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Analysis of Microsatellites and Parentage Testing in Saltwater Crocodiles
... As such, varying study designs and lack of null hypothesis testing lead to the question of potential upward bias based on the differences in predictive power, on the accuracy of inferring multiple paternity and number of contributing sires particularly in the absence of parental genotypes. To assess these implications, the data from Isberg et al. (2004) were re-analyzed using different scenarios that emulate a range of potential study designs incumbent with wild population studies. Isberg et al. (2004) showed the utility of 11 microsatellite markers to assign parentage of C. porosus in the context of improving the accuracy of genetic improvement programs. ...
... To assess these implications, the data from Isberg et al. (2004) were re-analyzed using different scenarios that emulate a range of potential study designs incumbent with wild population studies. Isberg et al. (2004) showed the utility of 11 microsatellite markers to assign parentage of C. porosus in the context of improving the accuracy of genetic improvement programs. Sixteen known-breeding pairs (one male and one female) that were housed together for numerous years were genotyped. ...
... Using the known dataset from Isberg et al. (2004), this study hypothesized that by limiting different elements of information provided to the different parentage assignment software used, the number of inferred sires could be appreciably upward biased. This included varying the number of microsatellites evaluated, excluding population allele frequencies and excluding parental genotypes. ...
Up to 10 males were reported to sire clutches of crocodilian eggs but review of the underlying study designs raised questions of potential upward bias of inferred sire numbers. To test this premise, different scenarios were explored using a published dataset of 16 known single- sire saltwater crocodile pairs and their offspring which were originally confirmed using a 11 loci microsatellite panel in CERVUS. Varying the number of microsatellites, omitting one or both parental genotypes and using different parentage analysis techniques revealed that total allele number, rather than number of loci, determined inferred sire accuracy in two opposing ways. Using the single- locus minimum method and GERUD, which both require prior knowledge of family group-ings (i.e., nests), fewer alleles (and loci) accurately inferred only one father. In contrast, CERVUS and COLONY required all 11 loci (65 alleles) and both parental genotypes to (a) assign correct family groups and (b) infer the correct sire number, except in one family where two sires were equally assigned based on their number of homozygous loci. When less genotype information was provided, CERVUS and COLONY inferred up to six and seven sires, respectively. Given this data is from confirmed single-sire matings, and yet up to seven sires could be inferred, the significance of inappropriate study design is clearly demonstrated. Consideration should be carefully given to gen-otype data, particularly those collected specifically for population diversity studies, which are also used to infer multiple paternity because the underlying data collection assumptions are not equivalent between the two outcomes.
... As such, varying study designs and lack of null hypothesis testing lead to the question of potential upward bias based on the differences in predictive power, on the accuracy of inferring multiple paternity and number of contributing sires particularly in the absence of parental genotypes. To assess these implications, the data from Isberg et al. (2004) were re-analyzed using different scenarios that emulate a range of potential study designs incumbent with wild population studies. Isberg et al. (2004) showed the utility of 11 microsatellite markers to assign parentage of C. porosus in the context of improving the accuracy of genetic improvement programs. ...
... To assess these implications, the data from Isberg et al. (2004) were re-analyzed using different scenarios that emulate a range of potential study designs incumbent with wild population studies. Isberg et al. (2004) showed the utility of 11 microsatellite markers to assign parentage of C. porosus in the context of improving the accuracy of genetic improvement programs. Sixteen known-breeding pairs (one male and one female) that were housed together for numerous years were genotyped. ...
... Using the known dataset from Isberg et al. (2004), this study hypothesized that by limiting different elements of information provided to the different parentage assignment software used, the number of inferred sires could be appreciably upward biased. This included varying the number of microsatellites evaluated, excluding population allele frequencies and excluding parental genotypes. ...
Up to ten males have been reported to sire clutches of crocodilian eggs but review of the underlying study designs raised questions of potential upward bias of inferred sire numbers. To test this premise, different scenarios were explored using a published dataset of 16 known single-sire saltwater crocodile pairs and their offspring which were originally confirmed using an 11 loci microsatellite panel in CERVUS. Varying the number of microsatellites, omitting one or both parental genotypes and using different parentage analysis techniques revealed that total allele number, rather than number of loci, determined inferred sire accuracy in two opposing ways. Using the single-locus minimum method and GERUD, which both require prior knowledge of family groupings (ie nests), fewer alleles (and loci) accurately inferred only one father. In contrast, CERVUS and COLONY required all 11 loci (65 alleles) and both parental genotypes to (a) assign correct family groups and (b) infer the correct sire number, except in one family where two sires were equally assigned based on their number of homozygous loci. When less genotype information was provided, CERVUS and COLONY inferred up to six and seven sires, respectively. Consequently, crocodilian maternal genotypes should at least be obtained either directly (blood, tissue) or indirectly (eggshells, eDNA) in addition to a representative population sample of adult genotypes before embarking on multiple paternity determination. Running different scenarios using different analytical methods and reporting on the lowest number of inferred sires is encouraged remembering these sires are hypothetical until proven by true genotype.
... 130 Table 5.1 Voucher codes for samples according to sampling area. 150 Table 5.2 Microsatellite primers used in this study (Isberg et al., 2004). 153 Table 5.3 Sequence characterization for the microsatellite markers. ...
... Microsatellite markers are ubiquitous in most eukaryote genomes and provide hyper-variable sequenced tagged single locus markers which capable of providing relatively contemporary estimates of migration and relatedness among individuals (Miles et al., 2009a). For these reasons, the markers have been widely used by many researchers to assess population structure and diversity, mating behaviour, hybridisation, as well as dispersal systems, in a variety species of crocodile (Isberg et al., 2004;Lewis et al., 2013;Hekkala et al., 2015;Lapbenjakul et al., 2017;Mauger et al., 2017). ...
... Polymerase Chain Reaction (PCR) was conducted using MyCycler TM Thermal Cycler and microsatellites analysis were follow Isberg et al. (2004) protocols. Three microsatellite markers were used in this study namely Cj101, Cj105 and Cj131 as shown in the Table 5.2. ...
This study is designed to gather information on historical exploitation and ongoing HCC; recent distribution and ecology of crocodile and genetic relationship of crocodile population in Sarawak, to aid sustainable crocodile management and finding solutions for mitigating the HCC. Historical data saw a connection between the exploitation of crocodile with decreasing trend of HCC in Sarawak from the Rajah Brooke era (1900 – 1941) until the post-war period (1946 – 1979), and an increasing trend of HCC from 1980 until 2017 in response to the recovery of the animal populations. Since 1900, crocodile attacks had been occurred in 22 major river basins (RB) in Sarawak, suggesting that the reptile has been widely dispersed throughout all major river basins in the state. For 118 years (1900 – 2017), the highest number of crocodile attacks were recorded in Lupar RB (22.2%) and the attacks had happened up to the inland areas of Belaga and Pelagus in Rajang RB. Further analysis of incidents show crocodile attacks were associated with the human activities pattern, where more attacks involved male victims (84.4%) and adults from age 31 to 40 years old (19.3%). The data also revealed that crocodile attacks in Sarawak could happen anytime regardless of the time, month, season, lunar cycle or tidal. However, more attacks were recorded during the daylight, in the months of March and April, during the Northeast monsoon, at the nights of the first quarter of the lunar cycle and at the time of high tide. Furthermore, fishing (25.2%) and bathing (24.4%) possess the highest risk of crocodile attack in Sarawak, clearly showed that crocodiles are more likely to attack when the victim is in water. Crocodile survey in selected tributaries in Rajang RB showed the distribution of the reptiles throughout the river basin with higher crocodile density at the lower region, the highest density was in Igan River (1.37 individuals/km); while in the middle and upper regions had recorded relatively low density with the lowest density recorded was in Katibas River (0.06 individuals/km) and no crocodile was spotted in Kanowit River. Four out of eight surveyed rivers in Rajang RB recorded increase in the density of crocodile compare to previous survey suggesting that the crocodile population in the river basin is experiencing recovery. The presence of crocodile in different regions (lower, middle and upper) of Rajang RB indicated that C. porosus in Sarawak live in wide range of habitats; from large salt water river system and small tidal tributaries (near to estuary) in lower region into hypo-saline or fresh water non-tidal tributaries in the middle and upper regions. Variation in term of density and distribution of crocodile between the different regions are mainly influenced by the saline characteristic of the river, habitats and the abundance of food sources for crocodile. Based on the analysis of DNA microsatellite sequence data, distinctive subpopulations of C. porosus according to geographical area (river basin) could be observed. High gene flow (Nm) among the crocodile subpopulations suggests frequent movements of the reptile happen across the river basins throughout Sarawak. In general, populations of C. porosus in Sarawak are experiencing expansion as supported by the mismatch distribution and evolutionary neutrality test data, suggesting that populations of crocodile in Sarawak are panmictic population. The findings of the present study imply that increasing of crocodile attacks is associated with the recovery and increased distribution of the reptile in Sarawak, thus crocodile management should emphasis on mitigating HCC and simultaneously continue the efforts for conservation of crocodile and its habitat.
