Fish genomics: From genetic manipulation to genomic analyses

Fish genomics: From genetic manipulation to genomic analyses
Thorgaard 1, G.H., K.M. Nichols 1,2, A. Felip 1,3, A.M. Zimmerman 4,5, K.H. Brown 1,6, R.E. Drew 1, B.D.
Robison 6, K. Sundin 1,7, J. Brunelli 1, R.B. Phillips 8, S.S. Ristow 9 and P.A. Wheeler 1
1
School of Biological Sciences and Center for Reproductive Biology, Washington State University, Pullman,
Washington 99164-4236 USA
2
National Oceanic and Atmospheric Administration, Northwest Fisheries Center, 2725 Montlake Blvd. E.,
Seattle, WA 98112 USA
3
Present address: Instituto de Acuicultura de Torre de la Sal, Consejo Superior de Investigaciones Científicas
Dpto. de Fisiología y Reproducción de Peces, Ribera de Cabanes s/n, 12595 Castellón. SPAIN
4
Department of Animal Sciences, Washington State University, Pullman, Washington 99164-6332 USA
5
Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts
02139 USA
6
Present address: Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844-3051 USA
7
Present address: Signature Genomic Laboratories LLC, 44 West 6th Avenue Suite 202, Spokane, Washington
99204 USA
8
School of Biological Sciences and Center for Reproductive Biology, Washington State University, 14204 NE
Salmon Creek Ave., Vancouver, Washington 98686-9600 USA
9
Agricultural Research Center, Washington State University, Pullman, Washington 99164-6240 USA
Abstract
Genetic and genomic studies with fish models can present both distinctive advantages and challenges.
In many countries, there are fewer researchers and less funding available for investigation with fishes than for
some competing research models. At the same time, fish models have distinct advantages in ease of culture
and manipulation relative to some other vertebrate models. Species such as the zebrafish and fugu have been
the focus of large scale DNA sequencing projects, and among the aquarium fish research model species, the
zebrafish and medaka have well-developed genetic maps including the distribution of extensive numbers of
genes on chromosomes. Among aquaculture species, the channel catfish, tilapia, Atlantic salmon and rainbow
trout also have relatively well-developed genetic maps which emphasize polymorphic microsatellites.
The tolerance of fishes to genetic manipulations such as androgenesis (induced all-paternal
inheritance) and gynogenesis (induced all-maternal inheritance) has made it possible to rapidly produce clonal
lines in several species (e.g., zebrafish, medaka, ayu, tilapia, common carp, rainbow trout). We will review
these methods and their advantages for genetic and genomic studies in fishes with a special emphasis on recent
advances in the rainbow trout model.
The amenability of fishes to genetic manipulations was first demonstrated by studies in which
polyploidy was successfully induced using temperature shock treatments.
Methods became increasingly
successful and were expanded to include hydrostatic pressure treatments and the use of gamma and UV
irradiation of the eggs or sperm to induce androgenetic or gynogenetic progeny, respectively. Streisinger
proposed that it should be possible to generate homozygous clonal lines of fishes by two successive cycles of
gynogenesis and reported using this approach successfully in zebrafish in 1981. In this manner, research lines
analogous to the inbred lines which have been produced in mice using many generations of sibling mating can
be generated in fishes (and amphibians) in two generations.
The potential for utilizing this approach was
seemingly unlimited. Although the approach has been applied successfully for producing clonal lines of tilapia
and medaka, as well as zebrafish, the largest application has been in our laboratory to the rainbow trout model.
With the widespread development of methods for analyzing genetic markers such as microsatellites and AFLPs
(amplified fragment length polymorphisms), the stage is set for widespread utilization of clonal fishes in
genetic and genomic studies.
Research with clonal fishes clearly has tremendous potential.
Genetic uniformity over time is
beneficial for many types of genetic studies, and production of consistent, homozygous experimental material
is particularly advantageous for many mapping and sequencing studies in which interpretations are facilitated
by homozygosity.
For example, large-scale BAC (Bacterial Artificial Chromosome) fingerprinting and
sequencing is more reliable when variation due to allelic heterozygosity is eliminated by using homozygous
clonal material. Similarly, microarray studies may be more repeatable if clonal animals are utilized. When
androgenesis is utilized, there is also the potential to produce individuals with identical nuclear genotypes but
which vary in their mitochondrial genotype.
Challenges for working with clonal lines can be substantial, as implied by the relatively limited use of
these methods. Optimizing treatments and the use of high-quality gametes are critical to success of the
manipulations. Fertility problems have been observed among homozygous individuals, especially females.
Proper identification and inventory of individuals over time is also critical to research programs using these,
and other, research lines.
In spite of these challenges, our lab group has had considerable success in utilizing clonal lines of
rainbow trout as research models for genetic and genomic research. We have modeled our approach on that
originally outlined by Streisinger but selected androgenesis, rather than gynogenesis, as the primary
manipulation method. The two main advantages of utilizing androgenesis rather than gynogenesis have been
(1) the potential for producing male, as well as female, clonal lines in this male heterogametic fish species, and
(2) the potential for using sperm cryopreservation for storing lines. The production of male, as well as female,
clonal lines has proved advantageous because the fertility problems in males do not seem to be as severe as
those in females. We have used the natural geographic diversity within the rainbow trout species as a source of
genetic variation in producing our clonal lines. Although caution is warranted in extrapolating our results
broadly because a clonal line may not always be representative of the population from which it was derived,
studies to date have shown consistencies between the clonal line and donor population for the traits of
development rate and resistance to the parasite Ceratomyxa shasta.
The following steps have been used in our studies: (1) Detect phenotypic variations among the clonal
lines by direct study of the clones, or by comparing the characteristics in crosses to common outbred
individuals. (2) Analyze segregation of the variation among doubled haploid (homozygous diploid) progeny
produced by androgenesis from hybrids between the lines we are comparing. (3) Evaluate the statistical
association between phenotypes/ traits and molecular markers (e.g., AFLPs or microsatellites) in these doubled
haploids using QTL (quantitative trait locus) analyses. To date, the following traits have been mapped by QTL
analysis using this approach: embryonic development rate to hatch, variation in the numbers of several
meristic elements, resistance to the pathogen Ceratomyxa shasta and variation in natural killer cell-like activity.
Resistance to the infectious hematopoietic necrosis virus and domestication-related behaviors also show
differences among lines but have yet to be elucidated in QTL analyses. We are also developing a congenic line
of rainbow trout in which a QTL for rapid embryonic development from one clonal line is being introgressed
into another clonal line with slow development.
