 

Ploidy normally can be induced shortly before 1st cleavage but rarely before the 2nd or 3rd cleavage. Shock applied to a cleaving egg inhibits cytokinesis. Consequently the zygote undergoes genomic replication but fails to undergo cytoplasmic division.  Physical inductors namely temperature, pressure or electricity cause the retention of polar body or inhibition of cytokinesis, depending on the development stage at which the shock is given.

Methods used for cofirmation of ploidy in fish 

Chemical inductors used are- cytochalasin B, colchicines and anaesthetics.  Use of cytochalasin B frequently results in mosaicism, Colchicine arrests cytokinesis, while anaeshtetics are capable of retaining polar body.

Advantages of chromosomal manipulations
Triploidy and Tetraploidy
1- Triploids are normally sterile which may lead to extended growth. 2- Sterility is advantageous where control of reproduction is desirable. 3- Triploids hybrids are more viable than diploid hybrids and may help in combining desirable characters from two species in a sterile hybrid. 4- Another application is related with the fact triploids have higher hetrozygosity than diploids. Heterozygosity is increased by using polyploids in plant breeding programs.

Gynogenesis
1- As a method of sex control. Disadvantage is that gynogenetic offspring are inbred. 2- A method for the rapid production of inbred lines. Valuable when totally homozygous gynogenetic diploids are used as female parents to produce an µinbred line¶ of genetically identical offspring. 3- Partially inbred gynogenetic diploids produced by retention of 2nd polar body are less valuable in the rapid generation of inbred lines as same loci remain heterozygous in a high proporation of offspring. 4- A novel possible application of gynogenesis is as a gene transfer technique in cases in which sperm inactivation is incomplete and some residual paternal inheritance takes place.

Androgenesis
1- Where males have shorter generation times than females, androgenesis might make more rapid genetic improvement possible. 2- Inbred lines might be stored in the form of cryopreserved sperm and recovered through androgenesis. 3Monitoring the effects of mitochondrial genotype on performance. It would be possible to compare the fish with identical nuclear genotypes and different mitochondrial genotypes. One way extrachromosomal maternal inheritance can be evaluated.

Cryopreservation of gametes Preservation of gametes
Prolonged viability of gametes is advantageous in many ways. 1- It can improve the induced breeding technology and would assist in the selective breeding programme. 2- The problem of asynchronous maturity time of males and females during artificial breeding can be alleviated. 3- Different stocks / strains maturing at different times can be interbred. 4- The gametes can be transferred to different hatcheries more conveniently. 5- Making of sperm bank, useful for ex-situ conservation of genetic diversity. Short term preservation Long term preservation

Short-term preservation 
Short±term preservation of gametes can be done for some hours to few days at temperature between 4 to 00C.  Both undiluted and diluted semen of common carp and rainbow trout could be preserved for some days at 4 to 00C.  Oxygenation and addition of antibiotics further prolongs spermatozoa survival.  In salmonids, spermatozoa in undiluted semen survives upto one month at 1-40C with addition of antibiotics and oxygen.  The semen plasma is not a good medium for in vitro storage as it contains a variety of protease enzymes, which can induce alteration in the semen. 

Ova stripped out soon after ovulation & stored in vitro in undiluted state retain fertilizability for a few hours.  The fertilizability of eggs can also be preserved by keeping them in ovarian fluid or in an isotonic electrolytic solution for few hours to a few days at cold temperature above O0C.  Viability of eggs kept in ovarian fluid at 40C was in temperate fishes like Salmo trutta for ten days Clupea harengus for two days.  In Fundulus heteroclitus and Tilapia, the fertility of eggs remained better in ovarian fluid at 10-150C.

Long term preservation Cryopreservation 
Crypreservation is a branch of biology that elucidates the principles and methods of preservation of living things at ultracold temperature (-1960C).  At this temperature, the metabolic activities of the cells are arrested but they remain viable.  Their normal functions can be reactivated after proper thawing.  Cryopreservation involves basic steps: freezing, storage and thawing.