Keywords: Crocodylus porosus, human-crocodile conflict, recovery, expansion.
... porosus) were used to assess various pedigree information scenarios. Details of the animals can be found within Isberg et al. (2004) but briefly, genotypes for all 32 parents (16 sires and 16 dams) and their 101 offspring were available. Using an 11-microsatellite locus panel, Cervus 2.0 (Marshall et al. 1998) was able to assign these offspring as 16 full-sibling families when both parental genotypes were known in 100% of cases. ...
... In addition, despite a 3% loss of exclusionary power when only one parent was known, Cervus 2.0 was also able to assign parentage in 100%. Knowing this, the data from Isberg et al. (2004) were reanalysed in COLONY (Wang 2016) and GERUD 2.0 (Jones 2005) which are the two most commonly used in the crocodilian parentage literature listed in Table 6.2. ...
... For scenarios B and C, the sire and dam genotypes were used to estimate population allele frequencies where necessary. To illustrate the exclusionary power of number and polymorphic content of microsatellite loci used, all 11 microsatellites from Isberg et al. (2004) were used in COLONY, although GERUD 2.0 can only accommodate for a maximum of ten loci so the least polymorphic (Cj 122) was removed, and compared to a sevenmicrosatellite panel. The seven microsatellites retained were those left after removing the two most and least polymorphic from Isberg et al. (2004;Cj101, CUD68, Cj16, Cj18, Cj105, Cp10 and Cj119). ...
This book aims to be a comprehensive review of the literature on the conservation genetics of the New World crocodilians, from the biological and demographical aspects of the living species to the application of molecular techniques for conservation purposes. It covers the current status of the molecular genetics applied to phylogenetics, phylogeography, diversity, kinship and mating system, and hybridization, as well its implications for decision making with regards to the conservation of these species at academic and governmental levels. This book can be used as a guide for graduate and undergraduate students to understand how conservation genetics techniques are carried out and how they can help preserve not only crocodilians but also other living species.
... This also appears to have been the case for a range of other crocodilians across the globe; a lack of a recent bottleneck has also been demonstrated in C. niloticus (Flint et al. 2006) In zoological terms, kinship refers to a direct blood relationship that can be the result of either sexual or asexual reproduction. The identification of relatedness or parentage between individuals has many applications across a range of species and is of particular interest to the crocodile farming industry (Isberg et al. 2004). Parental relations in crocodilians are complicated by the fact that many clutches of eggs consist of the progeny of multiple fathers. ...
... johnstoni) (Miles et al. 2008). Loci have been identified that measure multiple traits, including population diversity and mating behaviour (Isberg et al. 2004;Miles et al. 2008). The latter authors, for example, identified 253 new microsatellites for the estuarine crocodile, and these, because of the high levels of genetic diversity exhibited in many microsatellite loci (Bruford et al. 1996;Wright & Bentzen 1994), can be utilised to identify not only the genetic diversity present in a given population, but also to model the history of the population and to estimate effective population size. ...
... Most population genetics' studies of the estuarine crocodile, both on captive and wild populations (for example; Isberg et al. 2004;Jaratlerdsiri et al. 2012;Miles et al. 2010;Miles et al. 2008;Miles et al. 2009), have been carried out in Australia. There have been numerous studies examining the population genetics of various species of crocodilian, such as Morlet's crocodile (C. ...
The project sought to examine the effects of habitat loss and fragmentation on the ecology and population genetics of the estuarine crocodile (Crocodylus porosus). Additionally, the role played by humans in this anthropogenically-altered landscape was examined. Through the utilisation of a host of technologies, some previously established, some completely novel in crocodilian research, a new insight into how the landscape is utilised by these cryptic predators was developed. This project represents a first detailed look at Sabah’s crocodilian population, as well as being the first active crocodile research carried out in Sabah’s longest river. Male crocodiles were found to adhere to one of two behavioural strategies, territorial and nomadic, mirroring findings of Campbell et al. (2013). Territory sizes were, however, found to be smaller than those described in Australia, this was attributed to increased prey availability and ecosystem productivity. Only two females were tagged and appeared to also display differences in behavioural strategy. However, due to the small sample size, further work is required to confirm this. Both males and females were found to avoid barriers and were unwilling to pass beyond the barrier, despite no physical obstruction. Nests were detectable aerially through the use of drones and medium-large scale surveys shown to be feasible. Nests were found to all display a number of similarities in terms of habitat characteristics, allowing for refined modelling of survey locations. This allows for a larger survey area to be completed given a limited number of flights, highlighting its cost effectiveness versus traditional methods of nest surveying. Genetic analysis suggested that there was no evidence of a genetic bottleneck following the population recovery that has occurred over the last 30 years. Geographically indistinct haplogroups were discovered, as well as limited levels of inbreeding. The project also indicated that the population studied had undergone a population expansion that seems to have coincided with the onset of the last ice age and is likely attributable to changes in climate.
... The resource pedigree used in this study consisted of 133 individuals from 16 known family groups from Janamba Croc Farm (Northern Territory, Australia), as described in Isberg, Chen, et al. (2004) . Thirty-two wild-caught adults were maintained as known-breeding pairs in unitized (one male and one female) pens. ...
... Thirty-two wild-caught adults were maintained as known-breeding pairs in unitized (one male and one female) pens. There was an average of 6.3 progeny per breeding pair, and their pedigree was confirmed in Isberg, Chen, et al. (2004) . Blood sampling and DNA extraction techniques are described in Isberg, Chen, et al. (2004). ...
... There was an average of 6.3 progeny per breeding pair, and their pedigree was confirmed in Isberg, Chen, et al. (2004) . Blood sampling and DNA extraction techniques are described in Isberg, Chen, et al. (2004). ...
The first evidence of genetic linkage and sex-specific recombination in the order Crocodylia is reported. This study was conducted using a resource pedigree of saltwater crocodiles consisting of 16 known-breeding pairs (32 adults) and 101 juveniles. A total of 21 microsatellite loci were available for analysis. Ten of the 21 loci showed linkage with 4 linkage groups: 3 pairwise (Cj131/Cj127, CUD68/Cj101, and Cj107/Cp10) and 1 four-locus (Cj122, CUD78, Cj16, and Cj104) being found. Linkage analysis on the 21 loci revealed evidence of sex-specific differences in recombination rates. All 5 nonzero interlocus intervals were longer in females than in males, with the 4-loci linkage group 3-fold longer in females than in males (41.63 cM and 14.1 cM, respectively). This is the first report of sex-specific recombination rates in a species that exhibits temperature-dependent sex determination.
... The markers have been widely used by numerous researchers to evaluate population structure, mating behavior, diversity, dispersal systems, as well as hybridization in a range of crocodile species (Hekkala et al., 2015;Isberg et al., 2004;Lapbenjakul et al., 2017;Lewis et al., 2013;Mauger et al., 2017). At the same time, a sufficiently substantial collection of microsatellite primers for different species of crocodiles has been produced for research purposes and it has assisted numerous researchers on crocodilians' genetic studies. ...
... Total genomic DNA was extracted from crocodile samples (tissues and scutes) using the 2% CTAB protocol (Doyle & Doyle, 1987) and QIAamp DNA Investigator Kit (QIAGEN, Germany) following manufacturer's protocol, then subjected to 1% Agarose Gel Electrophoresis (AGE). Standard Polymerase Chain Reaction involved the 13 sets of microsatellite loci following Isberg et al. (2004), using a thermocycler (MyCyclerTM) with one negative control for every batch. The thermal profile used was as follows: initial denaturation at 94°C for 15 seconds, followed by 35 cycles of annealing at 54.6°C for 30 seconds, extension at 72°C for 50 seconds, and then a final 4-minute extension step at 72°C. ...
