A single locus PCR amplified minisatellite region as a hypervariable genetic marker in gadoid species

Aquaculture ELSEVIER

Aquaculture 137 (1995) 31-40

A single locus PCR amplified rninisatellite region as a hypervariable genetic marker in gadoid species P. Galvin *, D. McGregor, T. Cross Department of Zoology, University College Cork, Lee Makings, Prospect Row, Cork, UK

Abstract Recent concerns over the status of wild gadoid populations together with the increasing cultivation

of a number of gadoid species has prompted the search for new genetic markers. Minisatellite containing clones were isolated by screening a size selected whiting (Merlangius merlangus L.) genomic DNA library with Jeffreys’ 33.15 and 33.6 probes. A partial sequence of one of these clones revealed a minisatellite repeat with a core sequence of 31bp. PCR primers complimentary to both sides of the minisatellite region were designed and following optimisation of PCR amplification conditions, either one or two products per individual were amplified with sizes ranging from 460 to 1870bp. Familial analysis has confirmed the Mendelian inheritance of the products. Screening of whiting populations from the Irish Sea (n = 50), Baltic Sea (n = 50)) Atlantic coast of Norway (n = 100)) south North Sea (n = 100) and north North Sea (n = 50) revealed a minimum of 24 resolvable alleles per population with a mean heterozygosity value of 0.94. UPGMA cluster analysis based on Nei’s genetic distance indicated that geographically adjacent populations were more similar. A similar highly variable region in cod (Gadus morhua L.) and haddock (Merlanogrammus aeglejinus

L.) is also amplified using these primers.

Keywords:

PCR; Minisatellite DNA; Gadoids; Merlangius merlangus

1. Introduction Marine gadoid species are characterised by very large effective population sizes and vast dispersal capabilities. Understanding of the population structures of these species has been hindered by the inadequacies of allozyme and mitochondrial DNA markers (e.g. Cross and Payne, 1978; Mork et al., 1985; Grant, 1987; Smith et al., 1989). More discriminatory techniques are required to provide a more reliable assessment of gadoid population structure. Development of single locus minisatellite DNA probes has * Corresponding author. 0044-8486/95/$09.50

0 1995 Elsevier Science B.V. All rights reserved

S.SD10044-8486(95)01108-O

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revolutionized salmonid genetics (Taggart and Ferguson, 1990b; Bentzen et al., 1991; Prodiihl et al., 1993), through substantial improvements in the ability to resolve differences both at the individual and population levels. In recent years, the versatility of the Polymerase Chain Reaction (PCR) method has been incorporated into many aspects of molecular biology. A number of PCR amplifiable minisatellite loci have been described for human studies (Jeffreys et al., 1988; Horn et al., 1989), but to our knowledge, only a single such locus has been previously isolated in any fish species (McGregor, Galvin and Cross, unpublished data). The main aims of this study were to combine the power of minisatellite single locus analysis with the versatility of the PCR approach, in order to investigate population differentiation of whiting (Merlangius merlungus L.) in the east Atlantic. However, as microsatellite oligonucleotide primers are known to amplify homologous regions even in distantly related species (Rico, Rico and Hewitt, unpublished data), and minisatellite primers for humans amplify polymorphic regions in primates (Gray and Jeffreys, 1991) , a further aim was to test the applicability of a pair of whiting minisatellite primers to cod (Cc&us morhua L.) and haddock (Melanogrammus aeglifinus L.) as genetic markers for both population genetics and breeding studies, since these species are currently being cultured in Norway and Canada.

2. Materials and methods 2.1. Sample collection and DNA extraction Samples of whiting were collected from five locations in Western Europe (see Fig. 1). DNA extraction was carried out as described by Taggart et al. ( 1992). Alcohol (99% ethanol) preservation was preferred to frozen storage ( - 20°C) due to its convenience for

Fig. 1. Regions in the north-east Atlantic from which samples were collected: Irish Sea (n = 50). Baltic Sea (n=50), Borgensfjord (n= 100). south North Sea (n= 100) and noahNorth Sea (n=50).

