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International Council for theExploration of the Sea

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C.M.1985/F:20/Ref. K and MMariculture Cttee SessionRef: Shellfish and Anadromous

and Catadromous Fish Cttees

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Subverting Random Segregation of Genes to Produce Clonesof Superior Performing Mollusks, Fish or Crustaceans

A. Crosby Longwell

National Marine Fisheries ServiceNortheast Fisheries Center

Milford LaboratoryMilford, Connecticut 06460

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ABSTRACT

Development of totally tetraploid fish, crustacean and molluscan individuals

or mosaics should offer a hitherto unconsidered possibility for direct, mass,

cornmercial production of diploid or tetraploid, heterozygous hybrid or non-hybrid

clones of the best-performing or most scientifically important females in either

selectively bred or wild stocks. This is so because preferential pairing of

perfectly identical, artificially duplicated homologous chromosomes would subvert

their random segregation in meiosis and hence that of the genes encoded in them.

All gametes and products of female meiosis of any tetraploid or of the tetraploid

gonad sectors of any diploid would then be genetically identical even when crossing­

over occurred. Gynogenetic stimulation and development of such identical eggs

would yield diploid clones, and when this was followed by polyploidization of the

female gamete or first embryo division the outcome would be tetraploid clones.

Chromosome pairing can be erratic in some auto- and allo-tetraploids, but it is

stable in others with preferential pairing of identical over non-identical homol­

ogous chromosomes. Chromosome pairing has yet to be studied nuch in aquaculture

organisms. Still, the great economic and scientific utility' of such an approach as

described, and .its power relative to selective breeding and/orgynogenetic techniques

~. alone make exciting the possibilities this creates for aquaculture researchers,

fishery biologists andhatchery producers... ..

RESUME

Le developpement des poissons, des crustaces, et des molluscs, totalement

tetraplo'de, soit sous la forme des individus, soit des mosaics, devrait offrir

une possibilite inconnue jusqu1a maintenant pour la production cornmerciale, directe

et en masse, des "clones" soit diplo'de, soit tetraplo'de heterozygotique hybride

ou non-hybride des femelles meilleur en performance, ou la plus importante scien-.

tifiquement en stocks soit selectionne, soit sauvage. C1est ainsi parce-que

l'assortissement preferentielle des chromosomes identiques, dupliques

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artificiellement homologue subvertirait leur segregation au hasard en meiose et

alors celle des genes qui sont code la-dessus. Tous les gametes et les produits

de la meiose femelle des tetraplo~des ou des secteurs gonadique des diplo~des

serraient identique genetiquementm~me quand on avait la phenomene de "crossing­

over". La stimulation gynogenetique et le developpement de ces oeufs identique

donnerait des clones diplo~des, et quand ces processus sontsuivis par la poly­

plo~dization des gametes femelles ou la premiere division d'embryon, la resultat

. serrait des clones tetraplo~des. L'assortissement chroffiffiomique peut~tre erra­

tique en quelques auto- et allotetraploYdes, mais c'est stabile pour des autres

avec l'assortissement preferentielle des chromosomes homologues identiques ou

non-identiques. L'assortissement chromosomique n'etait pas beaucoup etudie

jusqu'a maintenant pour les organismes en aquaculture. Mais la grande utilite

economique et scientifique de ce processus ici decrive, et sa puissance relative

a la production selectionne etjou les techniques gynogenetiques eux meme font

excitant les possibilites crees pour les rechercheurs en aquaculture, les biolo­

gistes des pecheries, et les producteurs des hachuries .

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INTRODUCTION

The purpose of this report is to poJnt out a combination of simple procedures

which can directly result in the production of clones or exact diploid or 'tetrap1oid

copies of non-inbred, heterozygous, hybrid or non-hybrid fema1es of fish, she11fish

or crustaceans. A1though some imp1ications of gene segregation in hybrid tetra­

p10ids have been brief1y a11uded to in the aquaculture literature, the fu11

exciting imp1ications of tetrap10id chromosome pairing, which forms the physica1

basis for gene segregation, seem to have escaped recognition until pointed out for

4It' the first time earlier this year (Crosby Longwe11, 1985). One reason for this is

probab1y inattention to more recent, basic cytogenetic findings on genetic contro1

over chromosome pairing.