Of the recognized 24 crocodilian species, two species are found in Sarawak: the saltwater crocodile (Crocodylus porosus) and Malayan false gharial (Tomistoma schlegelii). C. porosus is the most commonly found crocodile and currently listed in Appendix II of the Convention on International Trading in Endangered Species of Wild Flora and Fauna (CITES), which allows harvesting wild populations for commercial purposes. To strengthen conservation efforts, ecological and genetic data are needed to inform management decisions. Thus, this study was designed to estimate relationship coefficients between crocodiles in thirteen river basins using 13 microsatellite markers. Fifty-eight wild crocodile samples were obtained and finally analysed by clustering of PCR products. Of the 60 samples amplified, one marker (Cj35) was polymorphic and showed double bands, whereas the other seven markers (Cj127, Cj131, Cj122, Cj101, Cj119, CUD68 and Cj16) revealed a single band. Microsatellite loci (Cj105, Cj18, Cj104, Cp10, and Cu4-121) displayed multiple bands. Using the unweighted pair group with the arithmetic mean (UPGMA) clustering method, an unrooted phylogenetic tree was obtained, with coefficients ranging between 0.51 and 1.00. We successfully assessed population genetic structure and resolved genetic relationships among six clades (Clade A to F) out of the total seven clades. DNA microsatellites are a promising resource for determining the relationships among crocodiles in Sarawak. The findings are useful for future sustainable utilization of the wild crocodile population.
... Detection and correct estimation of multiple paternity or male/female fidelity is highly dependent on the sampling effort (McVay et al. 2008), information on genotypes of both the parents (Zucoloto et al. 2009) and hatchlings as well as the duration of the study (Lance et al. 2009). Correct background information on the sampled animals is important to avoid erroneous results from genetic software programs (Isberg et al. 2004). Collection of information on mating adults is not effective as many species mate in water or in large groups and adult animals other than actual parents also contribute in parental care. ...
... Bashyal et al. (2014),Bishop et al. (2009),Campos et al. (2018),Dalzell et al. (2009), Fitzsimmons et al. (2001,Hinlo et al. (2014),Isberg et al. (2004),Lapbenjakul et al. (2017),Miles et al. (2009a, b, c),Russello et al. (2007),Serna-Lagunes et al. (2012),Shirley and Austin (2017), Velo-Anton et al.(2014),Lafferriere et al. (2016),Hekkala et al. (2010Hekkala et al. ( , 2015,Ghosh et al. (2020) and Van Asch et al. (2019) Alligator mississippiensis ami, csu, cala, asi, cacr, mni, ppa, tsc, gga, ote, cni, crhAmavet et al. (2008Amavet et al. ( , 2017,Davis et al. (2001aDavis et al. ( , b, 2002, DeThoisy et al. (2006),Glenn et al. (1998),Huang and Wang (2004), Shafiei-Astani et al. (2015), Verdade et al. (2002), Villela et al. (2008), Lance et al. (2009), Muniz et al. (2011), Zucoloto et al. (2006, 2009), Wang et al. (2017), Rotstein et al. (2002), Hu et al. (2010) Crocodylus johnstoni cac, csu, cpo, cmo, cni, cpa, crh, csc, csi, cin, cjo, cmi, cno, ote Lafferriere et al. (2016), Bishop et al. (2009), Campos et al. (2018), Cunningham et al. (2016), Dever and Densmore III (2001), Dever et al. (2002), Fitzsimmons et al. (2001, 2002),Mauger et al. (2017), Milián-García (2011), Pacheco-Sierra et al. (2016, 2018), Rodriguez et al. (2008, 2011), Russello et al. (2007), Serna-Lagunes et al. (2012), Shafiei-Astani et al. (2015), González-Trujillo et al. (2012), Velo-Anton et al. (2014), Weaver et al. (2008), Isberg et al. (2004, 2006), Lewis et al. (2013), McVay et al. (2008), Cunningham et al. (2016), Hekkala et al. (2010, 2015) and Van Asch et al. (2019) Crocodylus acutus cmo, csi, cac, crh, cpo, cin, csu, cjo, cmi, cni, cpa, ote Lafferriere et al. (2016), Fitzsimmons et al. (2001, 2002), González-Trujillo et al. (2012), Pacheco-Sierra et al. (2016), Rodriguez et al. (2008), Weaver et al. (2008), Budd et al. (2015), Isberg et al. (2004), Lewis et al. (2013), McVay et al. (2008), Milián-García et al. (2016), Velo-Anton et al. (2014), Campos et al. (2018) Crocodylus palustris ptr, ami, crh, si, cacr, tsc, ote, cac, gga Aggarwal et al. (2015) Caiman latirostris cala, csu, caya, mni, ccr, ppa De Thoisy et al. (2006), Godshalk (2006), Villela et al. (2008), Amavet et al. (2008, 2012, 2015, 2017), Muniz et al. (2011), Ojeda et al. (2017), Zucoloto et al. (2009) Crocodylus siamensis csi, cpo, cacr, tsc Chaeychomsri et al. (2008a), Yu et al. (2011) and Shafiei-Astani et al. (2015) Paleosuchus trigonatus ppa, cacr, mni Muniz et al. (2019) Caiman crocodilus cacr, caya Oliveira et al. (2014) Alligator sinensis Asi Zhu et al. (2010) and Pan et al. connections in Mauritiana revealed high genetic differentiation and viable genetic diversity even when population sizes were as small as five individuals (Velo-Anton et al. 2014). ...
Microsatellite DNA is among the several molecular tools used for determining genetic variability and inbreeding depression in threatened populations. It has been used extensively for population genetics studies in conservation of crocodilians. Even though single nucleotide polymorphisms have higher precision compared to microsatellite DNA markers, microsatellites offer a cost advantage which is particularly important to researchers in the global south. Here, we review the role of micro- satellite DNA as a conservation tool in crocodilians. Employing appropriate keywords in three online databases, we studied 78 publications, where microsatellite DNA had been used to study crocodilian species. We found that 504 species-specific markers were designed for 13, out of a total of 24 crocodilian taxa. Genus Crocodylus had the highest number of species- specific markers and was the most studied taxa using microsatellites. Moreover, microsatellite markers developed for C. porosus were successful in cross amplification of microsatellite markers in 19 other crocodilian species. Microsatellite based studies had highest focus on analyses of multiple populations of a single species. Based on our review of microsatellite based studies on populations of crocodilian species, we recommend that microsatellite DNA markers are an effective conservation tool that can provide critical information on population structures of threatened crocodilian species.
... In addition, some crocodile farms contain breeding pens to produce a constant supply of eggs and hatchlings. These swamp-like environments provide ideal conditions for mosquito breeding and attract large populations of these water birds [15]-the perfect scenario for WNV KUN transmission. However, an additional role for infected crocodiles in transmitting the virus to mosquitoes, i.e., as amplifying hosts, has not been investigated. ...
... Thirty-nine hatchlings were obtained from four wild clutches incubated under standard conditions (32 ± 0.5 • C; 95%-100% humidity) at Darwin Crocodile Farm, Noonamah, Northern Territory, Australia [21][22][23]. On the day of hatch, each animal was scute cut for individual identification [15] and randomized between three pens. Each pen was 200 cm wide and 202 cm in length including a feed deck 30 cm wide tapering to a maximum water depth of 19.5 cm. ...
West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2–17 dpi and in-contact animals 11–21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies.
... When using microsatellite markers for paternity analysis, the credibility of the results is highly dependent on the exclusion probability [14,23]. As revealed by previous studies, paternity results are credible when PE I and PE II values exceeded 99.9% and 99%, respectively [14,[23][24][25][26]. ...
... When using microsatellite markers for paternity analysis, the credibility of the results is highly dependent on the exclusion probability [14,23]. As revealed by previous studies, paternity results are credible when PE I and PE II values exceeded 99.9% and 99%, respectively [14,[23][24][25][26]. In this study, PE I and PE II values were 99.99% and 99.93%, respectively, indicating that the 21 microsatellite loci we used were appropriate for parentage analysis. ...