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skeletal muscle tissues using this method proved to be extremely difficult (very low yields of DNA). This difficulty was not experienced when extracting DNA from the same tissue from non-gadoid fish species (i.e. Atlantic salmon (S&no salur L.) and turbot (Scophthalmus muximus L.) ) in our laboratory. In contrast, DNA extraction from gill tissue presented no such difficulty and provided high yields of undenatured DNA. DNA was also extracted from a whiting family consisting of both parents and 8 day post-hatch larvae offspring, which were kindly provided by A. Geffin, Port Erin Marine Laboratory, Isle of Man, as well as gill tissues from five cod and five haddock from the Irish Sea. 2.2. Minisatellite

locus isolation

20 pg of DNA was pooled from 20 whiting individuals, digested with Mbol restriction enzyme and fragments were separated by electrophoresis on a 1% agarose gel. DNA fragments of 1.O to 4.0kb were excised from the gel and Prep-a-Gene (BioRad@) purified. Ligation into pUC 18, transformation of ‘supercompetent’ XLl-Blue E.coZi cells (Stratagene@) and detection of positive recombinants with possible minisatellite-containing inserts followed the methods described by Prodijhl et al. ( 1993). Following hybridization, partial sequencing of inserts from positive recombinants was used to identify and characterize the minisatellite region, and to enable the design of primers for the flanking regions of the minisatellite locus as described by McGregor et al. ( 1995). 2.3. PCR ampli$cation

and electrophoresis

Approximately 100 ng DNA were amplified in a volume of 20 ~1 containing X 1 Reaction Buffer IV (Advanced Biotechnologies@) (0.75M Tris pH 9.0,200 mM (NH4)$04, 0.1% Tween 20, 15 mM MgC&), 0.25 mM dNTP, 0.5 PM of each primer and 0.5 units of Thermostable DNA polymerase (Advanced Biotechnologies@) . Using a Hybaid@ thermal cycler, reactions were given an initial denaturation ‘Hot Start’ at 95°C for 5 min before the addition of the enzyme. Amplification was then carried out using 25 cycles of denaturation (95°C for 1 min), annealing (57°C for 1 min) and extension (72°C for 2 min), with a final extension period of 10 min at 72°C added at the end of the 25 cycles. PCR products were separated by electrophoresis on 200 mm X 300 mm X 6 mm 1% agarose gels in TBE buffer (containing 0.5 pg ml- ’ ethidium bromide) for approximately 14 h at 5V cm- ‘. Electrophoresis was terminated when the smallest amplified products approached the anodal end of the gel. The gels were then placed on a UV transilluminator and photographed. A further record was made by overlaying the gels with Saran Wrap@ transparent film and recording the positions of the bands on the gel with red ink marks on the film. Photocopying of this transparent film then provided a permanent full size record of the gel to facilitate characterization of the PCR products. Fragment sizes were determined by comparison with the 100 bp size ladder 2.4. Statistical analysis The BIOSYS-1 computer package was used to calculate allele and genotype frequencies, and heterozygosity estimates. Genotypic frequencies were tested for conformance with

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Hardy-Weinberg expectations using the CHIHW program of Zaykin and Purdovkin ( 1993). In order to use the program, it was necessary to reduce the data set to 26 allele classes by pooling all alleles larger than 1210. As alleles larger than that size were quite rare, such pooling was not expected to largely impact the overall result. Homogeneity x2 analysis of allele frequencies among samples was performed by another program (CHIRXC) by the same authors. These programs use Monte Carlo simulations as described by Roff and Bentzen ( 1990) to avoid the problems associated low numbers of individuals in some classes. A UPGMA dendrogram based on Nei (1972) genetic distance was constructed using the PHYLIP package (Felsenstein, 1993).

