© 2004 The American Genetic Association
Brief Communication |
Analysis of Microsatellites and Parentage Testing in Saltwater Crocodiles
From the Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, NSW, 2006, Australia. (Isberg, Chen, and Moran), and Janamba Croc Farm, P.O. Box 496, Humpty Doo, NT 0836, Australia (Barker).
Address correspondence to Christopher Moran at the above address, or e-mail: ChrisM{at}vetsci.usyd.edu.au.
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Abstract |
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Fifteen microsatellite loci were evaluated in farmed saltwater crocodiles for use in parentage testing. One marker (C391) could not be amplified. For the remaining 14, the number of alleles per locus ranged from two to 16, and the observed heterozygosities ranged from 0.219 to 0.875. The cumulative exclusion probability for all 14 loci was.9988. The 11 loci that showed the greatest level of polymorphism were used for parentage testing, with an exclusion probability of.9980. With these 11 markers on 107 juveniles from 16 known-breeding pairs, a 5.6% pedigree error rate was detected. This level of pedigree error, if consistent, could have an impact on the accuracy of genetic parameter and breeding value estimation. The usefulness of these markers was also evaluated for assigning parentage in situations where maternity, paternity, or both may not be known. In these situations, a 2% error in parentage assignment was predicted. It is therefore recommended that more microsatellite markers be used in these situations. The use of these microsatellite markers will broaden the scope of a breeding program, allowing progeny to be tested from adults maintained in large breeding lagoons for selection as future breeding animals.
The Australian crocodile industry is a new and emerging industry based primarily on the production of skins from the saltwater crocodile (Crocodylus porosus). For this industry to meet the demand for a high-quality product, the incorporation of a genetic improvement program into farm management is essential. The program will be based on the selection of candidates according to their own and their relatives' phenotypic performances for selection criteria related to defined selection objectives (Isberg et al. 2003). Obviously, the implementation of a successful breeding program will require correct pedigrees. Errors in assigned parentage may lead to incorrect genetic evaluation of candidates, resulting in real genetic improvement being less than expected (Visscher et al. 2002).
Only a small number of adult crocodiles on Australian crocodile farms are kept in unitized breeding pens (one male: one or two females). The majority of adults are maintained in breeding lagoons containing many males and females (Webb 1989). In these situations, matings are neither observed nor recorded. It would be advantageous for industry, and for the success of a genetic improvement program, to include these animals and their offspring in a multitrait selection program.
A common method of marking a crocodile for identification is to cut scutes in a unique sequence. Scutes are vertical, triangular osteoderms on the dorsal midline of the posterior tail that bifurcate into two rows of more laterally flattened scales in about the middle third of the tail and continue cranially (Richardson et al. 2002). In captive breeding, this method of marking is usually done on the day of hatch. This method, as described by Richardson et al. (2002), is one of the few permanent and practical identification methods available, although it is not without problems. Errors can result from incorrect cutting of the scute sequence at hatch, reading the numbers incorrectly later, or the changing of numbers over time due to the regrowth of incorrectly cut scutes. Other methods include toe tagging and microchips, which have their own disadvantages.
If parentage errors are relatively high, the success of a multitrait selection program utilizing records from relatives will be seriously compromised. Previously published microsatellite markers for crocodiles (FitzSimmons et al. 2001) have enabled a parentage determination kit to be assembled and evaluated in this study. The markers evaluated provide an indispensable complement to a selection program for farmed saltwater crocodiles, enabling confirmation of the pedigree records of juveniles from pens of known parents. Further, they widen the scope of the program by enabling the pedigrees of offspring produced from mass matings in lagoons to be deduced.
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Materials and Methods |
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Animals and Sampling
One hundred thirty-nine individuals were sampled from Janamba Croc Farm (Northern Territory, Australia). There were 16 known family groups consisting of 32 parents, with an average of 6.7 offspring per family. Parents were long-term, known-breeding pairs housed in unitized pens. The parents were wild-caught, and there was no evidence of relationship among them. Offspring from these pairs were collected as clutches of eggs, developed in an incubator, and uniquely marked at hatching by scute cutting. With use of clutch-specific scute cuts for identification, blood for DNA preparation was collected at various opportunities during the production system (either at the end of their first year or at slaughter). Blood samples were taken from eight adults and all of the juveniles by the occipital venous sinus technique described by Lloyd and Morris (1999), and tissue samples were taken from the remaining adults with a specifically designed biopsy punch at any accessible location along the tail.
Experimental Protocol
We extracted DNA, using standard phenol/chloroform protocols (Sambrook et al. 1989). Fifteen of the 26 microsatellites developed by FitzSimmons et al. (2001) were randomly chosen for this study with primer sequences shown in Table 1. For every microsatellite locus, the amplification reaction took place in a total volume of 15 µl. PCR reagents included 1 unit of Taq DNA polymerase (various sources), 1x PCR buffer (Promega), and final concentrations of 0.1 mM dNTPs, 0.3 mM each of forward and reverse primer, and 1.3–3.3 mM MgCl2 and approximately 50–100 ng of template DNA. Standard PCR conditions included a touchdown protocol with an initial denaturation at 95°C for 15 min; then three cycles of 95°C for 40 s, 63°C for 1 min, and 72 °C for 1 min 30 s; then five cycles of 95°C for 40 s, 61°C for 1 min, and 72°C for 1 min 30 s; and then 35 cycles of 95°C for 40 s, 59°C for 1 min, and 72°C for 1 min 30 s, finally being held at 72°C for 20 min. We ran PCR products on a denaturing 6% polyacrylamide gel, using an ABI 373 sequencer (Applied Biosystems), and alleles were scored with GeneScan and Genotyper software (Applied Biosystems).