The potential for wider use of clonal lines for genetic and genomic research in fishes is clearly great.
The application has moved beyond that originally visualized by Streisinger for analysis of induced mutations
but his foresight in advocating this research approach was nevertheless fundamental to the progress which has
been made. Perhaps the biggest lesson is the importance of time, patience and continuity for developing and
maintaining clonal lines if this approach is to see more widespread use.
Genetic and genomic studies with fish models present both distinctive advantages and challenges. In
spite of the importance of fishes as food sources and research models, in many countries there are fewer
researchers and less funding available for investigations with fishes than for other animal research models. At
the same time, fish models have distinct advantages in ease of culture and manipulation relative to some other
vertebrate models and there is the possibility to conduct the research in a more efficient manner as a result of
those differences. Fishes generally can be reared at lower cost and in larger numbers than mammalian or avian
species. Species such as the zebrafish and fugu have progressed far in DNA sequence information (Clark
2003), and among the aquarium fish research model species, the zebrafish and medaka have well-developed
genetic maps of the distribution of known genes on chromosomes (Woods et al. 2000; Naruse et al., 2004).
Among the aquaculture species, the channel catfish (Waldbieser et al., 2001), tilapia (Kocher et al. 1998),
Atlantic salmon (Moen et al. 2004) and rainbow trout (Young et al. 1998; Sakamoto et al., 2000) have
relatively well-developed genetic maps, emphasizing polymorphic microsatellites.
Tolerance of fishes to genetic manipulations
The tolerance of fishes to genetic manipulations such as androgenesis (induced all-nuclear paternal
inheritance) and gynogenesis (induced all-maternal inheritance) has made it possible to rapidly produce clonal
lines in some species (e.g., zebrafish (Streisinger et al. 1981), medaka (Naruse et al. 1985), ayu (Taniguchi et al.
1994), tilapia (Sarder et al. 1999), common carp (Komen et al. 1993) and rainbow trout (Young et al. 1996)).
Although such approaches are technically possible in amphibians, they have not been widely applied. These
approaches do not appear to be technically possible in birds or mammals. We will review methods for
producing clonal fishes and their advantages for genetic and genomic studies with a special emphasis on recent
advances in the rainbow trout model.
The amenability of fishes to genetic manipulations was first demonstrated by studies in
which polyploidy was successfully induced using temperature shock treatments (Swarup 1959).
These studies followed earlier similar studies in amphibians (Fankhauser 1945).
Methods for inducing
retention of the second polar body and for blocking the first cleavage division became increasingly successful
and were expanded to include hydrostatic pressure treatments and the use of gamma and UV irradiation of the
eggs or sperm to induce androgenetic or gynogenetic progeny, respectively (e.g., Purdom 1969). A significant
conceptual advance came when Streisinger et al. (1981) proposed that it should be possible to generate
homozygous clonal lines of fishes by applying gynogenesis to two successive generations of fish and reported
using this approach successfully in zebrafish in 1981. In this manner, research lines analogous to the inbred
lines which were already established as research models in mice (Silver 1995) could be rapidly developed. In
mice, developing such lines requires many generations of sibling mating. However, with the limited resources
available for studying fish models and the relatively long generation times of some species, the production of
inbred lines by sibling mating was not a practical alternative for fishes. Using chromosome set manipulation,
clonal lines can thus be generated in fishes (and amphibians) in two generations. The potential applications for
this approach outlined by Streisinger were seemingly unlimited. Although the approach has been applied
successfully for producing clonal lines of tilapia, medaka, ayu and common carp as well as zebrafish, the
broadest application to date has been in our laboratory to the rainbow trout model. With the widespread
development of methods for analyzing polymorphic genetic markers such as microsatellites and AFLPs
(amplified fragment length polymorphisms), the stage is now set for widespread utilization of clonal fishes in
genetic and genomic studies.
Clonal lines: potential, challenges, advantages
Research with clonal fishes clearly has tremendous potential. As amply demonstrated in the inbred
mouse model, genetic uniformity over time is beneficial for many types of genetic studies. Consistency of
research over time and location becomes possible using such lines.
Production of uniform, homozygous
experimental material is particularly advantageous for many mapping and sequencing studies in which
interpretations are facilitated by homozygosity.
For example, large-scale BAC (Bacterial Artificial
Chromosome) fingerprinting and sequencing can benefit when variation due to allelic heterozygosity is
eliminated by using homozygous clonal material. Similarly, microarray studies may be more repeatable if
clonal animals are utilized. When androgenesis is utilized, there is also the potential to produce individuals with
identical nuclear genotypes but which vary in their mitochondrial genotype (Brown and Thorgaard, 2002).
Androgenesis typically involves inactivation of maternal nuclear genetic material, but does not inctivate all of
the mitochondrial genomes present in the egg. This provides opportunities for detailed studies of phenotypic
effects resulting from mitochondrial differences as well as analysis of mitochondrial/ nuclear interactions.
Challenges for working with clonal lines can be substantial, as implied by the relatively limited use of
these methods and especially by the few instances of ongoing research with these models. In spite of the
history of producing clonal lines in a number of species, the work with some species has not continued. Recent
research on the zebrafish model, for example, no longer utilizes clonal lines. At this point the common carp
and rainbow trout are the two clonal line models which are continuing to show the most progress. Our
laboratory has emphaized the rainbow trout model because of the broad knowledge of and interest in this
species (Thorgaard et al., 2002).
A variety of factors may have limited the utilization of clonal lines. Optimizing treatments and the use
of high-quality gametes are critical to initial success of these manipulations. Suboptimal treatments can
hamper the success of these methods. The availability of gametes in sufficient numbers and over a length of
time which allows successful experimentation is of obvious importance. Fertility problems have been observed
among homozygous individuals, especially females. It is perhaps notable that the species for which the most
ongoing success has been seen (common carp and rainbow trout) both have relatively high fecundities. In
addition, androgenesis has been utilized in combination with gynogenesis in propagating clonal lines in these
species, which may have allowed the propagation of a wider range of lines and the loss of lines due to fertility
problems in the homozygous female. Proper identification and inventory of individuals over time is also
critical to research programs using these, and other, research lines. Lines should be periodically genotyped to
confirm their identity and homozygosity (Young et al. 1996; Robison et al. 1999). Maintaining lines over time
requires continuing commitments of space and funding which can be challenging to sustain.
Development of rainbow trout clonal lines
In spite of these challenges, our lab group at Washington State University has had considerable success
in utilizing clonal lines of rainbow trout as research models for genetic and genomic research. We have
modeled our approach on that originally outlined by Streisinger but have selected androgenesis, rather than
gynogenesis, as the primary manipulation method. The two main advantages of utilizing androgenesis rather
than gynogenesis have been the potential for producing male, as well as female, clonal lines in this male
heterogametic fish species, and the potential for using sperm cryopreservation as a method for storing lines.