Social behaviors are poorly known for the critically endangered Yangtze finless porpoise (YFP, Neophocaena asiaeorientalis asiaeorientalis). Here, group composition and dispersal patterns of the YFP population living in the Poyang Lake were studied by parentage-based pedigree analyses using 21 microsatellite loci and a 597 bp segment of the mitochondrial DNA control region. In this study, 21 potential mother-offspring pairs and six potential father-offspring pairs (including two potential parents-offspring pairs) were determined, among which 12 natural mother-offspring groups and a maternal group of three generations were found. No genetically-determined fathers were found associated with their offspring. This study also found that maternally related porpoises at the reproductive state tend to group together. This suggest maternal relationship and reproductive state may be factors for grouping in the YFP population. In natural mother-offspring groups, male offspring were all younger than two years old, which suggest male offspring may leave their mothers at approximately two years of age, or at least they were not in tight association with their mothers as they may have been under two years old. However, female offspring can stay longer with their mothers and can reproduce in the natal group.
... Microsatellites have been used to investigate population structure and gene flow in wild populations of Morelet's Crocodile Crocodylus moreletii Duméril & Bibron, 1851 (Dever & Densmore 2001;Dever et al. 2002), American Alligator Alligator mississippiensis Daudin, 1802 (Glenn et al. 1998;Davis et al. 2002) and Black Caiman Melanosuchus niger Spix, 1825. Microsatellites have also been useful in parentage analysis in Saltwater Crocodiles C. porosus Schneider, 1801 (Isberg et al. 2004), in determining and maintaining genetic variability in crocodiles bred for the leather trade (Flint et al. 2000;FitzSimmons et al. 2002) and to build the scaffolding for a genetic linkage map (Miles et al. 2009a). ...
... The heterozygosity estimates from our data for C. acutus, C. niloticus, C. porosus and C. siamensis fall within the ranges of estimates previously reported for captive purebred C. siamensis, Ho = 0.42±0.17 (FitzSimmons et al. 2002), farmed C. porosus, Ho = 0.59 (Isberg et al. 2004) and in wild populations of C. niloticus, He = 0.27-0.61 (Hekkala et al. 2010) and Ho = 0.51 (Bishop et al. 2009), C. moreletti, Ho = 0.49 (Dever et al. 2002) and Melanosuchus niger, Ho = 0.47-0.70 . ...
Limited information is available on the Philippine Crocodile, Crocodylus mindorensis, concerning levels of genetic diversity either relative to other crocodilian species or among populations of the species itself. With only two known extant populations of C. mindorensis remaining, potentially low levels of genetic diversity are a conservation concern. Here, we evaluated 619 putative Philippine Crocodiles using a suite of 11 microsatellite markers, and compared them to four other crocodilian species sample sets. The two remaining populations from the island of Luzon and the island of Mindanao, representing the extremes of the former species’ distribution, appear to be differentiated as a result of genetic drift rather than selection. Both extant populations demonstrate lower genetic diversity and effective population sizes relative to other studied crocodilian species. The 57 C. mindorensis and C. porosus, Saltwater Crocodile, hybrids identified earlier from the Palawan Wildlife Rescue and Conservation Center were revalidated with a suite of 20 microsatellite loci; however, the timing of the event and the prevalence of hybridization in the species had yet to be fully determined. We defined the hybrids as one first cross from a C. porosus female and a C. mindorensis male and 56 C. mindorensis backcross individuals. This hybridization event appears to be confined to the PWRCC collection.
... Una de las desventajas de los microsatélites es el alto costo que implica el desarrollo de primers especie específicos; no obstante, pueden utilizarse primers heterólogos de especies estrechamente relacionadas (Selkoe y Toonen, 2006). Diversos estudios han demostrado que entre las especies del género Crocodylus hay un gran éxito en la amplificación cruzada de loci microsatélite; primers desarrollados para C. johnstoni (FitzSimmons et al., 2000) amplifican también en C. moreletii (Dever y Densmore, 2000;Mcvay et al., 2008), C. porosus (Isberg et al., 2004) y por último C. rhombifer y C. acutus (Weaver et al., 2008). ...
... Sin embargo, se encontró que para algunos de estos juveniles existen varios padres y/o madres potenciales; no se pudo establecer con certeza una madre o padre ya que este tipo de análisis de parentesco tiene mayor poder si se conoce con seguridad el genotipo de uno de los padres (Kalinowski et al., 2007). Por tal razón, cuando no se conoce ninguno de los padres, es recomendable el uso de un mayor número de loci altamente polimórficos (Isberg et al., 2004). En el presente estudio, el bajo polimorfismo de cuatro de los seis loci pudo haber incidido en la presencia de varias madres y padres potenciales para un juvenil, lo que hizo necesario llevar a cabo una aproximación por verosimilitud para establecer la pareja de padres más probable para cada uno de estos 89 juveniles. ...
A genetic study of Crocodylus intermedius ex situ population was made. This population is located in the Estación de Biología Tropical Roberto Franco. The aim of this study is to establish the ex situ population genetic potential and propose mating pairs for its management. Heterologous primers were used for microsatellite loci amplification; three of them were monomorphic and six polymorphic with 2 to 16 alleles. Adults were parent candidates of a different number of juveniles, which suggest differences in reproduction. Also, 121 juveniles did not have adult parent candidates, for that reason they were considered from external origin. Between adults and juveniles there were not differences in He and A, which means that genetic diversity loss was not found. Mating pairs are proposed between individuals with a r=0 coefficient to increase heterozigosity. Every individual should reproduce to avoid allelic richness loss and genetic population differentiation from wild population.
... Microsatellites have been isolated from a number of crocodilians including Alligator mississipiensis (Glenn et al. 1998), Caiman latirostris , Crocodylus moreletii (Dever and Densmore 2001), Crocodylus johnsoni and Crocodylus porosus (Fitzsimmons et al. 2001). These markers have been employed in a range of species to assess genetic diversity, mating behaviour, hybridisation, as well as dispersal systems (Glenn et al. 1996(Glenn et al. , 1998Fitzsimmons et al. 2001;Dever et al. 2002;Dever and Densmore 2001;Davis et al. 2002;Dessauer et al. 2002;Verdade et al. 2002;Isberg et al. 2004;Zucoloto et al. 2006). ...
... The saltwater crocodile (Crocodylus porosus), which is distributed throughout much of South East Asia, is relatively uncharacterized genetically. Microsatellite markers have previously been generated in C. porosus for use in parentage and species identification (Fitzsimmons et al. 2001(Fitzsimmons et al. , 2002Isberg et al. 2004). Using the 21 microsatellites available for C. porosus, Isberg et al. (2006) recently demonstrated the first evidence of genetic linkage in the order Crocodylia, as well as within the class Reptilia. ...
Genomic elucidation and mapping of novel organisms requires the generation of large genetic resources. In this study, 253
novel and polymorphic microsatellite loci were isolated and characterized for the saltwater crocodile (Crocodylus porosus) by constructing libraries enriched for microsatellite DNA. All markers were evaluated on animals obtained from Darwin Crocodile
Farm in the Northern Territory, Australia, and are intended for future use in the construction of a genetic-linkage map for
the saltwater crocodile. The 253 loci yielded an average of 4.12 alleles per locus, and those selected for mapping had an
average polymorphic information content (PIC) of 0.425.
... Modern DNA techniques can help to identify the parentage (PI, parental identifi- cation). This principle has been shown by Isberg et al. (2004), Lee and Cho (2006) and Rohrer et al. (2007). Especially the work of Isberg et al. (2004) on saltwater crocodiles is a good example on how complicated the parental identification and/or verification can be in non-commercial conditions. ...
... This principle has been shown by Isberg et al. (2004), Lee and Cho (2006) and Rohrer et al. (2007). Especially the work of Isberg et al. (2004) on saltwater crocodiles is a good example on how complicated the parental identification and/or verification can be in non-commercial conditions. If the prob- lem of erratically assigned parents can be solved for animals kept under natural condi- tions, e.g. ...
Proper knowledge about individual origin and pedigree is a major point for animal breeding and genetic livestock improvement. Mainly for goat, but also for sheep, breeding in Sicily information on these points is limited, and sometimes even misleading. Sheep and goat milk production is, in many regions and especially on Sicily, a rural enterprise, where males and females graze together and pedigree of offspring can be largely unknown. Genetic improvement in this situation is challenging. Modern DNA techniques can help to identify the parentage (PI, parental identification). In this paper we discuss (1) our set of microsatellites markers in relation to the ECONOGENE set, (2) accuracy of microsatellite genotyping for a goat and a sheep breed, (3) the principle of verification and identification as tested on one goat breeding farm, and (4) direct use of the technique in a running sheep or goat breeding program. For a practical application we suggest the use of around 20 quality microsatellite markers, and the use of primiparous milk analysis.