FREEZING 
During freezing several physico-chemical changes take place within the cell and its surrounding area.  Plasma membrane consists of a lipid-protein bilayer that controls the transport of metabolites and ions.  Lipid remains in a liquid state at normal temperature but gets frozen at low temperature which affects the permeability and structural integrity of the membrane.  Initially ice crystal formation occurs in the extra-cellular medium due to freezing and as a result it becomes hypertonic to the cell.  To maintain the osmotic balance, the intra-cellular water comes out leading to the reduction of the cellular volume. 

These changes cause mechanical damage to the cell.  The temperature range between 0 to -400C is most critical during freezing since most of the cryoinjuries take place in this temperature range.  Below-1200C the freeze concentrated residual liquid in the extra-cellular solution and the dehydrated cytoplasm vitrify and further cellular damage does not take place.

Vitrification 
Vitrification is the process that transforms intracellular water to noncrystalline solids after freezing. This occurs under two conditions. I- If the cooling rate is very high, it does not allow sufficient time for the water molecules to crystallize. II- The solution is so concentrated that the high viscosity at low temperature does not allow water molecules to get crystallized.  The temperature at which vitrification begins is called as glass transformation temperature, which is -130C for water.  Cryoinjury can be reduced in the presence of some protective chemicals called cryoprotectants and a diluting solution, called extender. 

During freezing intra-cellar water should be allowed to come out of the cell. Rapid cooling fails to provide sufficient time for the cells to dehydrate and avoid the detrimental effects associated with intra-cellar ice crystal formation.  In contrast, slow cooling results in excessive dehydration and reduction of cellular volume to the extent that is not reversible.

Cryoprotectants 

Cryoprotectants help to prevent or reduce the intra-cellular ice crystal formation during freezing and thawing. 

A good cryoprotectant should be non-toxic, water soluble and capable of altering the physico-chemical properties of water during freezing.

There are two types of cryoprotectants. i- Permeating- Glycerol, dimethyl sulphoxide (DMSO), ethylene glycol and methanol can permeate into the cell. ii- Non-permeating- Polyvinyl pyrrolidone (PVP), glucose, sucrose, egg yolk, serum and skimmed milk form a coating externally around the cell that prevents ice formation in vicinity.  The non-permeating cryoprotectant in conjunction with a permeating one, helps to depress the freezing point and prevent ice crystal formation in the vicinity of the cell.  The adjuvant like egg yolk provides additional strength to membrane stability.

Extender 
Defined as a solution of salts and some times including some organic compounds, which helps to maintain the viability of cell during refrigeration.  An ideal extender provides osmotic stability by buffering action, protects the membrane from injury and preserves the biological activity of the cell. STORAGE  For long-term storage, liquid nitrogen (-1960C) is the most useful medium.  Freezing at any temperature below -700C will generally maintain the viability of the cells for a longer period.  The storage can be done in cryovials, straws or in the form of pellets.  The heat transfer capacity of these containers are different for which the freezing and thawing rates should be specifically modulated.

THAWING 
Thawing is required to bring back the cell to its normal temperature for resuming its usual function.  During thawing the same physico-chemical changes occur as that of freezing but in reverse order.  Slow thawing produces large ice crystals which damage the cell structure leading to its death.

Cryopreservation of fish spermatozoa and embryos 
Spermatozoa can be cryopreserved more easily for their structural simplicity and small size.  Eggs are more complex than the spermatozoa. They are more sensitive to freezing injury.  Fish eggs are large in size, possess thick chorion and high quantity of yolk which pose problem during cryopreservation.  The cryoprotectants, particularly glycerol and DMSO, can¶t penetrate easily into the eggs.  Due to above preservation of fish eggs and embryos have not yet been feasible.  Spermatozoa of more than 250 species of fishes could be preserved.  Successful cryopreservation of spermatozoa depends upon extender composition, cryoprotectant type, extender-semen dilution ratio cooling and thawing rates.  The time gap between spermatozoa collection and freezing also affects the success rates of cryopreservation.