We have successfully applied established cryopreservation methods for salmonids (Wheeler and Thorgaard
1991) for storing and recovering clonal lines by androgenesis. The availability of male, as well as female,
clonal lines has proved advantageous because the fertility problems in males do not seem to be as severe as
those in females. At this point we are propagating two female (XX) lines and seven male (YY) lines of
rainbow trout (Table 1).
We have used the natural geographic diversity within the rainbow trout species as a source of genetic
variation in producing our clonal lines. Rainbow trout have been distributed widely around the world, but are
naturally distributed along the Pacific coast of North America from northern Mexico to Alaska, and on the
Kamchatka Peninsula of Russia (MacCrimmon 1971).
At this point, we are rearing clonal lines of rainbow
trout originating from the states of Alaska, Washington, Idaho, Oregon and California in the United States
(Table 1). The OSU (Oregon State University) line is a female (XX) line, and is of primary importance in our
research program as a line to which our various male lines have been crossed. The OSU line was derived from
a strain of California origin which historically was propagated at the Mt. Shasta hatchery of the California
Department of Fish and Game. We are seeking to capture significant genetic variation representative of natural
populations within the rainbow trout species in our sample of clonal lines. The Swanson line, originally
derived from the Swanson River on the Kenai Peninsula of Alaska, and the Clearwater line, originally derived
from the Clearwater River in Idaho, are examples of lines which appear to have a number of distinctive
characteristics that distinguish them from the OSU line and that merit further study. The lines we are
propagating have varying histories of domestication. The OSU, Arlee and Hot Creek lines have longer histories
of rearing in hatcheries than do the Swanson, Clearwater, Klamath (Oregon), Skookumchuck (Washington) and
Whale Rock (California) lines.
Quantitative behavioral differences are evident among the lines we are
propagating (Lucas et al. 2004). The Clearwater and Swanson lines, consistent with their rearing histories,
show differences in behavior from the Arlee and Hot Creek lines. Efforts to better understand the genetic
control and mechanistic basis for these behavioral differences are ongoing. BAC libraries have been developed
from DNA of the OSU and Swanson clonal lines (Thorgaard et al., 2002)
It is important to note that the clonal lines which we are rearing represent a single haploid genome
extracted from the population from which they were derived. Caution is warranted in extrapolating our results
to make broad inferences about the source populations because a clonal line may not always be representative
of the population from which it was derived. However, studies to date have shown consistencies between the
clonal line and donor population for the traits of development rate (Robison and Thorgaard 2004) and
resistance to the parasite Ceratomyxa shasta (Nichols et al. 2003a). It is possible that genes associated with
traits which are under significant selection may be more likely to become homozygous within natural and
cultured populations. This may increase the likelihood that the clonal lines that we develop from a single
haploid genome are indeed representative of their source populations.
Genetic analyses of divergent phenotypes using clonal lines
The following are the steps we have used to date in genetic analysis of trait differences among our
lines. As outlined below, we hope to extend these studies to understand the genetic and molecular mechanisms
responsible for differences among natural and cultured populations of rainbow trout.
(1) Detect phenotypic variations among the clonal lines by direct study of the clones, or by comparing
the characteristics in crosses to common outbred individuals. The studies by Robison et al. (1999) comparing
development rate among the clonal lines and by Lucas et al. (2004) examining behavioral differences among
the lines are good examples of this initial level of analysis. We have found that it is difficult to propagate
clonal individuals in the numbers which we would like for comparative studies. The comparisons of numbers
of meristic elements described by Nichols et al. (2004) represent an exception in which we have made direct
comparisons among clonal individuals. However, it appears that for many traits, meaningful differences can be
detected by using sperm from clonal males to fertilize eggs from common, outbred females.
Although
differences among lines which are recessive in nature might not be detected with such a design, traits showing
predominantly additive or dominant inheritance should be detectable. Crosses to outbred individuals have the
further advantage that groups derived from the various clones can be compared directly and efficiently. By
utilizing common eggs, maternal effects are minimized and the lots being compared can be reared at the same
time and under common conditions. The magnitude of the developmental (Robison et al.1999) and behavioral
(Lucas et al. 2004) differences detected using this approach, even when female parents are held in common,
support its value for making initial comparisons among the lines.
(2) Analyze segregation of genotypes and phenotypes the variation in the doubled haploid
(homozygous diploid) progeny produced by androgenesis from hybrids between the lines we are comparing.
Robison et al. (2001) first utilized doubled haploids for QTL (quantitative trait locus) analysis in a cross of two
clonal lines to examine the genetic control of development rate. This approach is similar to approaches which
have been utilized in some plant genetic studies (Burr and Burr 1991).
The principal advantages of this
approach appears to lie in the wide genetic diversity present among the segregating individuals (greater than in
backcross or F2 mating designs) and the potential for scoring efficient, dominant markers such as AFLPs in
such a cross because all of the progeny are homozygous. Potential disadvantages of scoring in doubled
haploids are the difficulty of producing sufficient numbers of individuals and the possibility of unusual
phenotypic expressions related to homozygosity. Overall, the success of detecting QTLs using this approach
(discussed below) seem to validate analysis in doubled haploids.
(3). Evaluate the statistical association among the phenotypes/ traits and molecular markers (e.g.,
AFLPs or microsatellites) in doubled haploids using QTL analyses. A number of traits have now been
successfully mapped by QTL analysis using this approach. Three different crosses (OSU X Swanson, OSU X
Clearwater and OSU X Hot Creek) have been utilized to date for QTL studies and other crosses appear to have
good potential for such studies. The number of successful QTL studies to date is impressive considering the
relatively limited resources and number of individuals committed to these studies.
The OSU X Swanson cross was the first to be used for QTL analysis in studies by Barrie Robison. A
difference in embryonic development rate to hatch was initially found between the OSU and Swanson lines
(Robison et al. 1999) and a hybrid clone was produced between the OSU female and Swanson male lines.
Hybrid clones have been found to be quite vigorous relative to homozygous clones (Young et al. 1995). Our
original intention was to backcross the OSU X Swanson hybrid clone to the OSU line and to examine marker
and trait segregation in the backcross. However, when eggs were not available at the same time as sperm from
the hybrid clone, we proceeded to produce doubled haploid progeny from the hybrid clone by androgenesis.