Dependent on the degree of genetic variabi1ity in chromoscime pairing, obtaining

clones of major maricu1tured species cou1d be a simple matter of app1ying gyno­

genetic and po1yp1oidizing techniques current1y used on sa1monids, carp, other,

cu1tured finfish and mo11usks* to the ripe eggs of'artificial1y synthesized tetra­

p10id hybrid or non-hybrid fema1es or to those of the fu11y or,partial1y tetrap10id

germ line of otherwise 1argely diploid hybrid or non-hybrid fema1e~. Th~ specific

basis for this procedure is competitive chromosome pairing in tetrap10id oocytes •

A yet entire1y different procedure for obtaining clones is being researched in our

1aboratory by S. Stiles and J. Choromanski (also see Stiles et~. 1983), and will

be reported by Stiles and Choromanski next yea~.

Essential to understanding, appreciating and using a cloning procedure as

described here is a review of the general significance of chromosome pairing and

bivalent orientation on the meiotic I spind1e and of natura11yoccurring departures

* In carps these techniques are as described in John et a1., 1984; Nagy and Csanyi,1978; in sa1monids, Chevassus, 1983; Chourrout, 1983; Linco1n and Hardiman,1982;Purdom, 1983; Veda et al. 1984; Utter et al., 1983; in shel1fish, Arai et ~., 1983and 1984;' Longo, 1972;St~nley et Al.,T98T; Stiles, 1978; Stiles et al::1983;Tabarini, 1984.

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from this. This is reviewed here prior to a description of the cloning process.

Possibi1ities of de1iberate1y manipu1ating chromosome pairing or segregation are

discussed, a10ng with recent findings on genetic contro1 over chromosome pairing.

Fo11owing an out1ine of the cloning process; is a critica1 appraisa1 ofthe 1ike­

1ihood that the basic conditions for this might be met in cu1tured she11fish and

fish. A1together this makes a strong case for better appreciation of the relevance

to aquacu1ture breeding of basic cytogenetic mechanisms as they have been amp1y

described in a variety of anima1 and plant forms. Higher p1ants,vertebrate and

invertebrate anima1s and man all possess chromosomes essentia1ly simi1ar in structure,

function and behavior. Hence chromosome phenomena central to genetic variation

e1ucidated in various other groups are informative on the possibilities of inducing

simi1ar phenomena in a variety of maricu1ture types, as here in relation to a

process for multiplying identica1 genotypes.

PRESENTATION OF PROCEDURE AND ITS RATIONALE

Chromosome synapsis and bivalentorientation'as the physical',basis forrandomsegregation of genes

How the chromosomes become arranged in relationship to the poles of the meiosis I

spind1e in gametogenesis determines their distribution, and accordingly the shuffling

of materna1 and paternal genetic factors into new combinations (as in Sybenga, 1975a;

Khush, 1978; Smith, 1978; Rothwel1, 1979; Lima-de-Faria, 1983). Orientation of

paired chromosomes (biva1ents) on the spindle is usua11y a matter of chance. The

inf1uence of bivalent orientation on the genetic composition of the polar body and

meiosis 11 metaphase group in oogenesis can be visua1ized in Figure 1.

Aprerequisite for the orientation of chromosomes on the spind1e is the asso­

ciation of homo1ogous parental chromosomes into pairs or biva1ents. Whatever the,

physica1 basis for chromosome pairing, it is very exact. Whi1e chromosomes in

gametogenesis are paired in prophase of meiosis even genes linked together on the

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same chromosomes can become mixed through exchange of physical segments between" ,

the two paired parental chromosomes. The significance of this phenomenon (crossing-'

ove:), along with that of ,independent assortment of chromosomes at meiosis I, is, '

the formation of new combinations of genes in gametes. Unlike the two.genetically

identical nuclei of a mitotic division, the four nuclei resulting from the two

normal meiotic'divisions in a diploid are all dissimilar. On fertilization such

gametes yield almost limitless new genetic combinations the raw material of

either natural or artificial selection.

Naturally occurring subversions of the:physical'basis'for random segregation of'genes

Even though the physical process just described isthe usual occurrence in. .

meiosis, the whole process of random segregation of chromosomes and crossing-over of

genes is subverted in some organisms. See again as in Sybenga; 1975a; Khush, 1978;

Smith, 1978; Rothwell, 1979; and Lima-de-Faria, 1983. There are even species where

all the chromosomes from oneparent move to the'same spindle pole. More,relevant

to the discussion here, however, are cases of apomixis where meiosis is suppressed,

and a single mitotic division replaces the two meiotic divisions. Resultant eggs

are no different from somatic cell nuclei of the female. Parthenogenetically devel­

oped offspring of these are natural clones of the sole female parent .