EXTENDERS 
Undiluted milt with or without cryoprotectant is unsuitable for cryopreservation, therefore a suitable extender or diluent is added to the milt.  Generally the solutions like Ringer¶s or Hank¶s medium mixed with some adjuvant like egg yolk, skimmed milk or lecithin are used as extender.  The dilution ratio of milt with extender is highly variable and requires standardization for every species.  The volume of extender varies between 3 to 10 times the volume of the semen.

CRYOPROTECTANT 
The choice of cryoprotectant and its concentration are of much importance.  Equilibration time- time allowed for the penetration of cryoprotectant into the cell before freezing, is not necessary for fish spermatozoa for their small size.  Most commonly used cryoprotectants are- DMSO (7-10%), glycerol (10-20%), methanol (5-15%), ethylene glycol (7%), propen-diol (7-10%).  Glycerol as a cryoprotectant was suitable in Mugil cephalus, Gadus marhua, Silurus glanis but it was toxic to salmonids and Epinephelus tauvina for which DMSO was used.  Methanol was used successfully to cryopreserve the spermatozoa of Tilapia but had no cryoprotective effect in Sillago cillata and Lates calcarifer.

Freezing and thawing protocal 
Freezing and thawing rates are very critical cryopreservation. The cooling rate shoud be moderate. during  Cooling rate of about 20-300C / min was suitable for salmonid fishes.  Fast freezing by directly immersing the sample in liquid nitrogen causes lethality. However, fast thawing rate generally protects the fertilizability.  Slow thawing causes cell death due to ice-crystal formation around and inside the cell.  After thawing the cryopratectant should be removed by centrifugation. 

In come species, spermatozoa motility has to be induced by activator solution after thawing. Sodium bicarbonate, caffeine or theophylline in saline water are good activators. The time gap between thawing and fertilization should be as short as possible to enhance fertilization rate. Viability of cryopreserved spermatozoa are assessed by % of motility Duration and intensity of motility Fertilization and hatching rates Time of collection of milt is of much relevance for success of cryopreservation. Cryopreservation may prove easier in those species where spermatozoa swim for a longer time in external medium. 

 i) ii) iii)  

Population genetics 
Fish are specially appropriate as a group for studies of genetics of natural populations because of their extreme diversity, large populations sizes and their poikilothermic physiology which increases their susceptibility to thermal effects of the environment.  Fish are also widely cultured, therefore are also subjected to manipulations of their genetical constitution intentionally or unintentionally and or naturally or artificially.  There are many other aspects of population genetics that are directly applicable to cultured populations of fish.  Almost all of our knowledge about the genetics of natural population of fish has been gained from electrophoretic data which incorporates the use of isozymic gene frequency data in the understanding of the genetic structure of fish populations.

Markers used for studies of Population Genetics

Protein

DNA

mt DNA

Nuclear DNA

Analysing the Proteins by Electrophoresis

NonNon-enzymatic proteins Protein Enzymatic proteins

Non-enzymatic proteins Transferrin, haemoglobin, eye-lens protein, parvalbumin (a muscle protein) and general muscle proteins have been analysed in some fishes.

Enzymatic proteins More than 50% of the enzymes are known to occur as isozymes and are formed generally due to genetic causes.  Isozymes are classified into two groups according to the number of gene locus / loci involved in their production.

MultiMulti-locus isozymes: isozymes: 
These are formed by the involvement of more than one gene loci. Multi-locus isozymes have considerable structural and functional differences and they can be distinguished readily by electrophoresis.

Allelic isozymes: 
These are formed by different alleles of a gene and called as allozymes. These isozymes differ by one or a few amino acid substitutions.  Isozymes are specific gene products. Electrophoretic analyses reveal qualitative differences in expression of isozyme locus/loci during the embryonic development and in different adult tissues within a species. These are regarded as good genetic markers for the following reasons:

1.