This proved to be fortuitous because the design was highly successful in detecting a major QTL for
development rate in the cross (Robison et al. 2001).
The OSU X Clearwater cross was the second used for QTL analysis in studies by Krista Nichols. The
Clearwater line has a number of trait differences relative to the OSU line and this cross has proved fruitful for a
number of studies. Being derived from the Clearwater River in Idaho, this line is a representative of the inland
form of rainbow trout which is recognized as a distinct group relative to the more widely propagated coastal
strain (Allendorf and Utter 1979; Behnke 1992). The Clearwater line is of anadromous (steelhead) origin and
shows differences in numbers of meristic elements, resistance to the pathogen Ceratomyxa shasta, and
development rate relative to the OSU line. Several QTLs were shown to be associated with differences in
meristic elements (Nichols et al. 2004) and resistance to Ceratomyxa shasta (Nichols et al. 2003a). Differences
in development rate and in smoltification (a trait related to the steelhead life history) are currently being
characterized. Because of the genetic distinctiveness of the Clearwater line relative to the OSU line as well as
the large number of trait differences between these lines, this cross appears to be a particularly promising one
for further genetic, mapping and mechanistic studies.
The OSU X Hot Creek cross was utilized for QTL analysis in studies by Ana Zimmerman. This cross
of two lines of domesticated origin was studied because of substantial differences in natural killer cell-like
activity between the lines. A single major QTL of very large effect related to natural killer cell-like activity
was localized to one linkage group among doubled haploids from this cross (Zimmerman et al. 2004). We
hope to pursue this very promising result further. Several loci were also found to be associated with a
difference in numbers of pyloric caecae between the lines in this cross (Zimmerman et al. in press).
Resistance to the infectious hematopoietic necrosis virus and behavioral variations related to
domestication also show differences among lines but have yet to be elucidated in QTL analyses. These traits
are likely to be complex in nature but we believe that we have good prospects for making progress in genetic
analysis of these traits. For such traits, we are examining the use of a progeny test design in which progeny of
doubled haploid males crossed to outbred females are used to better identify trait values and identify QTLs.
This approach is similar in principle to that used for defining the breeding value of sires in dairy cattle based on
the characteristics of their daughters.
Other applications and ongoing directions
Another application of the clonal lines has been in the isolation of Y-linked markers and the study of
sex chromosome evolution. Felip et al. (in press) was able to identify a number of AFLP markers linked to the
Y chromosome in a study of the OSU X Swanson cross. These markers proved to be useful in characterizing
the nature of Y chromosome differences among the lines (Felip et al. 2004). The rainbow trout model appears
to be a promising one for studies of sex chromosome evolution and differentiation. Our lines of rainbow trout
also exhibit differences in Y chromosome morphology (Felip et al. 2004; Phillips et al. 2004) which can be
further studied in the future.
Another promising avenue of research lies in development of clonal lines for studying physiological
effects of mitochondrial variation. Androgenesis allows individuals to be produced which inherit their nuclear
genes from the male but their mitochondrial genes from the female parent (May and Grewe 1993; Brown and
Thorgaard 2002). We have exploited this to produce lines of rainbow trout which are identical or near-identical
in their nuclear genome but which differ in their mitochondrial haplotype (Brown and Thorgaard, 2002). These
lines may be useful for dissecting the significance of mitochondrial haplotype variation for development,
organismal physiology and evolution.
Ultimately, we hope to better understand the genetic and molecular mechanisms responsible for the
differences among the lines. Toward the goal of better dissecting molecular mechanisms, we are developing a
congenic line of rainbow trout in which a QTL for rapid embryonic development from the Clearwater clonal
line is being introgressed into the slow developing OSU line (Sundin et al, in press). Congenic and advanced
backcross lines have proven to be very useful in the genetic dissection of traits in mice (Silver 1995) and plants
(Tanksley and Nelson 1996). By the third generation of the rainbow trout backcross, the association between
markers associated with this QTL and development rate was very notable (Figure 1). We have now produced
the fourth generation of this backcross. We believe that studying molecular variation using approaches such as
microarray analyses in advanced backcross/ congenic populations segregating for QTLs will be a fruitful
approach for dissecting molecular mechanisms (Jansen and Nap 2001; Wayne and McIntyre 2002).
Development of a high-quality genetic map will be also critical for increasing our mechanistic
understanding by making it easier for us to identify genes associated with QTLs. Doubled haploid panels have
proved to be especially valuable for mapping because their homozygosity allows dominant markers such as
AFLPs to be rapidly analyzed to produce a framework map (Young et al. 1998). The mapping of known genes
is also faciliated by the absence of heterozygosity in individuals from these panels (Nichols et al. 2003b). We
hope to greatly expand these efforts to improve our comparative genetic map. This should allow us to better
utilize map information being developed in other species through comparative studies.
The potential for wider use of clonal lines for genetic and genomic research in fishes is clearly great.
A variety of obstacles related to fertility, quality control and sustained commitment of resources represent
challenges to the approach. The application has moved beyond that originally visualized by Streisinger for
analysis of induced mutations but his foresight in advocating this research approach was nevertheless
fundamental to the progress which has been made. Perhaps the biggest lesson is the importance of time,
patience and continuity for developing and maintaining clonal lines if this approach is to see more widespread
use.
References
Allendorf FW, Utter FM. 1979. Population genetics. In: Fish Physiology, Vol. 8, Hoar WS, Randall DJ,
Brett JR (Eds) Academic Press, New York, pp. 407-454.
Behnke RJ. 1992. Native Trout of Western North America. American Fisheries Society Monograph 6.
American Fisheries Society, Bethesda, MD.
Brown KH, Thorgaard GH. 2002. Mitochondrial and nuclear inheritance in an androgenetic line of rainbow
trout, Oncorhynchus mykiss. Aquaculture 204: 323-335.
Burr B, Burr FA. 1991. Recombinant inbreds for molecular mapping in maize: theoretical and practical
considerations. Trends Genet. 7: 55-60.
Clark MS. 2003. Genomics and mapping of Teleostei (bony fish). Comp. Funct. Genom. 4: 182-193.
Fankhauser G. 1945. The effect of changes in chromosome number on amphibian development. Quart. Rev.
Biol. 20: 20-78.
Felip A, Fujiwara A, Young WP, Wheeler PA, Noakes M, Phillips RB, Thorgaard GH. 2004. Polymorphism
and differentiation of rainbow trout Y chromosomes. Genome 47: 1105-1113.
Felip A, Young WP, Wheeler PA, Thorgaard GH. An AFLP-based approach for the identification of sexlinked markers in rainbow trout (Oncorhynchus mykiss). Aquaculture: in press.
