© R.A. Dunham 2004. Aquaculture and Fisheries Biotechnology: Genetic Approaches
122 (R.A. Dunham)
Choice of Markers
Segregation analysis of polymorphic markers allows the assignment of isozymes, biochemi-cal markers, DNA fragments and genes to chromosomes, ordering of these genetic markers and genes, establishment of genetic linkages, mapping of important genes and identification of modes of inheritance of pro-duction traits by demonstrating the linkage of major genes, either singly or as part of polygenic systems (Liu and Dunham, 1998a).
Initially, most of this research was conducted with isozymes and easily scored qualitative traits; however, advances in DNA technology during the past decade have allowed rapid identification of very large numbers of mark-ers for producing genetic linkage maps – the mapping of genes on chromosomes.
Isozymes and other biochemical markers, such as genes associated with the immune system, have the probable advantages of being conserved across taxa and linked to quantitative traits, but have the disadvantage of being few in number. However, the advent of ESTs is rapidly overcoming this shortcom-ing for type I markers – actual genes – for gene mapping.
Assuming a recombination genome size to be 2000 centimorgans (cM), 200 evenly dis-tributed genetic markers would be required for a map with a resolution of 10 cM (or a map for any locus being within a 5 cM aver-age to the nearest marker) (Poompuang and Hallerman, 1997; Dunham and Liu, 2002).
Theoretically, a genetic map with 1000 molec-ular markers can place any locus in close
proximity to less than a million base pairs.
Thus, several thousands of markers may be needed to construct genetic linkage maps that can tightly localize specific genes for practical applications, such as gene isolation.
Microsatellites and AFLP markers are the most reliable, efficient and abundant mark-ers for detailed genetic linkage mapping in catfish (Liu and Dunham, 1998a,b; Liu et al., 1998a,b,c, 1999a,b,c,d,e,f, 2003; Waldbieser et al. 2001), and perhaps other aquatic organ-isms. Fine linkage mapping depends on the availability of large numbers of ESTs and the anchoring of well-ordered contigs of bacter-ial artifical chromosome (BAC) clones to linkage maps.
Conservation of microsatellites across a broad range of taxa in aquatic organisms has potentially important applications and implications (Liu et al., 2003) for gene map-ping, marker-assisted selection (MAS), cloning of genes and evolutionary studies.
High levels of genetic conservation allow comparative gene mapping, which is espe-cially important considering the rare avail-ability of type I markers (markers that encode genes) in fish (Z.J. Liu, unpublished results). Comparative gene mapping would facilitate rapid advancements in gene mapping of major aquaculture species.
Conservation of microsatellite loci across a broad range of species has been demonstrated among various taxa (Moore et al., 1991; Deka et al., 1994; FitzSimmons et al., 1995; Fredholm and Wintero, 1995; Menotti-Raymond and O’Brien, 1995; Coote and Bruford, 1996; Rico et al., 1996; Sun and Kirkpatrick, 1996;
Zardoya et al., 1996; de Gortari, et al., 1997;
Surridge et al., 1997; Liu et al., 2001c, 2003). In the majority of these studies, primers designed from microsatellite-flanking regions of one species were successfully evaluated in closely related species. For example, primers from humans were tested in other primates (Coote and Bruford, 1996), or primers from one species member of a family were evalu-ated for their conservation among other species in the family (Fredholm and Wintero, 1995; Menotti-Raymond and O’Brien, 1995).
Microsatellites are highly conserved among species of fish. Most channel catfish, Ictaluridae, primers amplified microsatellites from Cichlidae (Liu et al., 1999e).
Homologous microsatellite loci have endured for approximately 300 million years in turtle (FitzSimmons et al., 1995) and for about 470 million years in fish (Rico et al., 1996). Microsatellite-flanking sequences of fish may evolve at a slower rate than those of mammals (Liu et al., 2003) based on fish gene maps generated to date. The identification of homologous chromosome segments in siluri-form, cyprinodontiform and salmoniform fishes supports the hypothesis (Morizot, 1994) that teleostean gene arrangements may have diverged more slowly from those of the vertebrate ancestor than those same gene arrangements in mammalian orders. Gene map locations in one teleost may be highly predictive of map locations in other fish (Dunham et al., 1998).