Isozyme gene expression pattern is co-dominant type. This helps in analysing the genotypes more conveniently from the gel phenotype. Unlike the morphological characters, the phenotypic expression of isozymes is not influenced by the environmental factors. Any detectable change in isozyme electrophoretic pattern between different tissue types or individuals reflect certain genetic variation. For these characteristics, isozymes are considered superior to the morphological markers. Lactate dehydrogenase, malate dehydrogenase, glucosephosphate isomerase are some of the extensively studied isozyme systems in fishes.

2.

3.  

Electrophoresis of soluble (enzymatic) proteins Basic technique of electrophoresis 
Optimal resolution for a given protein system involves a number of variables including buffer system, distance of migration in a given buffer system, tissue in which protein is expressed and species.  Type of matrix used and electrical potential also play important role in resolution of proteins.  These variables are to be dealt effectively to avoid the loss of genetic information.

Advantage of electrophoretic methodology
1- It is relatively easy in application, inexpensive and it is an efficient technique. 2- Depicts direct relationship between appropriately chosen protein variants and the gene.

Simplified representation of the electrophoretic phenotypes of monomeric, dimeric and tetrameric molecules in homozygous and heterzygous states

Biochemical complexities 
 Biochemical complexities arise because of the structure of proteins. The simplest system is where the protein is a single molecule arising as a direct product of a gene, This monomeric structure generates a phenotype in which each allele is represented as a single band in the homozygote and as two bands in the heterozygote. Proteins also comprise complex molecules, polymers, in which two or more subunits produced by individual genes combine together. Where the protein is built of two molecules, a homozygote again shows the single band phenotype but the heterozygote shows three bands by random combination of the two different molecules.   

If the molecule-produced by one allele is A and that by the other is B, the heterozygote protein molecules will be AA, AB and BB in the ratio 1:2:1 just as for random assortment of genes but in this case involving intracellular proteins, not genes.  For these dimeric proteins it is important to note that the two units of the finished protein are produced by the one locus.  For more complex molecules, such as the tetrameres with four subunits, the options are wider because the subunits themselves may derive from one or to loci, depending upon the protein.  The most straightforward cases are those where only one locus is involved.

‡ If we again use A and B as depicting the subunits produced by each of the two different alleles, the finished molecule may be of five types. A4, A3B. A2B2, AB3 and B4. ‡ These would be produced in the ratio 1:4:6:4:1 under random association. ‡ But stereochemistry may influence the relative frequencies. ‡ Nevertheless, five-banded phenotypes indicate the existence of a tetrameric protein for which subunits are coded at a single locus. ‡ Where two loci code for subunits of a tetrameric protein, highly complex phenotypes are possible with patterns involving several bands.

Determining genetic models 
In the absence of breeding data the postulation of genetic status for variation in population data can proceed stepwise. 

The first step is to ensure that the variation is stable and

repeatable and not an artefact of the electrophoretic or sampling techniques. to the known or postulated subunit structure of the protein molecule. 

A second step is to assess the banding patterns in relation 

Thus for a monomeric model the simple single-banded patterns should prevail, whereas for a dimeric molecule, three-banded and single-banded patterns should be seen. 

Thirdly, the genetic model must be met, i.e. patterns must conform

to the principle that only two allelic expressions at most can occur in a diploid individual. 

The interpretation of the locus / allelic relationships between multiple bands on an electrophoretogram where population data or only limited experimental data are available involves simple inspection of the phenotypes to create hypothetical genotypes which can then be tested numerically.  For example, if three bands or fewer occur, designated a, band c, and an array of phenotypes abc; ac; bc exists, it is simple to postulate that a and b are alleles at one locus whilst c is produced by another locus.  The corresponding genotypes of the three phenotypic assays would be a:b c:c; a:a c:c; b:b c:c and this model could be tested against population distributions or segregation ratios from matings. 