Jansen RC, Nap JP. 2001. Genetic genomics: the added value from segregation. Trends Genet. 17: 388-391.
Kocher TD, Lee WJ, Sobolewska H, Penman D, McAndrew B. 1998. A genetic linkage map of a cichlid fish,
the tilapia, Oreochromis niloticus. Genetics 148: 1225-1232.
Komen J, Eding EH, Bongers ABJ, Richter CJJ. 1993. Gynogenesis in common carp (Cyprinus carpio). 4.
Growth, pheontypic variation and gonad differentiation in normal and methyltestosterone-treated homozygous
clones and F(1) hybrids. Aquaculture 111: 271-280.
Lucas MD, Drew RE, Wheeler PA, Verrell PA, Thorgaard GH. 2004. Behavioral differences among rainbow
trout clonal lines. Behavior Genetics 34: 355-365.
MacCrimmon HR. 1971. World distribution of rainbow trout (Salmo gairdneri). J. Fish. Res. Board Canada
28: 663-704.
May B, Grewe PM. 1993. Fate of maternal mtDNA following 60Co inactivation of maternal nuclear DNA in
unfertilized salmonid eggs. Genome 36: 725-730.
Moen T, Hoyheim B, Munck H, Gomez-Raya L. 2004. A linkage map of Atlantic salmon (Salmo salar)
reveals an uncommonly large difference in recombination rate between the sexes. Animal Genetics 35: 81-92.
Naruse K, Ijiri K, Shima A, Egami N. 1985. The production of cloned fish in the medaka (Oryzias latipes). J.
Exp. Zool. 236: 335-341.
Naruse K, Tanaka M, Mita K, Shima A, Postlethwait J, Mitani H. 2004. A medaka gene map: the trace of
ancestral vertebrate proto-chomosomes revealed by comparative gene mapping. Genome Research 14: 820828.
Nichols KM, Bartholomew J, Thorgaard GH.
2003a.
Mapping multiple genetic loci associated with
Ceratomyxa shasta resistance in Oncorhynchus mykiss. Dis. Aquat. Org. 56: 145-154.
Nichols KM, Young WP, Danzmann RG, Robison BD, Rexroad C, Noakes M, Phillips RB, Bentzen P, Spies I,
Knudsen K, Allendorf FW, Cunningham BM, Brunelli J, Zhang H, Ristow S, Drew R, Brown KH, Wheeler PA,
Thorgaard GH. 2003b. A consolidated linkage map for rainbow trout (Oncorhynchus mykiss). Animal
Genetics 34: 102-115.
Nichols KM, Wheeler PA, Thorgaard GH.
2004.
Quantitative trait loci analyses for meristic traits in
Oncorhynchus mykiss. Env. Biol. Fishes 69: 317-331.
Phillips RB, Noakes MA, Morasch M, Felip A, Thorgaard GH. 2004. Does differential selection on the 5S
rDNA explain why the rainbow trout sex chromosome heteromorphism is NOT linked to the SEX locus?
Cytogenet. Genome Res. 105: 122-125.
Purdom CE. 1969. Radiation-induced gynogenesis and androgenesis in fish. Heredity 24: 431-444.
Robison BD, Wheeler PA, Thorgaard GH. 1999. Variation in development rate among clonal lines of rainbow
trout (Oncorhynchus mykiss). Aquaculture 173: 131-141.
Robison BD, Wheeler PA, Sundin K, Sikka P, Thorgaard GH. 2001. Composite interval mapping reveals a
major locus influencing embryonic development rate in rainbow trout (Oncorhynchus mykiss). J. Hered. 92:
16-22.
Robison BD, Thorgaard GH. 2004. The phenotypic relationship of a clonal line to its population of origin:
rapid embryonic development in an Alaskan population of rainbow trout. Trans. Amer. Fish. Soc. 133: 455461.
Sakamoto T, Danzmann RG, Gharbi K, Howard P, Ozaki A, Khoo SK, Woram RA, Okamoto N, Ferguson
MM, Holm L-E, Guyomard R, Hoyheim B.
2000.
A microsatellite linkage map of rainbow trout
(Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates. Genetics 155:
1331-1345.
Sarder MRI, Penman DJ, Myers JM, McAndrew BJ. 1999. Production and propagation of fully inbred clonal
lines in the Nile tilapia (Oreochromis niloticus L.). J. Exp. Zool. 284: 675-685.
Silver LM. 1995. Mouse Genetics: Concepts and Applications. Oxford University Press,
New York.
Streisinger G, Walker C, Dower N, Knauber D, Singer F. 1981. Production of clones of homozygous diploid
zebra fish (Brachydanio rerio). Nature 291: 293-296.
Sundin K, Brown KH, Drew RE, Nichols KM, Wheeler PA, Thorgaard GH. Genetic analysis of a development
rate QTL in backcrosses of clonal rainbow trout. Aquaculture: in press.
Swarup H. 1959. Production of triploidy in Gasterosteus aculeatus (L.). J. Genet. 56: 129-142.
Taniguchi N, Han HS, Tsujimura A. 1994. Variation in some quantitative traits of clones produced by
chromosome manipulation in ayu, Plecoglossus altivelis. Aquaculture 120: 53-60.
Tanksley SD, Nelson JC. 1996. Advanced backcross QTL analysis: a method for the simultaneous discovery
and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor. Appl. Genet. 92:
191-203.
Thorgaard GH, Bailey GS, Williams D, Buhler DR, Kaattari SL, Ristow SS, Hansen JD, Winton JR,
Bartholomew JL, Nagler JJ, Walsh PJ, Vijayan MM, Devlin RH, Hardy RW, Overturf KE, Young WP,
Robison BD, Rexroad III C, Palti Y. 2002. Status and opportunities for genomics research with rainbow trout.
Comp. Biochem. Physiol. B., 133: 609-646.
Waldbieser GC, Bosworth BG, Nonneman DJ, Wolters WR. 2001. A microsatellite-based genetic linkage
map for channel catfish, Ictalurus punctatus. Genetics 158: 727-734.
Wayne ML, McIntyre LM.
2002.
Combining mapping and arraying: An approach to candidate gene
identification. Proc. Nat. Acad. Sci. 99: 14903-14906.
Wheeler PA, Thorgaard GH. 1991. Cryopreservation of rainbow trout semen in large straws. Aquaculture 93:
95-100.
Woods IG, Kelly PD, Chu F, Ngo-Hazelett P, Yan YL, Huang H, Postlethwait JH, Talbot WS. 2000. A
comparative map of the zebrafish genome. Genome Res. 10: 1903-1914.