... The expected heterozygosity obtained in the currently living crocodiles of the EBTRF is similar and even higher than that reported for wild populations of other species of the genus Crocodylus, evaluated with the same loci (e.g., Isberg et al. 2004;McVay et al. 2008;Mauger et al. 2017). This shows that, although the captive population of the Orinoco crocodile experienced an allele loss compared with the founder population, it maintains an important part of the variability in Table 5. Past and present reproductive combinations for four ex-situ subpopulations of Crocodylus intermedius in Colombia. ...
A purpose of ex-situ populations is the preservation of genetic variation, but this is a challenging task since genetic diversity is commonly lost through each generation, and so the establishment of management guidelines should be a high priority. Fifty years ago, the National University of Colombia began a breeding program in the Roberto Franco Tropical Station (in Villavicencio, Meta) to conserve the critically endangered Orinoco crocodile Crocodylus intermedius. Despite the large number of individuals raised and kept in captivity, the Station has not been able to release individuals due to a lack of a complete genetic characterization that could determine whether the population is genetically viable. In this study we used a panel of 17 microsatellite loci to overcome this problem. We estimated from the founder animals and the live crocodiles the inbreeding, heterozygosities, the number of alleles, and their richness, and frequencies to understand the effects of managing a captive breeding program without considering genetic profiles. Our results revealed that the living population maintains much of its founder diversity with high levels of heterozygosity and low overall inbreeding, making it suitable for maintaining captive breeding and for implementing wild releases. We estimated the individual genetic diversity of the living crocodiles, as well as their relationships. This information, combined with the size, sex, and location, allowed us to propose combinations and to restructure the breeding groups. We demonstrated that molecular data could be used to improve the management of ex-situ conservation programs well beyond what could be achieved with pedigree information alone.
... The 17 loci screened exhibited high polymorphism and resolution in paternity identification with mean PIC of 0.566 (> 0.50) (Botstein et al., 1980). CPE (the combined probability of exclusion) value, an important index in determining genetic parent of an offspring (Sherman et al., 2004), has been successfully used in paternity test for many species (e.g., Crocodylus porosus: Isberg et al., 2004;Ursusarctos: Itoh et al., 2012;Ailuropoda melanoleuca: Huang et al., 2015). However, CPE value was influenced by the number of loci genotyped and heterozygosity of each locus. ...
... Population genetic studies have been conducted on several species of crocodilians to address issues like hybridization with sympatric con-generic species (Machkour-M' Rabet et al. 2009;Weaver et al. 2008); testing for multiple paternity ; elucidating matting patterns ( Davis et al. 2001;; Rodriguez 2007); and assessing genetic diversity and gene flow Davis et al. 2002;Dever et al. 2002;Glenn et al.1998;Hekkela et al. 2010). Population genetics also have equally contributed to commercial breeding programs to facilitate the parentage assignment in order to identify individuals with desired phenotypic traits for breeding purposes ( Isberg et al. 2004) and to quantify the genetic variation in farm bred crocodiles ( Flint et al. 2000). ...
Population genetic studies provide information on the various aspects of
genetics of species and therefore have a wide spread application in conservation
research. This study assesses the genetic diversity, population subdivision, and genetic
structure within the populations of the American crocodile (Crocodylus acutus) from
eight localities in three sampling regions (mainland, northern and southern Coiba Island)
in south -western Republic of Panama. A total of 143 individuals were characterized
genetically by amplifying highly polymorphic microsatellite loci developed for
Crocodylus. Microsatellite loci utilized in this study showed considerable allelic
variation with a total of 61 alleles reported for nine loci across all localities with an
average of 6.8 alleles per locus. A model based clustering analysis revealed the
presence of three spatially overlapping genetic clusters among the sampled populations.
All three estimates of population subdivision (FST, RST, and ΦPT) are consistent with the
finding that the fine scale analysis of sampled populations revealed an overall presence
of genetic subdivisions among and within the sampled localities. The Panamanian
government should consider this information when designing conservation programs for
the American crocodile in CNP
... were allelic variants corresponding to a single locus Cj16. This locus was mapped to the genome with Cj16 primers (Isberg et al. 2004) using the in silico primer mapping algorithm in Geneious v 10.0.9 (Kearse et al. 2012). The Cj16 forward and reverse primers mapped to a single region in contig SciaK46_869 and were included in the alignment file for further analysis. ...
Crocodilians are an economically, culturally, and biologically important group. To improve researchers' ability to study genome structure, evolution, and gene regulation in the clade, we generated a high-quality de novo genome assembly of the saltwater crocodile, Crocodylus porosus, from Illumina short read data from genomic libraries and in vitro proximity-ligation libraries. The assembled genome is 2,123.5 Mb, with N50 scaffold size of 17.7 Mb and N90 scaffold size of 3.8 Mb. We then annotated this new assembly, increasing the number of annotated genes by 74%. In total, 96% of 23,242 annotated genes were associated with a functional protein domain. Furthermore, multiple non-coding functional regions and mappable genetic markers were identified. Upon analysis and overlapping the results of branch length estimation and site selection tests for detecting potential selection, we found 16 putative genes under positive selection in crocodilians, ten in C. porosus and six in A. mississippiensis. The annotated C. porosus genome will serve as an important platform for osmoregulatory, physiological and sex determination studies, as well as an important reference in investigating the phylogenetic relationships of crocodilians, birds, and other tetrapods.
... were allelic variants corresponding to a single locus Cj16. This locus was mapped to the genome with Cj16 primers (Isberg, et al. 2004) using the in-silico primer mapping algorithm in Geneious v 10.0.9 (Kearse, et al. 2012). The Cj16 forward and reverse primers mapped to a single region in contig SciaK46_869 and was included in the alignment file for further analysis. ...
Crocodilians are an economically, culturally, and biologically important group. To improve researchers' ability to study genome structure, evolution, and gene regulation in the clade, we generated a high-quality de novo genome assembly of the saltwater crocodile, Crocodylus porosus , from Illumina short read data from genomic libraries and in vitro proximity-ligation libraries. The assembled genome is 2,123.5 Mb, with N50 scaffold size of 17.7 Mb and N90 scaffold size of 3.8 Mb. We then annotated this new assembly, increasing the number of annotated genes by 74%. In total, 96% of 23,242 annotated genes were associated with a functional protein domain. Furthermore, multiple non-coding functional regions and mappable genetic markers were identified. Upon analysis and overlapping the results of branch length estimation and site selection tests for detecting potential selection, we found 16 putative genes under positive selection in crocodilians, ten in C. porosus and six in A. mississippiensis . The annotated C. porosus genome will serve as an important platform for osmoregulatory, physiological and sex determination studies, as well as an important reference in investigating the phylogenetic relationships of crocodilians, birds, and other tetrapods.
... Tissue samples have previously been retrieved from captured, farmed or deceased animals, and museum specimens (Gratten 2003, Isberg et al. 2004); however, remote biopsy sampling of wild crocodiles is poorly studied. Capturing salt water crocodiles (Crocodylus porosus) is logistically challenging and potentially dangerous even with animals confined within crocodile farms. ...
Biopsy sampling is a powerful tool for furthering our understanding of animal populations. Knowledge of diets and trophic energy flows, genetic structure and connectivity between populations, and uptake of environmental contaminants can all be elucidated through analysis of animal tissues, which is particularly useful for animals that are elusive and difficult to observe. Crocodylians are an aquatic group for which biopsy sampling is an appropriate tool; however, sample collection usually requires undertaking the arduous and dangerous process of capturing individual crocodiles. We describe a biopsy system that employs a modified version of a standard biopsy needle and pole kit to consistently extract crocodile tissue with greatly reduced effort and increased safety. This system takes advantage of the behavioral trait of crocodiles to “freeze” under the glare of a high‐powered spotlight, enabling an approach to within a few meters. We field‐tested the system in 2 remote river systems in northwestern Australia, during July–August 2015, collecting tissue samples from 69 crocodiles (Crocodylus porosus; size range = 0.9–4.4 m total length) with approximately 95% success. The pole biopsy system increases sample collection efficiency by removing the need to handle individual crocodiles. Moreover, this decreases stress on the crocodile population and significantly reduces the number of field crew required. Biopsy sampling is a powerful tool for furthering our understanding of animal populations, however for many large and elusive marine animals, obtaining sufficient tissue samples remains problematic. Motivated by obtaining a better understanding of connectivity between saltwater crocodile populations, we developed a biopsy pole system that enables multiple samples to be taken from a crocodile population in a timely and safe manner with minimal impact on the well‐being of individual crocodiles.