In the most successful ,vertebrate group where apomixis occurs - lizards - the

mechanism enabling parthenogenesis to occur is a premeiotic doubling of the chromo­

somes withouttheir division (endoreduplication). This is followed by an apparently

normal meiosis with only bivalent chromosomes.

The genetic consequences of this reproductive mechanism depend on the manner in

which the bivalent chromosomes are formed. If only endoreduplicated, identical

sister chromosomes ,pair in meiotic prophase, as is likely in most cases because of

the proximity of these to each other as the nucleus enters prophase of meiosis, eggs

developing without fertilization are also identical to their sole female parent.

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This is just as in those species where meiosis is suppressed'entirely and

replaced by mitosis. An examination of Figure 2 will make clear that, irrespective

of the orientation of chromosomes on the meiosis I spindle and irrespective of

crossing over, pairing of identical sister chromosomes assures that all four products

of meiosis are identical to another. Also they are identical to the female. Endo-·

reduplication also has the advantage here that chromosome number is maintained

without fertilization:

If in this form of parthenogenesis bivalent chromosomes are formed of homol­

ogous parental, not endoreduplicated chromosomes, offspring will be dissimilar to

one another and to their parent. Genetic homozygosity will increase. In contrast,

pairing of endoreduplicated chromosomes maintains whatever heterozygosity is present

in the female parent.

In gynogenetic development (a form of automixis as now used by fish and shell­

fish breeders) meiosis is normal. The diploid chromosome number is sometimes

restored either by fusion ofthe female pronucleus with apolar body, or by fusion

of two early cleavage nuclei. The exact genetic consequences of this depend on

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(as described in.~aker et ~., 1976), the deliberate search for these, or on

a basic understanding of the meiotic mechanism so' as to facilitate a better

manipulation of it than presently possible. Deliberate selection for increased

incidences of rare sporadic occurrence of parthenogenesisis another possibility.

These all seem worthy goals of basic research that ought to underlie applied mari­

culture studies (Longwell, 1985).

For now the synthesis of artificial tetraploids offers, with some initial ex­

perimental effort, a rather immediate means of achieving the same genetic product.

Even to a greater extent than in diploids, chromosome pairing in tetraploids is

known to be genetically regulated, and the process is also known to be genetically

variable.

The near-sterility of triploids and pentaploids is clearly due in'part to the

production of genetically unbalanced gametes as a result of irregularsegregation

of chromosomes without pairing partners. In tetraploids, multivalent chromosome

configurations canbe formed by the association of 4 or 3 chromosomes. However,

there is a higher probability of normal bivalent formation. Anaphase segregation,

therefore, results in a much.larger proportion of gametes with a balanced chromosome

composition. New tetraploids often suffer little reduced fertility initially .. .

Old, established tetraploid species or varieties can even have' increased fertility.

Whenever these bivalent configurations of new tetraploids come about through

preferential .pairing of the synthetically created, identical sister chromosomes

over the pairing of non-identical homologous chromosomes, gametes and all four

meiotic products in the female will be identical. This .is just as in the endo­

reduplicated naturally parthenogenetic lizards. Again see Figure 2 as to why this

must be so.

New allotetraploids - those formed from the artificial doubling of the chromo-

somes of a sterile interspecies hybrid - should not show multivalent pairing. This

is because of the dissimilarities of the parental chromosomes from the two species

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parents. Autotetraploids - formed from the artificial doubling of the chromosomes

of a non-hybrid - might be expected.to show multivalents because of the similarity

of the parental chromosomes. Sometimes, however, good bivalent pairing occurs in

diploid species hybrids, and also in their well-adjusted synthetic tetraploids

(called for reason of their diploid-like chromosome pairing, amphidiploids). Auto~

tetraploids which first exhibit quadrivalent pairing with loss of fertility and

uncertain genetic outcome later shift to bivalent pairing (Riley and Law, 1965;

Riley, 1968; Sybenga, 1975a and b; Waines, 1976). This was the case in maize which

shifted over a ten-year period (about 10-15 breeding generations for maize).