Mapping Systems
Gene mapping can be accomplished by using either intraspecific or interspecific systems.
Interspecific hybridization systems are pow-erful for the construction of genetic linkage maps. Interspecific approaches to gene map-ping have frequently been used, in recent years utilizing both microsatellites and AFLPs (Agresti et al., 2000) and in the early fish gene maps generated with isozymes (Morizot and Siciliano, 1979; Pasdar et al., 1984; Johnson and Wright, 1984; Johnson et al., 1987) because of the power and high level of poly-morphism found in interspecific systems. Liu et al. (2003) utilized a channel catfish blue
catfish hybrid system for gene mapping. The F1hybrids are fertile and F2hybrids or back-cross progeny can be readily produced (Argue, 1996; Argue and Dunham, 1998). The primary advantages of using this hybrid sys-tem for genetic linkage analysis are the high level of polymorphism between channel catfish and blue catfish and the fact that few reference families are needed because single families can be generated that are heterozy-gous for virtually every genetic marker.
Additionally, there are large performance dif-ferences between species for production traits, which could expedite QTL mapping.
Conservation of microsatellite loci between closely related species, as demonstrated by Liu et al. (1999e) for ictalurid catfish, allows construction of unified maps among those generated from intraspecific and interspecific mapping systems. One individual channel catfish was heterozygous for 24 of 31 microsatellite loci. Similar results were gener-ated for blue catfish, white catfish and flat-head catfish. These results indicate that small numbers of interspecific or intraspecific refer-ence families would be sufficient for gene mapping of microsatellite loci, and microsatellite markers would be almost as numerous as RAPD and AFLP markers. The microsatellites would, in fact, be even more powerful because of their codominance and ability to identify heterozygotes.
Utilization of haploid gynogenesis is another powerful mapping strategy. Single sperm typing is now possible but haploid gynogenesis has advantages compared with single sperm typing (Lie et al., 1994). Cell divi-sion in eggs is activated by irradiated sperm, resulting in haploid individuals, representing a single maternal meiotic event with no pater-nal genome contribution. The haploid gyno-genesis strategy represents the female counterpart of sperm typing but has the advantages that no individual sorting is needed, repeated tests are possible on the same individual because of the large amount of cells and DNA, it is not entirely dependent on PCR and markers that can be mapped are not restricted to non-coding nuclear DNA and include isozymes, mtDNA and ESTs (Lie et al., 1994). Both single sperm typing and haploid gynogenesis allow resolution of
recombina-tion rates below 0.5% and discriminarecombina-tion of loose linkages above 45%, up to no linkage, 50%. Computer simulation indicates that lethal genes, which may eliminate specific haplotypes and cause segregation distortion of markers linked with such genes, do not interfere with the recombination estimate (Lie et al., 1994). Additionally, haploid gynogenesis indicates the location of putative lethal genes relative to the informative markers. This strat-egy is efficient in distinguishing between vari-ants, and allowed Lie et al. (1994) to detect segregation distortion of microsatellites in Atlantic salmon, probably due to preselection of eggs or embryos resulting in differential mortality of certain genotypes.
Linkage Disequilibrium
When conducting linkage or population genetic analyses, linkage disequilibrium is sometimes observed. Linkage (or gametic) disequilibrium is when there is a lack of fit for observed two-locus gametic frequencies compared with those expected, based on the product of the single-locus allelic frequencies (May and Krueger, 1990). The frequency of an A1B2gamete (loci A and B) in the popula-tion should be equal to frequency of the Al allele times the frequency of the B2allele.
Linkage disequilibrium should decay by 1r each generation for random mating, where r is the recombination rate between the two loci (May and Krueger, 1990). The value for r can vary from zero for complete linkage to 0.5 for no linkage; therefore, link-age disequilibrium will decay by one-half each generation for most pairs of loci.