... Determining whether jerky came from a farmed elk or a wild elk, for instance, can be a valuable tool for wildlife law enforcement officers. As with other population assignments, establishment of whether a sample is wild or farmed can rely in whole or in part on STR or SNP characterizations (109,133,134). If the genotypes of the farmed parents are known, or if the animals within the farm are genetically distinct from animals in the wild, then it is fairly simple to match genotypes of offspring back to the parents, but it becomes increasingly complex in species with many possible parents, such as fish in a hatchery (134,135). ...
Wildlife forensics has recently been recognized among the wide variety of forensic science disciplines. This review compares human and wildlife DNA forensics, which use the same genetic tools, but often for far different purposes. Human forensic genetics almost invariably attempts to identify individual perpetrators involved in a given crime. Wildlife forensics often determines whether a crime has occurred. In addition to techniques familiar in human laboratories, like individual matching with STRs, wildlife analysts may be asked to determine the taxonomic identity, geographic source, or sex of evidence items, or the familial relationships or minimum number of individuals among a group of samples. This review highlights the common questions, legal framework, databases, and similar validation requirements to foster understanding between disciplines. Based on this understanding, human and wildlife DNA practitioners may work together and learn from each other in order to elevate the discipline of forensic genetics.
... Applying these markers variability estimates on crocodilians were higher than values obtained using methods previously mentioned (Glenn et al., 1998;Davis et al., 2000Davis et al., , 2002Fitzsimmons et al., 2000Fitzsimmons et al., , 2002Dever et al., 2002;de Thoisy et al., 2006). Several authors have also used microsatellite amplification for genealogical and mating system studies on crocodilians (Davis et al., 2001;Isberg et al., 2004;McVay et al., 2008;Weaver et al., 2008;de Oliveira et al., 2010;Hu and Wu, 2010). ...
The knowledge about reproductive strategies of a species contributes to their conservation. Multiple paternity is a reproductive behavior increasing effective population size, which could increase genetic diversity particularly in populations submitted to bottlenecks events. In Argentina, wild populations of Caiman latirostris are subject of a management plan devoted to their preservation and sustainable utilization based on its commercial interest. This program started in response to the evident numeric reduction of the populations, as a consequence of hunting pressure and habitat modification; it had a remarkable success in population recovery allowing the commercial use of C. latirostris. Data on reproductive behavior of C. latirostris are limited because mating occurs in the water and the information about their genetic diversity is scarce too. Our specific aims were to study the mating system and population genetic structure applying microsatellite markers in twelve C. latirostris families. The obtained results showed highly significant difference among populations and a lack of correspondence between geographical distance and genetic differentiation suggesting that populations of C. latirostris represent unstable metapopulations. In the paternity analysis was detected more than one father in two nests, which could be explained by capacity of storage sperm, proposed in females of a related species. The behavior of multipaternity could contribute to maintain viable populations of C. latirostris, since the maintenance of genetic variability within populations could help increase their capacity to respond to selective pressure. Further studies employing genetic and behavioral framework are needed to better understand the reproductive biology of C. latirostris.
... Although the knowledge and skills of farm management is advanced, the application of genetic technologies to advance productivity has been relatively slow compared to that of other commercial abalone species around the world. For industry to meet the demand for good quality export products, the inclusion of a genetic improvement programme into farm management is necessary (Isberg et al. 2004). ...
... In the present investigation, panel of polymorphic microsatellite markers recommended by FAO for biodiversity analysis were evaluated for assigning parentage. The high exclusionary power of such a battery of genetic markers shows the utility of such markers for evaluating the parentage (Isberg et al., 2004, Kong et al., 2006Osman et al., 2006). The parentage assignment at a known level of statistical confidence (95%) clearly showed that the kids borne are the IVF kids. ...
The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ??g/ml FSH, 100 ??g/ml LH, 1 ??g/ml estradiol-17?? 50 ??g/ml gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ??l drops of maturation medium containing granulosa cell monolayer and incubated in a 5% CO2 incubator at 38.5??C for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ??g/ml heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were co-incubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg PGF2??/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.
... This is comparable to the study of the saltwater crocodile (C. porosus) by Isberg et al. (2004) who also reported 99% probability of paternity exclusion, using 14 loci developed by Fitzsimmons et al. (2001). Similarly using only 5 microsatellite loci, Hu and Wu (2010) found a 94% paternity exclusion probability, and an 8.496 × 10 -4 probability of genetic identity (0.08% chance of misidentifying individuals). ...
The mating system of the spectacled caiman (Caiman crocodilus) was investigated in the Piagaçu-Purus Sustainable Development Reserve, Amazonas, Brazil. We used 6 polymorphic microsatellite
loci to genotype 13 females and 174 hatchlings representing 20 nests sampled over 4 consecutive reproductive seasons (2007–2010).
Paternity was determined by 2 methods: simple counts, and statistical analysis using Gerud 2.0. Results were congruent between
the 2 approaches, and the null hypothesis of single paternity was rejected in 19 of the 20 nests analyzed, thereby demonstrating
that C. crocodilus females are polyandrous and that the breeding system of the species can be characterized as polygamous. The data suggest
that 1–4 fathers were responsible for the paternity of the offspring, and that the males contributed differently (9–100%)
to each of the 20 nests. A total of 53 males copulated with the 20 females mothering the nests. This is the first study on
the mating system of C. crocodilus and the frequency of multiple paternity is among the highest values (95%) reported thus far for crocodilians.
... However, further investigation may be warranted as a number of diversity studies, albeit using microsatellites, have shown moderate levels of mean heterozygosity (H E = 0.040-0.941) between populations of C. porosus [43], A. mississippiensis [44] and C. latirostris [45] and high sequence divergence (nucleotide diversity = 0.152) at a population of C. porosus using MHC class I markers [46]. Furthermore, numerous phylogenetic subdivisions of MHC class II b exon 3 and their orthology of sequences from both Crocodilidae and Alligatoridae may suggest a greater number of gene lineages in this exon, relative to the MHC class II a exons described above. ...
Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II α and β evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II β diversity, whilst diversity within MHC class II α is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II α sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II α sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II β sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85-90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia.
... To date, genetic studies undertaken on the saltwater crocodile have predominantly focussed on farmed populations. Notable works exploiting variation at microsatellite loci include genetic linkage mapping (Miles et al. 2009a) and quantitative trait loci analysis to identify genes underlying traits of economic importance (Miles et al. 2010), and the identification of microsatellite markers for parentage testing (Isberg et al. 2004; Miles et al. 2009b). With support from mitochondrial DNA (mtDNA) markers, other studies utilising microsatellites have been able to identify genetic differentiation in saltwater crocodile populations from the Indo-Malay Archipelago and the Western Pacific Ocean (Gratten 2003; Russello et al. 2007). ...
The saltwater crocodile is distributed throughout south-east Asia and Australia. In Australia, it is most abundant in the Northern Territory and Queensland, where it is sustainably farmed for its skins and meat. The aim of this study was to elucidate the relationships and genetic structure among saltwater crocodiles from the Northern Territory of Australia using mitochondrial control region sequences from 61 individuals, representing nine river basins and six of unknown origin, as well as published sequences from other regions. Eight mitochondrial control region haplotypes were identified among both published and novel sequences. Three of the haplotypes appear to be restricted to specimens from northern Australia, with a single haplotype being the most widely dispersed across all river basins. Although Analysis of Molecular Variance provides some support for differentiation among river basins, the frequency of shared haplotypes among these geographical units and median-joining network analysis do not support a clear genetic structure or phylogeographic pattern for saltwater crocodiles in the Northern Territory. The results of this study will assist in furthering our understanding of the genetic diversity of wild saltwater crocodile populations used for ranching in the Northern Territory, as well as providing a framework for assessing the origin of unknown specimens in the future.