In grasshoppers there is a preference for identical over .homologous but non-~

identical chromosome pairing (Giraldez and Santos, 1981; Santos et 21.,1983).

In different plants there can be a preference for either type of pairing (Sybenga,

1975a and b; Evans and Tay10r, 1976; Giri1dez and Santos, 1981; Aung and Evans, 1983;

Evans and Davies, 1983). This seems to be dictated by prior somatic association of

the chromosomes, by specific differences between chromosomes, or by particular genes.

Figure 3 is a verba1-diagrammatic summary of thegenetic outcome of competitive

identical pairing in the tetraploid eggs of tetraploid maricultured individuals or

of diploid individuals whose gonads were at least made partially tetraploid. These

... are stimulated to develop gynogenetically via fertilization with genetical1y inac­

tivated, irradiated sperm. All gametes would be similar and all identical to the

mother, but with half of the mother's chromosomes. Gynogenetic development would

restare the diploid chromosome number.

When these diploids- not tetraploid individuals and clones - are what is

desired, breeding individuals would be selected for outstanding performance as

diploids. Some or all of the germ ·line or gonad of these selected individuals

would then be made tetraploid prior to the onset of meiosis early in oogenesis

whi1e the gonad is still undergoing mitotic cell division. Gonad sectors failing

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to undergo doubling would p~oduce much smaller eggs than tetraploid sectors.

These smaller eggs could be easily sorted.

Oyster clones might, of course, also be produced from females made tetraploid

in their meiosis or cleavage. However, it is expected that individuals could have

a different commercial performance aso diploids and as tetraplaids. This would neces­

sitate judging the value of diploid clones based on tetraplaid performance, something

probably better avoided for ordinary commercial production, especially since it is

not necessary to the described procedure.

As also shown in Figure 3, tetraploid clones can be obtained from tetraploid

oocytes with competitive pairing of identical chromosomes when gynogenetic stimu­

lation with irradiated sperm is followed by use of a polyploidizing agent. With

all the products of meiosis alike, fusion of the female pronucleus with the second

polar body only res tores the chromosome number of the egg to that of the female

parent. The genetic outcome is a tetraploid clone, all individuals identical to

the breeding female. In this case, to avoid judging commercial performance as

tetraploids on diploid performance, it would be better to select breeders from

tetraploid individuals than from diploids with tetraplaid germ lines.

Tetraploid clones would have the distinct commercial advantage over diploid

clones of being able to be propagated and multiplied indefinitely via normal sexual

breeding. Sex reversal techniques could be used in fishto create artificial males.

In oysters where sex determination has a big environmental component at the least

and protandry is common, some male individuals are expected among the clones of

females, or might be induced.

For the sake of further review and clarification of points made, Figure 4

merely summarizes the different genetic outcomes of gynogenesis as applied to

diploids and already reported and practiced in fish, and gynogenesis in tetraploids

with competitive pairing of identical homologous chromosomes.

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DISCUSSION .

Information on pairing preferences of chromosomesdoes not exist for the

variety of species in mariculture or aquaculture that might be desirably cloned.

Indeed in most instances tetraploid individuals or tetraploid mosaics have yet

to be even synthesized although a few.have been reported for salmonids. There is

little doubt though that these can be produced. It has been proposed that the

simplest way to make either triploids or tetraploids in shellfish .is through the

doubling of the germ line of otherwise mostly diploid individuals (Crosby Longwell,

_ 1968, 1984.and 1985); This should provide larger numbers of polyploid shellfish

more regularly than present manipulations of spawned eggs.. .

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. An analysis of pairing competition between identical and non-identical homol­

ogous chromosomes in tetraploid oocytes (or in spermatocytes if these become the

better cytological material for study) needs to be made once tetraploids are avail­

able, or once gonad sectors of diploids have been made tetraploid. Frequencies

of multivalents at metaphase I of meiosis would first of all provide some indication .

of the degree of n~n-preferential pairing between identical and non-identical

homologues. Full analysis would be.easiest in allopolyploids where the chromosomes

of the two species parents were grossly differentiated. In the absence of such

differentiation, or in autotetraploids the frequent differences inbanding patterns

between homologous chromosomes provenient from maternal and paternal parents are

a basis for such an analysis ofpairing preferences. W~en meiotic chromosome

pairing is preceded by somatic chromosome association that predetermines or influ­

ences the nature of meiotic associations, an analysis of somatic pairing makes it

possible to predict how meiotic pairing can be expected to proceed. Pairing com­

petition could be indirectly analyzed through the segregation ratios of isozyme

markers in progeny of tetraploids, or on the basis of any other convenient

chromosome marker. (In very ancient tetraploids as the salmonids the nature of

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pairing is no 10nger such a determining factor in gene segregation.because dup1i­

cated chromosomes wou1d have acquired many mutations differentiating them fram

the original identica1 homo1ogue.)