3. Results A partial sequence of one clone revealed a minisatellite region consisting of a 3 1 bp core repeat sequence (TAT GGT GGT GGT GGT GGT GGT GAA GTG GGT C) . A pair of 22-base oligonucleotide primers were designed for the flanking sequences (see Table 1)) such that amplification using these primers would include the repeat sequence together with 160 bp of flanking sequence. This locus has been designated Mmer-AMP2 in accordance with the nomenclature of the previously isolated locus Mmer-AMP1 described by McGregor et al. ( 1995). It is notable that each minisatellite repeat in the clone sequenced, contains a trinucleotide microsatellite repeat occurring between four and seven times (mostly six) in each of the minisatellite repeats. Electrophoretic analysis of the amplified products revealed either one or two bands per reaction, indicating fragment sizes ranging from 460-l 870 bp (see Fig. 2). Familial analysis, using sibling 8 day post-hatch whiting larvae and their parents, indicated that at least four of these bands corresponded to differentially sized alleles, segregating according to Mendelian expectations (see Fig. 3). The exceptions in lanes eight and nine represent misfiled individuals from different crosses. In a total of approximately 350 whiting examined, 40 separate alleles were resolved by characterizing alleles to the nearest 30 bp increment, assuming that an average of six trinucleotides occurred per minisatellite repeat when the whole minisatellite region was considered. Separation by electrophoresis on the 30 cm agarose gel provided inter-allelic migration differences ranging from 1 mm (between alleles > 1200 bp) up to 3 mm (between alleles < 800 bp) . Allele frequency distributions from the five regions examined (Fig. 4(a)-(e) ) indicated that there were no major allelic frequency differences between whiting from separate regions, and that no private alleles (sensu Slatkin, 1985) occurred at frequencies greater than 0.05. Heterozygosity estimates ranged from 0.93 to 0.95. Although observed heterozygosity was in all cases lower than expected values, none of populations showed a significant deviation from Hardy-Weinberg proportions. Heterogeneity x2 analysis of allele frequencies indicated significant differences between samples taken from the Irish Sea and Table 1 22-base oligonucleotide Primer 1 Primer 2

primers used to amplify Mmer-AMP2 GCT TAG CCC ATG AAG CTA AAG C CAC CTG TCA ATC ACT TCC GTC A

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1,500bp

600bp

Fig. 2. Agarose gel showing allelic variation for ten individuals. ladders (40C1500bp).

Allele sizes were estimated from the 100 bp size

those of the North Sea (i.e. both north and south North Sea being significantly different from the Irish Sea sample). Analysis of F-statistics revealed that just 0.3% of the total genetic diversity was due to differences between regions. However, a UPGMA dendrogram based on Nei ( 1972) genetic distance (Fig. 5) linked the two North Sea samples together and the Baltic and Borgensfjord together, with the Irish Sea sample appearing as an outlier, suggesting some degree of geographic clustering. PCR reactions under the same conditions using cod and haddock DNA templates amplified what appears to be a homologous locus in both species. Out of five individuals tested

861bp

492bp

Fig. 3. Male and female parents in Lanes 11 and 12, with seven offspring in Lanes 1-5, 7 and 10 showing Mendelian inheritance. The individuals in Lanes 8 and 9 are misfiled larva from other crosses (this was confirmed through tests with a second locus). A 123 bp size ladder in Lane s ranges from 492 bp to 983 bp.

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(a) South North Sea

(b) North North Sea 1.5 10

5 01 (c) Baltic Sea 15’ 101 ;I (d) Irish Sea 15 10 5 O(e) Borgenfjord, Norway 15 10 1 5 0I

Fig. 4. Allele frequency

distributions

at Mmer-AMP2

of the five populations

screened

for each species, all cod individuals were heterozygous with eight alleles resolved, while four of the five haddock were heterozygous with three alleles resolved (see Fig. 6). 4. Discussion From the sample of approximately 350 whiting analysed in this study, 40 alleles were resolved at the single minisatellite DNA locus studied. Although this corresponds to 820

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South North Sea

r-i

I’

North North

Baltic

Sea

Sea

Borgenfjord

L 0.12

Irish Sea 0.05

0.00

Fig. 5. UPGMA dendrogram based on Nei ( 1972) genetic distance.

possible genotypes, very little genetic differentiation was evident. The high level of allelic diversity coupled with the limited differentiation among samples from separate regions is consistent with previous findings for marine gadoids and other marine fish species using protein electrophoresis (Gyllensten, 1985). However, despite the high degree of variability at this locus, it is unlikely that a small scale study of this nature would reveal the true picture of the genetic interactions of such a species, which is characterized by a complex population biology. Whiting have an extended spawning period spanning from early spring to mid summer, suggesting the possible occurrence of temporal variation in population groups spawning in the same areas. Unlike anadromous species, where it is possible to physically

1,500bp

600bp

Haddock Fig. 6. Variation of Mmer-AMP2 ranges from 400 bp up to 1500 bp.

in five haddock

(Lanes 2-6)

Cod and five cod (Lanes 8-12).