Young WP, Wheeler PA, Thorgaard GH. 1995. Asymmetry and variability of meristic characters and spotting
in isogenic lines of rainbow trout. Aquaculture 137: 67-76.
Young WP, Wheeler PA, Fields RD, Thorgaard GH. 1996. DNA fingerprinting confirms isogenicity of
androgenetically-derived rainbow trout lines. J. Hered. 87: 77-81.
Young WP, Wheeler PA, Coryell VH, Keim P, Thorgaard GH. 1998. A detailed linkage map of rainbow trout
produced using doubled haploids. Genetics 148: 839-850.
Zimmerman AM, Evenhuis JP, Thorgaard GH, Ristow SS. 2004. A single major chromosomal region
controls natural killer cell-like activity in rainbow trout. Immunogenetics 55: 825-835.
Zimmerman AM, Wheeler PA, Ristow SS, Thorgaard GH.
Composite interval mapping reveals three
quantitative trait loci associated with pyloric caeca number in rainbow trout. Aquaculture: in press.
Geographic
Line identification Sex
Origin
OSU
F
Domestication
Level/ history
California
Distinctive
Characteristics
High
Deficient in NK-like activity
IHNV resistant
Moderate growth rate
Hot Creek
M
California
High
High NK-like activity
High pyloric caeca number
Slow embryonic development
Differentiated Y chromosome
Arlee
M
California
High
IHNV susceptible
Relatively fast growth
Slow embryonic development
X-like Y chromosome
Swanson
M
Alaska
Moderate
Fast embryonic development
Relatively slow growth
Early sexual maturation
X-like Y chromosome
Clearwater
M
Idaho
Moderate
(steelhead)
Fast embryonic development
Ceratomyxa shasta resistant
Differentiated Y chromosome
Represents inland lineage
Skookumchuck M
Washington
Moderate
Characterization in progress
(steelhead)
Klamath
M
Oregon
Moderate
Characterization in progress
Whale Rock F
F
California
Low
Landlocked steelhead origin
Whale Rock M M
California
Low
Landlocked steelhead origin
Figure 1. Association of time to hatch (tth; degrees centigrade x hours, on the x axis) and allelic type for a
microsatellite in a rainbow trout backcross. The microsatellite (OMM1009) is closely linked to a QTL for
development rate. The backcross is the third generation of crossing the Clearwater (rapidly developing) form
of the QTL into the background of the OSU (slower developing) clonal line.
The number of individuals
hatching at a given time point are indicated on the y axis. Association of microsatellite type with development
rate is highly significant (p < 0.0001) and explains 17.9% of the variation in development rate in this backcross
(R2 = 17.9).
OSU
CW
25
frequency
20
15
10
5
0
696
712
720
728
736
744
752
760
Time to hatch (degree-hours)
768
776
784
FISH GENOMICS:
FROM GENETIC MANIPULATION
TO GENOMIC ANALYSES
GARY THORGAARD
WASHINGTON STATE UNIVERSITY
PULLMAN, WASHINGTON, USA
1
COAUTHORS AND COLLABORATORS
Krista Nichols 1, Alicia Felip 2,3, Ana Zimmerman 2,4,
Kim Brown 2,5, Rob Drew 2, Barrie Robison 5,
Kyle Sundin 2,6, Joe Brunelli, 2, Ruth Phillips 7, Sandra Ristow 2
Paul Wheeler 2
1
National Oceanic and Atmospheric Admin., Seattle
2 Washington State University, Pullman
3 Instituto de Acuicultura de Torre de la Sal, SPAIN
4 Massachusetts Institute of Technology, Cambridge
5 University of Idaho, Moscow
6 Signature Genomic Laboratories LLC, Spokane
7 Washington State University, Vancouver
2
PRODUCTION OF CLONAL LINES OF FISHES
PRESENTS A POWERFUL APPROACH FOR
GENETIC AND GENOMIC ANALYSIS
GEORGE STREISINGER,
FOUNDER OF ZEBRAFISH
GENETIC RESEARCH AND
DEVELOPER OF THE CONCEPT
OF PRODUCING CLONAL
LINES OF FISHES USING
CHROMOSOME SET
MANIPULATION
3
CLONAL RAINBOW TROUT
4
CLONAL LINES HAVE BEEN PRODUCED IN A
NUMBER OF FISH SPECIES, INCLUDING:
ZEBRAFISH
MEDAKA
CARP
TILAPIA
5
WHY STUDY CLONAL LINES?
REPEATABILITY
EASE OF LINE MAINTENANCE
RECOVER STRAINS FROM CRYOPRESERVED SPERM
USE ESTABLISHED METHODS FROM PLANT & MOUSE
GENETICS (E.G., DOUBLED HAPLOIDS,
RECOMBINANT INBREDS, CONGENIC LINES)
LIMITATIONS
MUST RECOGNIZE THAT A SINGLE HAPLOID GENOME
IS SAMPLED IN FORMING A CLONAL LINE
LOW FECUNDITY OF HOMOZYGOUS FEMALES
NUMBERS OF PROGENY THAT CAN BE PRODUCED
BY ANDROGENESIS ARE LIMITED
6
7
PAUL WHEELER
8
OUR LABORATORY AT WASHINGTON
STATE UNIVERSITY HAS CHOSEN TO
SAMPLE THE NATURAL GEOGRAPHIC
VARIATION OF RAINBOW TROUT
AMONG THE CLONAL LINES THAT WE
ARE PROPAGATING
9
10
11
12
13
STRATEGY FOR ANALYSIS OF TRAITS
1. IDENTIFY CLONAL LINES DIVERGENT
FOR TRAIT
2. PRODUCE HYBRID BETWEEN LINES
3. PRODUCE DOUBLED HAPLOID PROGENY
BY ANDROGENESIS FROM HYBRID
4. TYPE AFLP MARKERS AND TRAIT VALUES
IN DOUBLED HAPLOID PROGENY
5. PERFORM QTL ANALYSIS TO TEST FOR
ASSOCIATIONS
14
BARRIE BOBISON,
PH.D. STUDENT AT
WSU AND NOW
ASST. PROFESSOR
AT THE UNIV. OF IDAHO,
FIRST USED DOUBLED
HAPLOID CROSSES
FOR QTL MAPPING IN
TROUT
15
Collecting Hatching
Times
16
17
A SINGLE MAJOR QTL WAS ASSOCIATED
WITH DEVELOPMENT RATE IN THE OSU
X SWANSON DOUBLED HAPLOID CROSS
Robison et al., , J. Hered. 92: 16-22 (2001).