Even if in a new1y synthesized.tetrap1oid of a commercia11y aquacu1tured

species, non-identica1 pairing occurred in asignificant portion of the.oocytes,

the breeder wou1d gain considerab1y by having produced a 1arge number if not one

hundred percent copies of the exceptiona1 individua1s. In the un1ike1y event.that

pairing preferences of chromosomes in all a110tetrap1oidsor all autotetrap1oids of

~ maricu1ture species are all unfavorab1e for cloning these groups there remains the

possibi1ity of finding or inducing gene or chromosome mutations which promote

pairing of identica1 homo10gues and suppress.pairing of non-identica1 homo1ogues.

Any genes favoring the increase of normal bivalent pairing wou1d increase fecundity

and hence have immediate, high, favorab1e.se1ection pressure as indicated by Sears

(1976) •.

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With respect to this possibi1ity of obtaining such meiotic mutants it is

worthwhi1e considering the new major information on genetic contro1 over chromosome

pairing. This comes from hexap10id wheat which has three c1ose1y re1ated chromosome. .

sets from three different, c1ose1y re1ated, diploid ancestors. The partia11y homo1­

ogous (or homoeo1ogous) chromosomes from the three ancestors have through evolution

remained so simi1ar that they can compensate for one another in the absence of any

one pair. Yet wheat - which is afar 1ess ancient polyploid than the.sa1monids ­

behaves as a diploid in respect to chromosome pairing with regular bivalent pairing

at meiosis. For some time it was almost unanimous1y be1ieved that the three basic

ancestra1 chromosome sets of hexap10id wheat did not pair because they had accumu­

1ated sma11 chromosome rearrangements in evolution. However, independent observa-

tions by Sears and Okamoto in the U.S. and by Ri1ey and Chapman in the UK provided

conc1usive evidence in 1957 that pairing between ancestra1 chromosomes (partia11y

homo1ogous or homoeologous) was prevented, and pairing restricted to the most

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completely homologous duplicated chromosome members by a mutant gene on one of the

twenty-one chromosome pairs. See Sears' 1976 review of genetic control of chromosome

pairing.

This important discovery has since led to other studies of genes promoting and

suppressing pairing in common wheat, in its relatives, in other crop plants and in

other species, all with.some implications for research and breeding applications

in aquacultured groups. Whereas cloning via a combination of tetraploidy, gyno­

genetic and polyploidizing techniques as proposed here requires genes promoting

... pairing of identical chromosomes, wheat breeding benefits from the genetic state

promoting the opposite·situation. This is because non-identical chromosome pairing

is required for the transfer of valuable genes from wild grass to wheat chromosomes

brought together in hybrids for thatexpress purpose. As aquaculture breeding and

chromosome engineering of aquaculture species advance, this too is a possibility to

be considered as pointed out recently (Crosby Longwell, 1985).

Any large-scale commercial production of induced polyploid or gynogenetic

shellfish, fish, or commercial crustaceans could well lead to the chance discovery

of meiotic mutants of use in developing naturally parthenogenetic strains through

suppression of the segregation division of meiosis. Use of polyploids and hybrids

4It would compensate some, as it has in nature, for reduced range of genetic diversity

resulting from such asexual hatchery reproduction.

Capability of producing clones of individual fish, shellfish or crustaceans,

some of which might subsequently be reproduced by normal mea~s,should prove a major

breeding advantage to aquaculturists with an economic potential akin to - possibly

in the short term - exceeding that of selection, hybridization, inbreeding or

gynogenesis alone. This is an advantage not likely to be practically achieved in

less fecund, internally fertilized agriculture mammals. In the U.S. cloning might

lead to the advantage aquaculture needs to be more economically competitive with meat

industrjes. Even so the cloning procedure described in this paper might in theory be

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applied on small scale to any experimental mamma1 or invertebrate with a basically

normal meiotic mechanism.