... For these reasons, microsatellites have been widely used for population studies in a variety of wildlife species (Wilson et al. 2004). In crocodilians, microsatellites have been used to assess genetic diversity, mating behaviour, hybridisation, as well as dispersal systems, in a variety of species (Glenn et al. 1996(Glenn et al. , 1998Fitzsimmons et al. 2001;Dever et al. 2002;Davis et al. 2002;Dessauer et al. 2002;Verdade et al. 2002;Isberg et al. 2004Isberg et al. , 2006. Despite this, informative microsatellite markers still do not exist for many crocodilian species (Glenn et al. 1998). ...
Microsatellite DNA loci have emerged as the dominant genetic tool for addressing questions associated with genetic diversity
in many wildlife species, including crocodilians. Despite their usefulness, their isolation and development can be costly,
as well as labour intensive, limiting their wider use in many crocodilian species. In this study, we investigate the cross-species
amplification success of 82 existing microsatellites previously isolated for the saltwater crocodile (Crocodylus porosus) in 18 non-target crocodilian species; Alligator sinensis, Caiman crocodylus, Caiman latirostris, Caiman yacare, Melanosuchus niger, Paleosuchus palpebrosus, Crocodylus acutus, Mecistops cataphractus, Crocodylus intermedius, Crocodylus johnstoni, Crocodylus mindorensis, Crocodylus moreletii, Crocodylus niloticus, Crocodylus novaeguineae, Crocodylus palustis, Crocodylus rhombifer, Crocodylus siamensis, and Osteolaemus tetraspis. Our results show a high level of microsatellites cross-amplification making available polymorphic markers for a range of
crocodilian species previously lacking informative genetic markers.
... Generalmente l'uso dei marcatori molecolari si limita all'esclusione della paternità se il genotipo a uno o più marcatori dell'individuo risulta incompatibile con quella del padre presunto. Tuttavia, come dimostrato da Isberg et al.,(2004) e Rohrer et al,(2007, l'uso di marcatori molecolari può essere fondamentale anche per l' attribuzione delle relazioni di parentela tra gli individui. Come precedentemente esposto l'identificazione esatta della paternità, in soggetti che originano da accoppiamenti non controllati o da gruppi di monta con più riproduttori maschi, rappresenta un potente mezzo per migliorare le capacità decisionali e gestionali degli allevatori e dei selezionatori. ...
The aims of the present work were to get ready an economic, rapid and toxic solvent-free
method to extract DNA from sheep milk somatic cells and to set up a system of microsatellites for
paternity assignment in Sarda sheep breed. During the years, several techniques which describe the
isolation of genomic DNA from somatic cells of cow and goat milk have been reported. Milk is
preferred to blood as a source of DNA because its collection is routinely performed, less expensive
and more easily accomplished than blood collection. Moreover milk use limits stressful practices
such as capture, handling and venipuncture in animal management. A rapid alkaline method
resulted most suitable as method for DNA extraction from sheep milk because fast and economic.
Genomic DNA was extracted from somatic cells starting from 20 mL of sheep milk and
used as substrate for the polymerase chain reaction. A system of 10 highly informative
microsatellites (MCM058, LSCV06, BM6444, BMS2213, CSSM43, BMS2252, MCM120,
OLADRB, MCM373, BMS0360) was chosen and spread in 3 multiplex for paternity assignment in
the Sarda breed. The proposed system would be a useful alternative to traditional pedigree
recording in the Sarda breed. Not only it had a high probability of excluding incorrect parental
(Pe=0.99996) relationships but it also provided a high percentage of unequivocal paternity
assignment (P=1.0 in a simulated population). Its regular utilization would increase the number of
individuals with known ancestors in the Sarda breed, with favorable consequences on the number of
genetically evaluated animals and the precision of their breeding values estimation.
OLADRB, MCM373, BMS0360) was chosen and spread in 3 multiplex for paternity assignment in
the Sarda breed. The proposed system would be a useful alternative to traditional pedigree
recording in the Sarda breed. Not only it had a high probability of excluding incorrect parental
(Pe=0.99996) relationships but it also provided a high percentage of unequivocal paternity
assignment (P=1.0 in a simulated population). Its regular utilization would increase the number of
individuals with known ancestors in the Sarda breed, with favorable consequences on the number of
genetically evaluated animals and the precision of their breeding values estimation.
... For example, microsatellite markers have been applied in past studies of genetic variability and population structure of Crocodylus moreletii (Dever and Densmore, 2001;Dever et al., 2002) and A. mississippiensis (Glenn et al., '98, 2002;Davis et al., 2001aDavis et al., , 2002. Other authors have applied microsatellites in parentage testing in Cr. porosus (Isberg et al., 2004) and in analyses of mating patterns in A. mississippiensis (Davis et al., 2001b). Davis et al. (2001b) also inferred multiple paternity, ''the occurrence of offspring within a single clutch being fathered by more than one male,'' in seven of 22 clutches of A. mississippiensis. ...
Detecting multiple paternity in wild populations of the broad-snouted caiman (Caiman latirostris) has important implications for conservation efforts. We have applied microsatellite markers to examine genetic variation in C. latirostris and also have provided the first data concerning detection of multiple paternity in wild populations of this species. Blood samples from four nest-guarding C. latirostris females and their hatchlings were obtained from Santa Fe Province, Argentina. Amplified products were analyzed by electrophoresis on 10% polyacrylamide gels and visualized with silver staining. Four out of the eight markers tested reliably amplified and yielded useful data. Using polyacrylamide gels with silver staining provides high enough resolution to obtain individual genotypes. In order to assess the presence or absence of more than two parents in each nest, we used the single locus Minimum Method, and applied Cervus 3.0 and Gerud 2.0 software in parentage analyses. Our results indicate more than one father in at least two families. This behavior could be the consequence of high habitat variability in the area where our population was sampled. The ability to understand mating systems is important for maintaining viable populations of exploited taxa like C. latirostris.
Recent Trends in Livestock Innovative Technologies explores the most recent developments and developing trends in the livestock farming industry. The book delves into the application of innovative technologies in various aspects of livestock production, management, and health through edited chapters.
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Fresh methods for illness prevention and management, such as the use of vaccines, diagnostics, and biosecurity measures.
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Recent Trends in Livestock Innovative Technologies is an informative resource for researchers, professionals, and policymakers interested in staying up-to-date with the advancements and future directions of the livestock industry.
Next-generation sequencing has allowed us to explore new methods where comparative and population genomics can be used simultaneously. Keeping this in mind, we surveyed and analyzed the frequency and distribution of microsatellites in the Indian gharial (Gavialis gangeticus) and compared it with American alligator (Alligator mississippiensis) and saltwater crocodile (Crocodylus porosus) to enrich them with genomic resources. Indian gharial has a low frequency, relative abundance (RA), and relative density (RD) of microsatellites as compared to other crocodilians. RA and RD were positively correlated with the GC content of genomic and transcriptomic sequences. The genomic sequences were dominated by dinucleotide repeats, whereas the transcriptomic sequences had an excess of trinucleotide repeats. Motif conservation studies among the three crocodilians revealed conservation of 69.2% of motifs. Species-specific unique motifs identified in this study could be used as molecular probes for species identification. A total of 67,311 primers were designed in all three species to enrich the crocodilians with genomic resources. The genomic resources developed in this study could accelerate diversity analysis within its individuals to design a proper mating plan to reduce inbreeding stress and further improve the species.
Crocodilian mating systems are complex but molecular genetics is providing some of the tools necessary to begin their unravelling. The most significant unravelling has been the widespread occurrence of multiple paternity (more than one sire for a clutch of eggs) across the global crocodilian species. The advantages of this strategy are still being debated and may differ across the different species, being dependent on factors such as population density and habitat availability at the individual population level. This chapter reviews the available literature outlining the impact other complexities such as nest site selection, the potential for communal nesting and “alloprotection” (non-biological female exhibiting nest guarding of conspecific eggs) could have on defining the mating strategies of crocodilians. However, it is argued that any advantage of genetic gain, genetic variability or inbreeding avoidance that might achieved by multiple paternity is overcome by low embryo and offspring survival leading to low recruitment rates into the adult breeding population. The limitations of experimental design are also discussed which may be leading to upwardly biased estimates of hypothetical sire numbers.