18
ANA ZIMMERMAN, STUDENT WITH SANDRA RISTOW,
FOUND A MAJOR QTL FOR NATURAL
KILLER CELL-LIKE ACTIVITY IN A CROSS OF THE
19
OSU AND HOT CREEK CLONAL LINES
Identification of a single major QTL controlling NCC Activity
THREE QTLs OF MODERATE EFFECT
WERE ASSOCIATED WITH PYLORIC
CAECA NUMBER IN THE OSU X HOT
CREEK CROSS (A. ZIMMERMAN)
Zimmerman et al, Aquaculture (in press).
21
KRISTA NICHOLS HAS ANALYZED A
NUMBER OF TRAITS IN THE OSU X
CLEARWATER CROSS
22
Breeding design: genome-wide QTL detection
X
OSU rainbow trout clone
XX female
Clearwater (CW) River steelhead clone
YY male
X
Egg inactivation
F1
hybrid clones
XY male
outbred female
Heat shock
DOUBLED
HAPLOIDS
23
MERISTIC VARIATION (K. NICHOLS)
Moderate effect QTLs identified for six traits
No association of meristic QTLs with
development rate
Nichols et al., Env. Biol. Fishes 69: 317-331 (2004)
C. SHASTA RESISTANCE (K. NICHOLS/
J. BARTHOLOMEW)
About four loci associated with C. shasta
resistance
Nichols et al., Dis Aquat. Org. 56: 145-154 (2004)
24
OBJECTIVE:
DETERMINE IF MAJOR LOCI UNDERLYING
DEVELOPMENT RATE, GROWTH AND TIMING OF
SEXUAL MATUIRY ARE THE SAME
SIGNIFICANCE
IMPORTANT INFORMATION IN THE DESIGN OF
SELECTIVE BREEDING AND MARKER-ASSISTED
SELECTION PROGRAMS
25
Breeding design: fine mapping of detected QTL
OSU clone
Clearwater clone
X
CLONAL LINES
(XX female)
(YY male)
X
OC F1 (XY male)
X
X
BC1
ADVANCED
BACKCROSS
INTROGRESSION
LINES
BC2
BC3
26
OTHER STUDIES PLANNED IN
THE NEAR FUTURE:
-IMPROVED RESOLUTION USING
PROGENY TEST DESIGNS?
-MAPPING OF CHALLENGING
TRAITS (E.G., STRESS RESPONSE,
BEHAVIORAL TRAITS)
-EXAMINING MITOCHONDRIAL
EFFECTS IN ANDROGENETIC
TROUT
27
BEHAVIORAL DIFFERENCES AMONG LINES:
STARTLE RESPONSE
Startle Response
Freeze vs. Frenzy Behavior
Number of Fish Exhibiting Behavior
40
30
Freeze Behavior
20
Frenzy Behavior
10
0
Swanson
Arlee
Clearwater
Strain
Hot Creek
BEHAVIORAL DIFFERENCES AMONG LINES:
POSITION IN WATER COLUMN
Position in the Water Column
Percent Use of Sections 1, 2, 3, and 4
80%
70%
Mean Percent of Time
60%
Section 1
50%
Section 2
40%
Section 3
30%
Section 4
20%
10%
0%
Swanson
Arlee
Clearwater
Hot Creek
Strain
29
CAN CLONE CROSSES BE EXPLOITED AS
RESOURCES FOR FUNCTIONAL GENOMICS?
RNA EXPRESSION
IN DOUBLED
HAPLOIDS OR
ADVANCED
BACKCROSS
POPULATION
SEGREGATING
FOR A QTL
TEST MICROARRAYS
FOR EXPRESSION
DIFFERENCES
ASSOCIATED WITH
QTL
30
ADVANTAGES OF DOUBLED HAPLOIDS/ CLONAL
LINES FOR MAPPING AND GENOMIC STUDIES
-VARIANCE AMONG SEGREGATING PROGENY
IS GREATER THAN IN BACKCROSS OR
F2 DESIGNS
-EASE OF SCORING EFFICIENT DOMINANT
MARKERS (E.G., AFLPs)
-ADVANCED BACKCROSS DESIGNS ARE POSSIBLE
DUE TO LINE IDENTITIES
-HOMOZYGOUS MATERIAL IS IDEAL FOR
PRODUCING BAC LIBRARIES AND FOR
SEQUENCING. (HETEROZYGOSITY AND
DUPLICATE GENES ARE NOT
CONFUSED).
31
FUTURE PROSPECTS:
BALANCING POTENTIALS AND LIMITATIONS
OF GENOMIC STUDIES WITH CLONAL FISHES
DEFINED EXPERIMENTAL ANIMALS
EXPERIMENTAL REPEATABILITY OVER
TIME AND LOCATION
EASE AND EFFICIENCY OF QTL DETECTION
GENOMIC ADVANTAGES OF HOMOZYGOSITY
VS.
REPRODUCTIVE CHALLENGES
HOW TYPICAL ARE CLONES OF OUTBRED
POPULATIONS?
CHALLENGES IN MOVING FROM LABORATORY
TO APPLICATION?
32
The following discussion parts were transcribed from tape-recordings and edited by the
organizing committee.
Presentation One (1) by Dr. Gary H. Thorgaard
Title:
Fish genomics: from genetic manipulation to genomic analyses
Dr. Nakayama (Chairperson):
Thank you Dr. Thorgaard.
The session is now open for
discussion. Any questions?
Asking person 1:
First, Gary thanks for a really wonderful talk. With regards to the
androgenetic systems of your haploids it looks like your androgens are of the same donor line…
Dr. Thorgaard:
(interruption) The female line?
Asking person 1:
(continue question) …for your production of your irradiated eggs.
And I’m wondering, I know in Drosophila studies, recent Drosophila studies show that the effect of
the environment on the strength of the association of maternal trait is very strong.
For example,
in Drosophila, the genotypes have been applied in cases where it had strong influence on the
homozygous traits and so I’m wondering have you looked at the use of multiple female lines for
the production of the irradiated eggs to evaluate the chromosome?
Dr. Nakayama (Chairperson):
Dr. Thorgaard:
Okay.
Now, that’s a very good question.
female lines for irradiated eggs for statistics.
We actually do use different
When we do the crosses, we almost always use the
OSU line as our female source but we get probably majority of our eggs comes most often from
something called Trout Water.
Maples found.
That’s a major commercial producer.
But one thing that Chris
She did some experiment on its development mainly QTL in which she looked at
the effect of varying the female lines and she found that the major QTL was consistent across all
the different female sources that were used for the irradiated eggs… making the irradiated eggs
but that some of the other QTL’s seem to be kind of coming and going.