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Microsatellite and Population Genetic Analysis
Initially, only the parents were evaluated for all 15 markers to determine the degree of polymorphism. Only those loci with two or more alleles were included in the population genetic analysis.
Tests of linkage disequilibrium between loci were done with Arlequin 2.000 (Schneider et al. 2000). Allele frequencies for each locus were estimated with Cervus 2.0 (Marshall et al. 1998), as were the probabilities of exclusion. Cervus was used for parentage testing, with a typing error rate of 0.01, and strict and relaxed confidence levels specified as 95% and 80%, respectively. Of the estimated 250 breeding animals on Janamba Croc Farm, only 32 parents were sampled (12.8% of the Janamba population), and each had all 11 loci typed. There were three ways in which Cervus 2.0 was used to assign parentage for this study. First, since the family groups used in this study came from unitized breeding pens where males and females are permanently paired, the markers were used to confirm correct data entry, scute cutting, and animal identification, since mating errors were not possible. To simulate a situation in which parentage is unknown, such as in the breeding lagoons, two scenarios for parentage assignment were tested: (1) identifying the male parent when the female is known, and (2) identifying either parent with no prior knowledge of the other.
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Results |
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Of the 15 loci evaluated, only 11 were genotyped on all available samples. C391 did not amplify under conditions recommended by FitzSimmons et al. (2002) or various other conditions tried. Cj35, CUJ-131, and CU4-121, although scored for the adult samples, were considered insufficiently polymorphic for inclusion in a parentage determination kit, with each revealing only two alleles in the population sampled.
Descriptions for each locus are in Table 2. The number of alleles detected ranged from two to 16 (average 5.1), while the effective number of alleles ranged from 1.3 to 6.5 (average 2.9). Genotype frequencies of the adult animals were within expectations of Hardy–Weinberg equilibrium at each locus (P >.05), with the exception of Cj104 (P =.032). However, this locus was kept in the analysis because minor deviations from Hardy–Weinberg equilibrium at few loci are unlikely to bias likelihood estimates considerably across all loci (Marshall et al. 1998). In addition, because only 32 wild-caught adults from various locations were sampled, these animals may not represent a sample from a natural population of the Australian saltwater crocodile. Tests of pairwise linkage disequilibrium were significant for six from 120 tests, equivalent to the number expected by chance alone. Cj127 was the most informative locus, with a probability of exclusion of.705, while the least informative was Cj122 (probability of exclusion =.10). Considering all 14 loci, the combined probability of excluding the second parent when the first is known was 99.88%, and the probability of excluding the first parent when neither parent is known was 97.42%. After omitting the three loci that displayed only two alleles, the average probability of exclusion when one or neither parent is known is 99.80% and 96.78%, respectively.
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Parentage Assignment
Using the 11 markers, 5.6% of the juveniles were found to have genotypes incompatible with their recorded parentage. These individuals were retested to confirm the incompatibilies were not due to sample misidentification. In each case, the genotypes of at least four loci excluded these parents. Thus, the animals must have been marked incorrectly when the scutes were cut, the scutes must have been read incorrectly, or the scute cut sequence must have been modified by biting. Laboratory errors cannot be excluded, but great care was taken with labeling and identification of samples. The multiple locus exclusion in each case means that mutation need not be considered as a reason for incompatible genotypes. These animals were omitted from analyses aimed at predicting parentage, because the adults sampled came from unitized pens of known-breeding pairs. No other adult crocodiles on the farm were sampled, so the true parents could not be identified.
Simulating a Situation in Which One or Neither Parent is Known
Female parent is known
In some situations, the female parent of a clutch could be identified when one collected the eggs, because of maternal nest protection behavior. In these situations in which the mother was known, Cervus 2.0 (Marshall et al. 1998) provided the option of specifying one parent as known. All 16 males were then treated as potential fathers. For 99 of the juveniles used in this simulation, the known father was designated the most probable father. However, for two of 101 juveniles, the most probable father assigned was known to be incorrect. In these two cases, the true father, known to be located in the same pen as the mother, was the second most likely.
Neither parent is known
In some cases, the mother cannot be identified when the eggs are collected from the nest. To simulate a situation in which neither parent is known, the assignment of parentage was divided into two parts. First, the most likely parent was predicted from all possible parents available. For the animals used in this analysis, 100% were assigned correctly to the most likely parent. Second, the analysis was rerun with the most probable parent specified as known, after excluding all other potential parents of the same gender as the known parent. Again, all except for the two juveniles mentioned above, were assigned their correct other parent as the most probable other parent.