The commercial advantage to aquaculture wou~d be in the production from single.

high performance non-inbred heterozygous females, large cultures of organisms

genetically identical to one another and to the outstanding, sole female parent

without having to produce, maintain or test inbred lines for hybridization. Culture

uniformity would be assured inasmuch as this is genetically determined. When sex

is genetically determined these clones would be all female. Individuals for cloning

might be selected out of either natural wild populations, from hybrid cultures or

from highly selected, artificially bred individuals.

Problems inherent in selective breeding as low heritability of important traits,

inability to apply strong selection intensities effectively to a multiplicity of

important traits simultaneously, and problems with inbreeding could be avoided by

directly cloning heterozygous individuals. Identification of superior genotypes for

replication should be no larger a problem than in traditional breeding. Once ob~

tained, the clone would be an ideal direct confirmation of the genetic value of the

particular individual.

While profiting by the economic multiplication of a few superior individuals

breeders would have to take care to otherwise maintain a range of genetic variability.

This would not be inherent in cloned individuals even though non-inbred heterozygotes.

This though seems no problem at least for the present.

Lack of strictly controlled hatchery conditions in mariculture, and dependence

on natural conditions for grow-out particularly in shellfish suggest that conditions

prevailing even from month to month might favor different genotypes. These dif­

ferent genotypes would, however, be provided to some considerable extent through

use of several females for cloning. All successful agriculture seems to have

resulted in a narrowing of the wild gene pool during cultivation, domestication

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and artificial selection. It can be argued that profitable, intensive aquaculture

can be no different.

Aside from commercial uses in aquaculture, clones of heterozygous i~dividuals

of either fish or shellfish would furthermore facilitate the investigation of both

basic genetic and fishery biological problems in marine species. This is because

complications caused by inability to distinguish fully between environmental and

genetic influences and their interactions would be avoided should clones be avail­

able of heterozygous individuals. Stock enhancement programs utilizing any clones

would almostcertainly assure that these could be recognized from native stock v/ith

sufficient ease to make checking the successof such programs feasible. Replicated

. heterozygous wildtype organisms would in many aspects be ideal for bioassays of

contaminants effects. Should a recombinant DNA be successfully inserted in eggs

or embryos of aquaculture species and be successfully integrated into the

genome of a few individuals, their direct cloning would have a distinct, immediate

advantage.

In the male tao genetically identical gameteswill be produced by tetraplaid

meiosis with identical homologaus chromosome pairing. Such sperm might be used to

produce clones of male individuals through androgenetic development of fertilized

eggs in which the female pronucleus has been either removed or genetically inac­

tivated. Progeny would be diploid copies of the male parent, or when chromosomally

doubled in first cleavage, tetraplaid clones. Because it is usually more difficult

to stimulate successful androgenetic than gynogenetic development, this possibility

was not treated as part of the protocol described above. The identical sperm of

tetraploid males or of diploid males with tetraploid gonads or gonad sectors should

more certainly be of value in breeding or genetic studies where it is desirable to

have a perfectly uniform genetic contribution from a male parent.

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If the female germ line of synthetic triploid fish or shellfish could

tolerate a doubling to the hexaploid condition (six basic sets of chromosomes),

pairing of identical homologous chromosomes combined with gynogenetic techniques

could lead to the production of triploid clones. Alternately crossing of tetraploid

and diploid copies of the same diploid-tetraploid mosaic or tetraploid would also

produce triploid clones.

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References

Arait

K.t

F. Naito and K. Fujino. 1983. Present status ofbasicresearch for

chrornosorne engineering in the abalone. Otsuchi.Ma~ine Research Center Report t

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Arait

K.t

F. Naito, H. Sasaki and K. Fujino. 1984. Gynogenesis with ultraviolet

ray irradiated sperrn in the Pacific abaione. Bull. Jpn. Soc. Sei. Fish.

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Aung, T. and G.M. Evans. 1983. Pairing controlgenes in Loliurn. In P.E. Brandharn

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Baker, B.S.t

A.T.C. Carpenter, M.S. Esposito, R.E. Esposito and L. Sandler. 1976.

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Chevassus, B. 1983. Hybridization in fish. Aquaculture 33: 245-262.