100 bp size ladder

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tag or characterize genetically the juvenile stages, in order to determine the degree of homing to native spawning sites, studies of gadoid species have had to rely on tagging experiments and genetic characterization of post-larval life stages, which may have dispersed considerable distances from their native spawning sites, as the eggs and larvae are pelagic. Such studies are also unable to determine where these marked fish spawn, as their recapture is often dependent on returns from the commercial fisheries, which may be concentrating primarily on the feeding grounds. PCR based minisatellite DNA analysis offers the possibility of genetically characterizing the very early life stages, prior to their dispersal from the spawning areas by sampling post-hatch larvae. In the current study, DNA was successfully extracted from 8 day post-hatch larvae, which provided enough DNA template for several PCR reactions. By sampling adjacent spawning areas over time, it should be possible to obtain a much finer interpretation of the degree of reproductive isolation (if any) that occurs in marine gadoid species. The increasing importance of gadoid species in aquaculture prompts the need for genetic markers to assist in selective breeding, both in the identification of families in mass selection programmes and with the aim of identifying Quantitative Trait Loci (QTLs) . Mmer-AMP2 contrasts with Mmer-AMP1 (McGregor et al., unpublished data) in having high levels of heterozygosity in both cod and haddock. Artificial culture of cod has already become established as an important aquaculture industry in Norway. Development of this industry will seek to provide improved strains with high levels of disease resistance and favourable food conversion ratios, while wanting to avoid substantial reductions in genetic variability. Single locus minisatellite markers, such as Mmer-AMP2 offer several advantages over other types of genetic markers for these purposes. The high level of variability contrasts with that of allozyme and mitochondrial techniques, where relatively low numbers of variants have been detected. Although minisatellite loci cannot be assumed to be completely free of the forces of selection (Krontiris et al., 1993), it seems reasonable to assume, that the alleles are functionally equivalent, and can therefore be treated as being selectively neutral as defined by Kimura ( 1986). This contrasts with protein variants such as haemoglobin, where gadoid studies have implicated selection as the basis for genetic differentiation (Mork et al., 1984). The relatively small amounts of DNA required, combined with the reduced sensitivity of the method to partial degradation of the DNA, are particularly useful for gadoid studies where the tissue appears to be more prone to degradation and the juvenile stages are so small. Further advantages of this method are the ability to avoid the time and expense involved in Southern blotting, and the hazards of working with radioactive isotopes. Ethidium bromide staining on agarose gels enables improved separation of alleles, since the bands are sharper than those achieved with auto radiography following Southern blotting (Boerwinkle et al., 1989). Through the isolation of more of these loci in the near future, it is hoped to provide a comprehensive gadoid marking technique, which will lend itself to automation by multiplexing of loci together with an integral allele cocktail, using different fluorescent stains and computerization of gel scoring. This would increase the sensitivity of the technique, enabling even greater numbers of alleles to be consistently resolved, and also reduce both time and cost factors, so that studies involving large numbers of individuals over several loci become feasible. It is therefore envisaged that this technique will play an important future role in both aquaculture and fisheries genetics of gadoid species

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Acknowledgements Thanks are due to Alec Jeffreys (University of Leicester, England) fro permission to use the 33.6 and 33.15 minisatellite probes, and to Andy Ferguson, Paul0 Prodijhl (The Queen’s University of Belfast, Northern Ireland) and John Taggart (University of Stirling, Scotland) for advice on library construction and screening. The authors are very grateful to David Thompson and David James, MAFF, Lowestoft, England for providing the North Sea and Baltic samples; to Mike Armstrong, Department of Agriculture, Northern Ireland for providing the Irish Sea sample; to Jarle Mork, University of Trondheim, Norway for facilitating the collection of the Borgensfjord sample and to Audrey Geffin, Port Erin Marine Laboratory, Isle of Man who provided the whiting family. Useful discussions with Godfrey Hewitt and Ciro Rico, University of East Anglia, England were also very beneficial. This research was funded as part of the EC FAR programme (contract no. MA.3.781)

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