Cervus 2.0 uses a logarithm of the likelihood ratio (LOD score), as described by Marshall et al. (1998) to identify the most likely parent. After evaluation of the likelihood of each possible combination of parents for a given offspring, the resulting LOD score produced for each possible parent is ranked, and the parent with the highest LOD score is considered the most likely parent (Jones and Ardren 2003). Because parentage allocation is achieved with exclusion probabilities, a higher probability is given to a potential parent that is homozygous for the alleles present in the offspring across the greatest number of loci. Therefore, including extra microsatellites, even the three lowly informative markers, may have given the extra exclusion power required for 100% correct filiation. Alternatively, genotype information from siblings within the same clutch could be used to provide extra information about the true father's genotype.
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Discussion |
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Pedigree errors were shown to occur in 5.6% of cases tested in this study. This is within the ranges of pedigree errors revealed in other animal industries (8.7%–15.5% in sheep: Barnett et al. 1999; 2%–22% in cattle: references within Visscher et al. 2002) using microsatellite markers. The parentage errors detected in this study could have resulted from incorrect scute identification or, less likely, from sample misidentification, because the adults sampled were from known unitized breeding pens where mating errors were not possible. The 11 microsatellites evaluated in this study provide sufficient power (99.80%) to be useful in a parentage determination kit to confirm parentage of offspring from unitized breeding pens for selection as future breeding animals.
Not all of the adults on the study farm were sampled. Thus, the true parents of these 5.6% of misassigned individuals could not be determined. The remaining adults on the study farm had unrestricted and unobserved choice of mates. These 11 microsatellites have shown adequate exclusionary power to determine parentage of these clutches retrospectively. This finding will provide the opportunity to include the progeny from animals maintained in uncontrolled lagoon breeding situations to be included in a multitrait selection program. The possibility of multiple paternities in clutches from lagoons may require a greater power of exclusion, which would be provided by using more loci.
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Acknowledgments |
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This research is being supported by RIRDC (Project No. US-109A) in collaboration with Janamba Croc Farm, NT Australia. S.R.I. is supported by a University of Sydney Postgraduate Award (cofunded). Thanks to Wayne Gurney and John Nash for help in collecting DNA samples, and to Jaime Gongora, Zung Doan, and Adam Stow for their guidance and invaluable technical advice. Thanks also to Nancy FitzSimmons, Jake Gratten and Tony English for advice on designing the tissue biopsy punch, with special thanks to John Olsen and Björn Isberg for manufacture and donation of the tissue biopsy punch.
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Footnotes |
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Corresponding Editor: Stephen O'Brien
Received September 29, 2003
Accepted April 27, 2004
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References |
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Barnett NL, Purvis IW, van Hest B, Franklin IR, 1999. The accuracy of current dam pedigree recording strategies employed by stud Merino breeders. In: Proceedings of the Association for the Advancement of Animal Breeding and Genetics. Mandurah, Western Australia, July 4–7, 1999; 13:373–376.
FitzSimmons NN, Buchan JC, Lam PV, Polet G, Hung TT, Thang NQ, Gratten J, 2002. Identification of purebred Crocodylus siamensis for reintroduction in Vietnam. J Exp Zool. 294:373-381.[Medline]
FitzSimmons NN, Tanksley S, Forstner MR, Louis EE, Daglish R, Gratten J, Davis S, 2001. Microsatellite markers for Crocodylus: new genetic tools for population genetics, mating system studies and forensics. In: Crocodilian biology and evolution (Grigg G, Seebacher F, and Franklin CE, eds). Chipping Norton, Australia: Surrey Beatty; 51–57.
Isberg SR, Nicholas FW, Thomson PC, Barker SG, Manolis SC, Moran C, 2003. Defining breeding objectives for saltwater crocodile genetic improvement programs. In: Proceedings of the Association for the Advancement of Animal Breeding and Genetics. Melbourne, Australia, July 6–11, 2003; 15:166–169.
Jones AG, Ardren WR, 2003. Methods of parentage analysis in natural populations. Mol Ecol. 12:2511-2523.[CrossRef][Medline]
Lloyd M, Morris PJ, 1999. Phlebotomy techniques in Crocodilians. Bull Assoc Reptil Amphib Vet. 9:12-14.
Marshall TC, Slate J, Kruuk L, Pemberton JM, 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Mol Ecol. 7:639-655.[CrossRef][Medline]
Richardson KC, Webb GJW, Manolis SC, 2002. Crocodiles: Inside out. Chipping Norton, Australia: Surrey Beatty.
Sambrook JE, Fritsch EF, Maniatis T, 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Schneider S, Kueffer J, Roessli D, Excoffier L, 2000. Arlequin Ver. 2.000: A software for population genetic analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland.
Visscher PM, Woolliams JA, Smith D, Williams JL, 2002. Estimation of pedigree errors in the UK dairy population using microsatellite markers and the impact on selection. J Dairy Sci. 85:2368-2375.
Webb G, 1989. The crocodile as a production unit. In: Proceedings of the Intensive Tropical Animal Production Seminar. Townsville, Queensland, July 19–20, 1989.
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