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Evans, G.M. and I.B. Taylor. 1976. Genetic control of hornoeologous chrornosorne

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161-168.

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Khush, G.S. 1978. Cytogenetics of Aneup1oids. Academic Press, New York.

Lima-de-Faria, A. 1983. Mo1ecu1ar Evolution and Organization of the Chromosome.

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Linco1n, R.F. and P.A. Hardiman. 1982. The production and growth of fema1e

diploid and triploid rainbow trout. Intern. Symp. on Genetics in Aquacu1ture,

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Longo, F.J. 1972. The effects of cytocha1asin B on the events of ferti1ization

in the surf c1am, Spisu1a solidissima. I. Polar body formation. J. Exper.

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Longwe11, A. Crosby. 1968. Oyster genetics: research and commercia1 applications.

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Se1don, Long Is1and, New York, p. 91-103.

Longwe11, A. Crosby. 1984. Talk to New Eng1andShe11fish Hatchery Operators,

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Figure 1. Metaphase I in a NON-HOMOZYGOUS DIPLOID OOCYTE with ORDINARY PAIRING

of the materna11y- and paterna11y-derived members of each chromosome

pair. For purpose of illustration a haploid chromosome number of 2

is assumed. Each of the two configurations represents one bivalent

chromosome (chromosome pair). Each bivalent half is composed of 2

chromatids replicated in advance for the second meiotic division.