Finally, it is necessary that allelic frequencies are compatible with
the observed frequencies of phenotypes and their postulated genotypes. 

This is the application of the famous Hardy-Weinberg law which was established independently by the two named geneticists shortly after the rediscovery of Mendel's work at the turn of the century.  Indeed, had Mendel not worked with a self-fertilizing plant he may well have made the discovery himself, in which case we might now be calling it Mendel's third law.  The Hardy-Weinberg law states that in a panmictic population there is a genotypic equilibrium determined solely by the frequencies of alleles and expressed as the expansion of the binomial (p+q)2 where p and q are frequencies of two alleles A and B at a locus and p+q=1. With three alleles, the binomial would be (p+q+r)2 and so on. (p=q)2 = p2 + 2pq = q2  Where p2 is the frequency of genotype AA, q2 that of BB and 2pq that of the heterozygote AB. 

The simplest derivation of the law is that alleles segregate independently in the formation of gametes, random union occurs between spermatozoa and ova during fertilization and parents mate at random in the population.  The first two conditions equal Mendel's first law; the third is the condition of panmixia, i.e. a population is panmictic if individuals can mate at random within it.  Taken together, these three, conditions imply that the population is a mixture of alleles which unite at random.  That is, they imply that if allele A has a frequency of p in the population, the chance of two A alleles coming together during zygote formation is p x p or p2, for two B alleles q x q, i.e. q2, and for the heterozgote it is p x q and q x p, i.e. 2pq.

Inferences From Population Genetic Data 
The data on phenotype frequencies are processed arithmetically to generate allele frequency data, the total number of alleles scored being equal to twice the number of fish examined since each fish has two sets of genes.  Thus in a sample of 20 fish, if six individuals are homozyous for allele A, four for the other allele B and ten are heterozygotes AB, the allele frequencies are simply p=[(6 x 2) + 10] ÷ 40 for A and q=[(4 x 2) + 10] ÷ 40 for B namely p=0.55 and q=0.45, respectively. 

A check on compliance with the Hardy-Weinberg equilibrium is then achieved simply by computing the terms p2 2pq and q2, and multiplying them by 20, the sample size, to generate the theoretically expected numbers of individuals of genotypes AA AB and BB, respectively, which can then be assessed for goodness of fit against the observed numbers by appropriate statistical tests such as 2.  In the hypothetical example the numbers would be 6.05, 9.90 and 4.05 - a remarkably good fit.  In addition to the allele and genotype frequencies three other genetic parameters are often derived from the observations, namely the frequency of polymorphic as opposed to monomorphic loci, the degree of heterozygosity over the loci studied and, where more than one population is analysed, the genetic identity of each population in relation to the others, i.e. the so called genetic distances.

Inbreeding 
The loss of genetic variation in artificially cultured populations because of inbreeding is well known.  In the present context the loss in genetic variation is largely attributable to be a limited population size.  The magnitude of this loss in a single generation is inversely proportional to be size of that population as expressed by the following equation.
F = 1
2Ne 

Where F is inbreeding coefficient, which represents the proportional loss in genetic variation and Ne is the effective size of the population. 

The effective size of a population is rarely equal to the total number of reproductive individuals in that population.  A major fact which influences Ne is the relative number of males and females in a population.  The effective population size of population with different numbers of males and female is expressed by the relationship.
1
Ne

=

1
4Nf

+

1
4Nm 

Where Nf and Nm are the numbers of females and males in the population respectively. 

The important consideration is that the value of Ne is strongly influenced by the sex which is the least frequent.  For example a population consisting of 1 male and 1 female has an Ne of 2, while a population with one male and 100 females has an Ne of approximately only 4.  Another major factor in the loss of genetic variation is fluctuations in population size from generation to generation.  A greatly reduced population size for a single generation can have a drastic effect.

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