Linkage disequilibrium can be caused by mixtures in the sample of two or more popu-lations with different allelic frequencies, a founding population already in disequilib-rium, selection for certain heterozygous genotypes or random genetic drift to high frequencies of particular chromosome types (May and Krueger, 1990). In most population studies, the linkage disequilibrium is caused by mixing or founder effects. If a population has a bottleneck with a low effective popula-tion size, linkage disequilibrium might be expected for several generations.
For most population studies, variance components of the linkage disequilibrium values will only help indicate recent mixing (zero to two generations) of two highly divergent intraspecific gene pools (May and Krueger, 1990). Forbes and Allendorf (1989) found linkage disequilibrium values for link-ages that have not yet decayed through recombination for fixed alternate alleles for several mixed populations of two distinct subspecies of cutthroat trout, Westslope and Yellowstone, after five to 15 generations of interbreeding.
Isozyme Maps
Isozymes were the first biochemical or mole-cular markers utilized for gene mapping in fish. Pasdar et al. (1984) examined linkage relationships of nine enzyme loci – aconitase (ACON), esterase (EST), glucose-phosphate isomerase A and B (GPI), glycerate-2-dehy-drogenase (G2DH), malic enzyme (ME), phosphoglycerate kinase (PGK), phospho-glucomutase (PGM) and superoxide dismu-tase (SOD) – in backcrosses of reciprocal F1 hybrids between green sunfish (Lepomis cyanellus) and red-ear sunfish (Lepomis microlophus) to each of their two parental species. A three-point linkage map contain-ing G2DH, PGK and SOD was obtained, with frequencies of recombination between G2DH and PGK and between PGK and SOD at 45.3 and 24.7%, respectively. The remain-ing six loci assorted independently.
Although partial tetraploidy is wide-spread in the teleost genome, salmonids pre-sent one of the most obvious and most studied problems in fish gene mapping because of their tetraploid ancestry. Johnson and Wright (1984) and Johnson et al. (1987) examined the joint segregation analyses of isozyme loci in males and females of seven species and three fertile species hybrids of trout, charr and salmon, and identified 15 linkage groups. Johnson and Wright (1984) were proponents of the interspecific approach to gene mapping, and concluded that, since linkage groups are highly con-served among species and hybrids, they can be combined to form a common linkage
map, in this case one for salmonids.
Pseudolinkage was observed and was explained as preferential multivalent pairing and disjunction of metacentric (centrally fused) chromosome arms with homoeolo-gous arms of other chromosomes in male salmonids, in contrast to bivalent pairing in females. Five pseudolinkage groups were detected and were highly conserved among species and hybrids. Conservation of pseudolinkages among salmonids must have been a result of major chromosomal fusions in a common tetraploid ancestor before the radiation of salmonid species.
Earlier, Davisson et al. (1973) and Lee and Wright (1981) had observed the psuedolink-age found in some salmonid males. Both cytological and linkage analyses indicated that spontaneous centric fusion and fission could account for the curious patterns of pseudolinkage of two lactate dehydrogenase (LDH) loci in males of brook trout and in the F1, F2and backcross generations of lake trout
brook trout hybrids (Davisson et al., 1973).
Intraindividual polymorphisms for acrocen-tric and metacenacrocen-tric chromosomes in somatic and gonadal tissue of these fish are consistent with the proposed polyploid evolution in Salmonidae. Mitotic and meiotic analyses of the tetraploid-derivative species, brook trout, indicated that the process of diploidization was incomplete (Lee and Wright, 1981).
The diploid number was 2N = 84, with 16 metacentrics and 68 acrocentrics for both males and females from different sources, and no inter- or intraindividual Robertsonian polymorphism was present. Oocytes at pachytene had the expected 42 bivalents with eight metacentric and 34 acrocentric pairs.
However, variable numbers of tetravalents, with a total of 35–40 bivalent plus tetravalent elements, were observed in metaphase I cells of males. Each tetravalent was composed of two acrocentric and two metacentric chromo-somes. Variability in the number of tetrava-lents was found not only among different brook trout sources, but also among different cells of the same fish (Lee and Wright, 1981).