It’s because, which I raise
a lot of interesting questions you know, I mean there were detrimental effects, possibly
mitochondrial related, that were associated with development.
And so generally for the major
effect we see consistencies but we think that maybe some subtle effects maybe associated with
the various egg types.
Asking person 1:
And that there’s a whole other direction I think to pursue.
It occurs to me that this kind of work is a very powerful tool to
evaluate both genetic and non-genetic counter effects.
Dr. Thorgaard:
Yeah, I think there are some very good opportunities.
Asking person 2:
You mentioned the potential use of genomic methods.
do you think these methodologies could be applied?
How soon
And do you think it will speed up the
identification of particular genomes as oppose to the segment of the chromosomes?
Dr. Thorgaard:
Well, the resources are definitely coming along fairly quickly. There
are already BAC library for the OSU and Swanson line, one female and one male line. I think
they’re coming along with rainbow trout micro-array but they already have a very nice micro-array
for Atlantic salmon.
And we share some materials with people in Canada that are working on that
micro-array.
We haven’t yet though applied the combination of the QTL family studies with the
micro-array.
We are hoping to do that soon.
So I think the materials are largely in place I think.
Start to combine the possible mapping approaches with the newer genomic technologies.
Asking person 2:
Thank you!
Asking person 3:
Thank you again.
I have a short question about your clonal lines.
You can apply very briefly about your clonal lines but maybe I missed the chromosome feature of
each of your progeny.
I’m wondering if you have a chromosomal variation?
Dr. Thorgaard:
Let’s go back now.
Creek line has 60.
The OSU line has 60.
Robertsonian chromosome changes.
The Arlee line has 64 chromosomes.
The Hot
These differences in numbers are associated with
I think we need to do a little bit more of work which appears
to have 58 chromosomes. The Swanson has 58 chromosomes, the Clearwater has 58, the
Scupucheck I don’t think we have the information yet and Welrock we don’t yet have the
information.
I expect that it’s going to be 64 chromosomes. There definitely are some variations
in the chromosome numbers associated with the line.
Ruth Philips has just got a paper coming
out now actually in which she compares the OSU 60 chromosome with the Swanson and
Clearwater 58. And she has been able to identify the specific chromosome associated with the
arrangement.
It seems to be the same fusion in both the Swanson and Clearwater pair.
we’re getting some information.
So,
We could certainly use some more banding type of work on this.
But, I think it’s just coming along.
Asking person 3:
appropriate linkage basis?
Dr. Thorgaard:
I’m just wondering if you have the feature to define.
Do you get
Is it difficult marking?
That’s something that Ruth Philips is largely pursuing.
She is in the
process of associating the linkage group of some of the genetic map with the physical features of
the specific chromosomes.
progress on.
That’s something that I think Ruth is emphasizing and making good
I think she is supposed to submit a paper in Thailand.
Asking person 3:
Thank you, again.
Asking person 4:
Professor, thank you so much for your conference.
questions.
I ask two
My first question is related to the… you mention during the presentation, that software
for linkage group with a specific chromosome. How could they identify this linkage group for these
chromosomes? Which techniques they used?
Did the software identify the linkage group to a
specific chromosome?
Dr. Thorgaard:
Masu or… masu salmon or?
Asking person 4:
(interruption) Can you show me the presentation? You showed a
slide where there’s a linkage group, there is an allele, a specific allele which are related with a
specific chromosome.
Dr. Thorgaard:
Asking person 4:
Dr. Thorgaard:
You mean something like this here?
No, no, before.
Before. Ok.
Asking person 4:
Ah, this one.
Dr. Thorgaard:
Okay, well, this one wasn’t really a study here. This is one in which
some kinds of arbitrarily naming the group. But, what added in her study, she used micro-satellites,
two or three micro-satellites per linkage group to allow us to have the linkage groups in the relation
by half-breed cross correspond to linkage groups in the other crosses. So that the problem with
AFLPs is that it’s very difficult to compare from one study to another. And so we would like to use
the AFLPs to get a good coverage but we need to use other markers like micro-satellites to be
able to use the same names for the different linkages. I don’t know if that answers your question.
Asking person 4:
My question was related with a software to identify a linkage group.
My question is, how in this work they could identify the chromosome? They used a basic mapping,
using fish, for example.
Dr. Thorgaard:
The programs we’re using are able to look at the linkage of the
markers and the association at the same time. So we basically get the linkage map and at the
same time that we do the QTL analysis. So you know the programs we are using, and I could talk
to you about them later seem to be able to do that. But, the big problem we have is having our
studies correspond in one cross to another cross.
So for those we need to use markers that are
in common in order to relate our studies for example to the Sakamoto and Danzmann’s studies.
We need to be using common micro-satellites.
Dr. Nakayama (Chairperson):
group.
I think there is some confusion on the term of the linkage
This one is a linkage group.
Asking person 4:
Dr. Thorgaard:
Yeah, yeah, yeah, I know.
So you’re talking about the chromosome now?
And the
chromosome linkage group?
Asking person 4:
A specific linkage group was associated with a specific chromosome.
So my question is how they could identify the physical chromosome?
Dr. Thorgaard:
Okay, going from a linkage group to the chromosome.
somewhat what Dr. Abe did.
That involves the use of the BAC.
micro-satellite that we know was on a specific linkage group.
This was
For example, you find a
And then, Ruth Philips, what she
does is isolate the BAC that corresponds to that micro-satellite and makes the fluorescence in-situ
hybridization.
So maybe, that, I’m sorry that I wasn’t getting to that earlier. You’re right. You
need to use another level of technology in order to match the chromosomes and linkage.
Asking person 4:
There is time?
Dr. Nakayama (Chairperson):
Asking person 4:
Very shortly.
Okay, what is the future of fish improvement, chromosome
manipulation, the Mendelian procedure or using genetic engineering?
Which one will be the best
choice for the future of the fish improvement?
Dr. Thorgaard:
You know….As much as I love this type of research, I would have to
say that if I was a company, right now I would still put my money on classical quantitative genetic
selective breeding program.
I think that there may be opportunities in areas like disease
resistance where you have a particularly challenging problem in the breeding.
But I consider
much of these to really be more on the realm of basic research for understanding basic processes.
And eventually I think the information that we have will move into the breeding realm.
it’s going to take time.
Asking person 4:
It’s part of a long-term investment.
Thanks a lot to you. Okay.
Dr. Nakayama (Chairperson):
presentation.
But I think
Thank you, Dr. Thorgaard.
Now, it’s time to move to the next