Crossing-over of genes among these chromatids shuffles genes linked

on the same chromosome •. Numbers 1 and 2 refer to chromosome 1 and to

chromosome 2. The letters Mand P refer to materna11y-derived and

paternally-derived chromosomes. Dark round bodies are centromeres,

the spind1e attachment region of the chromosomes. Straight lines

radiating from the centromeres of each bivalent half are spind1e

fibers which effect the poleward movement of each bivalent half to

spindle poles. ORIENTATION OF BIVALENTS ON THE SPINDLE RELATIVE TO

ONE ANOTHER WILL DETERMINE GENETIC Cor~POSITION OF THE GAr1ETES WHICH

WILL NOT BE ALL ALIKE BECAUSE MATERNALLY- AND PATERNALLY-DERIVED

CHROMOSOMES CARRY DIFFERENT GENES. WITH CROSSING-OVER OF GENES AND

WITH LARGER CHROMOSOME NUMBERS MORE DIFFERENT NEW COMBINATIONS OF

GENETIC MATERIAL ARE POSSIBLE THAN IN THE SIMPLE ILLUSTRATION HERE.

~~~~~-~- - ~ ~~ -~~~~-

Figure 1. For legend see over1ay ..'

· \. ..,....

Figure 2. Metaphase I in a NEWLY DOUBLED NON-HOMOZYGOUS DIPLOID WITH COMPETITIVE

PAIRING OF IDENTICAL HOMOLOGOUS CHROMOSOMES. OBVIOUSLY,ORIENTATION

OF BIVALENTSON THE SPINDLE RELATIVE TO ONE ANOTHER CANNOT LEAD TO ANY

DIFFERENCES AMONG GAMETES, EACH OF WHICH IS GENETICALLY IDENTICAL TO

THE MOTHER EXCEPT IN TOTAL NUMBER OF CHRO~10S0MES. EVEN CROSSING-OVER

OF GENES WILL NOT RESULT IN ANY DIFFERENCES IN THE FOUR NUCLEAR

PRODUCTS OF MEIOSIS IN THE SEVERAL OOCYTES OF ANY SINGLE INDIVIDUAL.

M\

Figure 2. For legend see overlay.

F1gure 3

Produets of 1e10s1s by stage. genet1e Outeo;; of 9Ynogenes1sw1th and wtthout·fus10n to polar body nueleus when there 1s

eompet1ttve patr1ng of homo10gous 1nstead of ho-oe010gous ehro-os~s

1n allotetrapl01d ooeytes. or of tdentteal hOROlogues tnstead ofnon-1dentteal ha.ologues 1n autotetraplotd ooeytes

Mlterna1 - der1vtd 1.Materna1 ~ dertved 1*Paternal - dertved 1Patemal - dertved 1*Matemal - derhed 2Maternal - dertved 2*Paterna1 - dertved 2Paterna1 - dert nd 2* .

(*arttftetally duplteated tn "ktng thepolyplotdtndhtdual. genn 11ne or rnacells - elaet eopy of.tts homologue

Maternal - dertved 1FEMALE Patemal - dertved 1GAHETE Maternal - dertved 2

Paterna1 dertved 2

1st POLAR BODY,DIVISION _.

Maternal - dertved 1Patemal - dertved 1

POLAR Materna1 - dertved ZBODY Paterna1 - dertved Z

Matemal - dertv!d 1POLAR Patemal - dertved 1Booy Materna1 - dertved 2

Patemal dertved Z

1n every oOeyte .Maternal - dertYed 1

POlAR Patemal - dertved 1BODY Matemal - dertved 2

. '. Patemal - derhed 21n every ooeyte

METAPHASE 11DIVISION

Materna1 - dertnd 1Paternal - dertved 1Maternal - dertved 2Paterna1 - dertved 2

'~cr.rOduets of Metos1s 1Materna - ertved 1Patemal - derhed 1 ....Matemal - derhed ~

Patemal - derhed Z 'Four produets of Metes1s 11

POLARBODY

GYNOGENESIS •WITH IRRADIATED

SPERM

Maternal - dertved 1Paterna1 - dertved 1Maternal - dertved ZPatemal - dertved 2

GYN ESlSWITH IRRADIATED

SPERM WITH FUSIDNOF FEMALE GAMETE

AND POLAR BOOYNUCLEUS'"

Maternal - dertved 1Matemal - dertved 1Paternal - dertved 1Patemal - dertved 1Matemal - dertved ZMaterna1 - dertved 2Paterna1 ~ dertved 2Patemal - dertved Z

EVERY OOCTYE BECOMES AREPLICA EVERYOOCYTE BECOMES AOF A DIPLOID FEMALE WHOSE REPLICA OF THE FEMALE. OR

GERM LINE OR SECTORS OF IT WAS A TETRAPLOID RENDITION OFMADE TETRAPLOID. OR ADIPLOID , ADIPLOID FEMALE WHOSE

RENDITION OF.A TETRAPLOID GERM LINE OR SECTORS Cf ITFEMALE WAS MADE TETRAPLOID

IN EITHER OF TWO CASES JUSTABOVE. EXACT NON-IKBRED COPIES (NON-INBREDCLONES) ARE PRODUCED OF INDIVIDUAL FEMALES SELECTED rOR OUTSTANDING

COHHERCIAL PERFORMANCE OR ON BASIS OF SCIENTIFIC INTEREST

•• The genette outeome ts the same When the.tetraplofd chromosome' number tsrestored by 1nhtbit1ng the 1st eleavage fnstead of by fusion of the fe-alegagete wtth the polar bodJ nueleus.

Figure 4. Comparison of progeny of gynogenesis of oocytes from diploid indi-. .,

viduals with homo1ogous chromosome pairing to that of a11otetrap1oid

or autotetrap1oid individua1s or mosaics with preferentia1, dip1oid­

1ike pairing of identica1 homo1ogues

Tetraploid Oocytes

Without fusion of gamete to polar body or

without doubling of 1st c1eavage

With fusion of gamete to 2nd polar body .

With doubling of 1st cleavage division

DIPLOID CLONES* OF THE TETRAPLOID

FEMALE, or CLONES OF DIPLOID FEMALE

with tetrap10id gonad or gonad sectors

CLONES* OF THE TETRAPLOID FEMALE. or

TETRAPLOID CLONES OF DIPLOID FEMALE

withtetrap10id gonad or gonad sectors

Same as for fusion with 2nd polar body

except for low frequency of new mutation

in polar body chromosomes - TETRAPLOID

CLONES* of DIPLOID OR TETRAPLOID FEMALES

Diploid Oocytes

Without fusion of gamete to polar body

or doubling of 1st cleavage

Hitb fusion of gamete to 2nd polar body

With doubling of 1st cleavage division

HAPLOID INDIVIDUALS**.each genetically

different from the other and from the

fema1e parent

HIGHLY INBRED DIPLOID INDIVIDUALS**.

all genetically different from one

another and from the female parent

PERFECTLY HOMOZYGOUS INDIVIDUALS**.

all genetically different from each

other and from the female parent

* All individuals in the clones are as heterozygous or hybrid as the female pro­ducing the oocytes.

** When eggs of any one of these gynogenetic progeny are subje~t to gynogenesis,clones are the result, but all members of the clones are genetically homozygous