Differential homoeologous pairing was pro-posed to account for the variable number of tetravalents and to explain the occurrence of pseudolinkage in some salmonid males.
Some of the linkage relationships in salmonids are due to duplicated loci result-ing from the tetraploid ancestry. Hollister et al. (1984) observed variable genotypes for the duplicate loci encoding the enzyme pep-tidase D (PEPD) in lake trout, brook trout and their fertile hybrid (splake). Non-ran-dom assortment was observed among prog-eny of parents doubly heterozygous for the PEPD-1 and PEPD-2 loci, the duplicate loci encoding GPI and the locus sorbitol dehy-drogenase (SDH). Linkage groups were PEPD-1 with GPI-1 and PEPD-2 with GPI-2 with SDH. The results fitted and were con-sistent with the earlier-determined chromo-somal model involving preferential tetravalent pairing of homoeologous chro-mosomes – pseudolinkage.
Disney and Wright (1990) later observed extensive multivalent pairing in lake trout, which, along with data on hybrid splake (brook lake) trout, supported a meiotic model to explain pseudolinkage.
Additionally, C-banding of mitotic and meiotic lake trout chromosomes revealed an intraindividual polymorphism for a Robertsonian fusion, and silver staining showed that the chromosomes with active nucleolar organizer regions located proximal to a centromere were not involved in the fusion event.
Null alleles can also complicate linkage analysis in salmonids and in oysters.
Unusual phenotypic distributions were observed at the muscle-specific, duplicate aspartate aminotransferase (AAT) locus in a wild population of brook trout, and analysis of these phenotypic distributions eliminated disparate gene frequencies, non-random association between the two loci and inbreed-ing as possible explanations (Stonekinbreed-ing et al., 1981). Models incorporating a null allele and inheritance data from hatchery populations of brook trout fitted the data and confirmed a null-allele polymorphism. This AAT null allele, along with other null-allele polymor-phisms in salmonids, is evidence that loss of duplicate gene expression is still occurring;
however, there is no such evidence of ongo-ing loss of duplicate gene expression in the Catostomidae, another tetraploid-derivative lineage (Stoneking et al., 1981).
Early gene-mapping research was also conducted with Xiphophorus, utilizing inter-specific approaches. Morizot et al. (1977) obtained a three-point linkage group com-prised of loci coding for adenosine deami-nase (ADA), glucose-6-phosphate dehydro-genase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGD) for Xiphophorus (Poeciliidae) by utilizing reciprocal backcross hybrids from crosses between either Xiphophorus helleri guentheri or X. helleri striga-tus and Xiphophorus maculastriga-tus. The alleles at this linkage group assorted independently from the alleles at isocitrate dehydrogenase (IDH) 1 and 2 and glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) 1, and the latter assorted independently from each other. The linkage group was conserved in all populations of both species of Xiphophorus examined. Data from X. helleri guentheri back-crosses indicate the linkage relationship, ADA-6%-G6PDH-24%-6PGD, and ADA-29%-6PGD (30% when corrected for double crossovers), but results from backcrosses X.
helleri strigatus gave different recombination frequencies for the same gene order. Possible explanations include differences due to an inversion or a sex effect on recombination.
The linkage of 6PGD and G6PDH exists in species of at least three classes of vertebrates (Morizot et al., 1977).
Recombination data from backcross hybrids among three species and four sub-species of Xiphophorus indicate four addi-tional linked loci, linkage group (LG) II, (esterase) EST-2-0.43-EST-3-0.26- (retinal lactate dehydrogenase) LDH-1-0.19- (man-nose phosphate isomerase) MPI (Morizot and Siciliano, 1979). Interference was detected in the EST-3 to MPI region, and LG II assorted independently from the six loci of LG I and from GAPDH-2 and IDH-2.
Next, Morizot et al. (1991) analysed 76 polymorphic isozyme loci in backcross hybrid individuals from intra- and interspe-cific crosses of the genus Xiphophorus (Poeciliidae), identifying 17 multipoint link-age groups containing 55 protein-coding loci and one sex-chromosome-linked pigment-pattern gene. Gene orders were determined for ten linkage groups, and total genome length was estimated to be 1800 cM.
Comparisons of the Xiphophorus linkage map with those of other fishes, amphibians and mammals suggested that fish gene maps are remarkably similar and probably retain many syntenic groups (Morizot et al., 1991); this will be discussed in more detail later.
Six isozyme linkage groups have been established for ictalurid catfish, using the channel–blue catfish interspecific hybrid sys-tem. Gene–centromere distances were esti-mated for six loci in gynogenetic channel catfish (Liu et al., 1992) and for additional polymorphic loci in blue–channel triploid hybrids. At least 28 polymorphic isozyme loci were found and used to establish channel cat-fish multipoint LGs I–VI, comprising 18 loci (R.A. Dunham, B. Argue and D. Morizot, unpublished). Eleven unlinked loci may bring the total of isozyme-marked chromosomes to 17 of the 29 chromosome pairs of channel and blue catfish. The extensive genetic variability within and between blue and channel catfish at approximately 70 isozyme loci (Dunham and Smitherman, 1984; Hallerman et al., 1986;
Carmichael et al., 1992) could allow significant expansion of this isozyme-based gene map.
Ictalurus LG I is comprised of loci coding for glutathione reductase and PGM (Morizot et al., 1994). Three other loci assort indepen-dently from the LG I pair, providing isozyme markers for four chromosomes.
Three isozyme loci assigned to catfish linkage group LG II are also linked in LG II of poeciliid fishes, indicating the evolution-ary conservation of both neutral and physio-logical markers. Comparison of the gene maps of poeciliid and salmonid fishes sub-stantiates that this result is not a rare event.
Orthology is sometimes difficult to establish because poeciliids are diploid and salmonids are recently tetraploid-derived (Johnson et al., 1987). However, at least four cases of linkage-group conservation have been iden-tified, and linkage-group divergence has yet to be observed (Morizot, 1990), again illus-trating the strength of the linkage conserva-tion in fish. Within poeciliids, LGs II of Xiphophorus and Poeciliopsis are homologous (Morizot et al., 1989) and are homologues of Poecilia LG I (Narine et al., 1992).
Additionally, poeciliid and salmonid synte-nies can be identified in centrarchid fishes
and ranid frogs and are apparently even evo-lutionarily conserved in segments of human chromosomes 12, 15 and 19 (Morizot, 1990).
Morizot (1990, 1994) and Morizot et al. (1991) proposed that the chromosomal arrange-ments of duplicated genes in fishes suggest retention of patterns produced by three rounds of tetraploidization.
In Xiphophorus poeciliids, CA1 (ortholo-gous to catfish CAH-2) has been assigned to LG XXIV (the X. maculatus sex-chromosome linkage group), and GAPDH-2 (orthologous to catfish GAPDH-1) is linked to PEPC (orthology with catfish dimeric peptidases is uncertain) in LG U3. Sex-chromosome linkage groups vary widely even within orders for fish.
Catfish LGs II and III are strong examples of evolutionary conservation of linkage groups in fishes and between fishes and mammals. The orthologues of the three cat-fish LG II isozyme loci are also linked in Xiphophorus LG II, and GPI and MPI ortho-logues are also linked in salmonid LG 13.
Orthologues of MPI and -mannosidase are syntenic on human chromosome 15 (O’Brien, 1993). Catfish LG III shows homology with other fish linkage groups. Xiphophorus LG IV contains pyruvate kinse (PK), GPI, PEPD and probably a cytosolic IDH locus, which is orthologous to the catfish arrangement of
Orthologues of MPI and -mannosidase are syntenic on human chromosome 15 (O’Brien, 1993). Catfish LG III shows homology with other fish linkage groups. Xiphophorus LG IV contains pyruvate kinse (PK), GPI, PEPD and probably a cytosolic IDH locus, which is orthologous to the catfish arrangement of