The yacare caiman (Caiman yacare) is a reptile from South America and one of the two crocodilian species present in Argentina. The degradation of their natural habitat and strong hunting pressure led to a sharp numerical decline of wild populations. Nowadays C. yacare is included in the Appendix II of CITES, and at the same time it has been subjected to ranching practices in some areas in Argentina. In this context, it is important to better understand the population structure and mating system of the species. To do this, we amplified ten microsatellite markers (SSRs) in 148 individuals of 13 C. yacare nests. All of markers were polymorphic with 2-12 alleles per locus, with allelic sizes ranging between 154 and 400 bp and medium levels of polymorphism (Ho = 0.152-0.551 and He = 0.221-0.621). We were able to determine the maternal genotype in 9 out of 13 nests. In 6 of them we found more than one paternal genotype, being the maximum 3 fathers for a single nest. This study is the first to provide evidence of multiple paternity behavior. These findings will be useful to improve management and conservation strategies for the species.
Genetic population analysis using molecular markers is probably the most important issue in conservation genetics and today it is a very useful tool for the study of species subjected to sustainable use. Caiman latirostris (broad-snouted caiman) is one of the two crocodilian species cited for Argentina. Their wild populations were drastically reduced in the 1950s and 1960s due to commercial hunting and intense alteration of their habitat, and C. latirostris was included in the Appendix I of CITES. Since 1990, management plans that use ranching system (harvest of wild eggs, captive rearing and reintroduction to nature) began in Argentina. Through these management activities, Argentine caiman populations were numerically increased and transferred to the Appendix II of CITES that allows the regulated trade of their products. Genetic population studies are being
The saltwater crocodile (Crocodylus porosus) forms the basis of a crocodile farming industry for the international skin trade in Australia. For this industry, mortalities from stress and disease are common due to a compromise of the adaptive immunity. Currently the genetic understanding of the immune response is poor, which in turn impedes an understanding of genes, and hence genetic markers, affecting disease susceptibility. As a key component of adaptive immunity is the Major Histocompatibility Complex (MHC), this thesis characterises the MHC genes with an emphasis on the saltwater crocodile and assesses genetic diversity, evolutionary mechanisms that are influencing diversity and their roles in adaptive immunity. The genetic diversity among saltwater crocodiles showed the number of MHC variants within an individual ranging from one to seven, indicating that there are at least four gene loci in this species. An association between a certain MHC variant and Lymphoid proliferation/ Vasculitis/ Encephalitis in saltwater crocodiles was identified (P = 0.00007), suggesting genetic susceptibility to the disease. Phylogenetic analyses showed that MHC variants from 20 species of Crocodylia clustered at the genus or family level rather than in species-specific groups, indicating orthologous relationships. Selection detection analyses showed that balancing selection influenced some classes of MHC in Crocodylia. In addition, construction of Bacterial Artificial Chromosome scaffolds in the saltwater crocodile showed MHC class I genes located along with antigen processing genes and a framework gene. This would support structural variation of the saltwater crocodile MHC that differs from that expected in tetrapod ancestors. This project offers a better understanding of immunogenetics and immunogenomics in Crocodylia and presents recommendations for future research, where these findings could serve as a foundation in order to achieve a complete picture of MHC in Crocodylia.
The present status of existing crocodile species has increased the necessity to develop novel utilities for conservation. We have developed 18 microsatellite loci from the Indian Gharial (Gavialis gangeticus) intended for genomic explanation and applied them to study genetic variation. Polymorphism of each locus was assessed in 32 individuals for G. gangeticus India. The number of alleles per locus varied from 2 to 8 (mean 5.5) for Indian population. Observed and expected heterozygosity ranged from 0.73 to 1.00 and 0.50 to 0.81, respectively and the average polymorphic information content is 0.565. These selected markers are helpful for assessing population structure, intraspecific difference, and conservation and management of G. gangeticus.
Among the loss of genetic diversity due to population declines, population fragmentation and habitat loss, hybridization also stands as a threat to Morelet’s crocodile (Crocodylus moreletii) populations. Genetic surveys in Belize and the Yucatan Peninsula have detected evidence of hybridization with the American crocodile (C. acutus). Admixture between these two species is most likely driven by human-mediated translocations. Along the central gulf coast of Mexico, C. moreletii populations are presumed to be purebred. To test this, we use nine microsatellite loci and sequence data from the mitochondrial control region to detect if C. acutus alleles have introgressed into populations of C. moreletii from central Veracruz. In 2010, C. moreletii was transferred from Appendix I to II of CITES based on a whole species demographic analysis, which indicated that populations had recovered across its range. Our study shows that populations in central Veracruz are purebred, although they exhibit low levels of genetic diversity most likely caused by inbreeding. Our data also suggest there is fragmentation among populations of C. moreletii, which may lead to further loss of genetic variation. Due the purity and low genetic diversity of C. moreletii populations from central Veracruz, we recommend increased protection and active management practices that take genetic data into account.
Abstract The establishment and evaluation of family lines using pedigree information provides an advanced understanding of the variability that exists for complex, economically valuable traits and is a necessary step in the execution of an effective breeding programme. The aim of this study was to assign parentage to mass-spawned Haliotis midae juveniles using species-specific microsatellite markers. Screening of wild abalone individuals revealed that the nine loci selected complied with the minimum requirements for parentage analyses: a null allele frequency Keywords: Haliotis midae L; microsatellite marker; parentage assignment; pedigree Document Type: Research Article DOI: http://dx.doi.org/10.1111/j.1365-2109.2009.02428.x Affiliations: Molecular Aquatic Research Group, Department of Genetics, Stellenbosch University, Matieland, South Africa Publication date: August 1, 2010 (document).ready(function() { var shortdescription = (".originaldescription").text().replace(/\\&/g, '&').replace(/\\, '<').replace(/\\>/g, '>').replace(/\\t/g, ' ').replace(/\\n/g, ''); if (shortdescription.length > 350){ shortdescription = "" + shortdescription.substring(0,250) + "... more"; } (".shortdescription a").click(function() { (".originaldescription").slideDown(); return false; }); }); Related content In this: publication By this: publisher In this Subject: Aquaculture & Fisheries By this author: van den Bergb, Nicol-Candice ; Roodt-Wilding, Rouvay GA_googleFillSlot("Horizontal_banner_bottom");
Saltwater crocodiles are in high demand for the production of luxury fashion items. However, their susceptibility to disease incurs substantial losses and it is hoped to be able to genetically select these animals for disease resistance. So far, this has only been enabled by phenotypic selection. Investigating the major histocompatibility complex (MHC) could provide insight into the ability of an individual to respond to pathogens acting as a selective pressure on the host. Here, we assessed genetic diversity and a role of selection in shaping the diversity of MHC class I exon 3 among 42 saltwater crocodiles from nine river basins in the Northern Territory, Australia. We generated 640 sequences using cloning and sequencing methods and identified 43 MHC variants among them. Phylogenetic analyses clustered these variants into two major clades, which may suggest two gene lineages. We found the number of variants within an individual varying between one and seven, indicating that there are at least four gene loci in this species. Selection detection analyses revealed an elevated ratio of nonsynonymous to synonymous substitutions (mean = 1.152 per codon), suggesting balancing selection. Population differentiation analyses revealed that the MHC did not show structuring among the river basins, and there were some shared variants among them. This may be a result of possible gene flow and/or similar selection pressures among populations. These findings provide background knowledge to identify potential MHC markers, which could be used for selecting genetically variable individuals for future disease associations. All MHC class I exon 3 sequences reported in this paper were submitted to the GenBank database with following accession numbers: HQ008785-HQ008789, HQ008791-HQ008798, HQ008808-HQ008815, HQ008824, HQ008826-HQ008830, HQ008835, HQ008839, HQ008842-HQ008850, and JX023536-JX023540.
We examined 24 microsatellite markers to
select those most suitable for individual identification
and parentage tests of brown bears
(Ursus arctos)
based on statistical parameters and experimental error
using liver samples (38 issues) from nuisance bears killed
in the Akan–Shiranuka Region, eastern Hokkaido, Japan from
1996 to 2006. We found seven microsatellite markers suitable
for identifying individuals (G1A, G10B, G10L, UarMU5, UarMU23,
UarMU50, and UarMU51; probability of identity
P
id=3.17
×10−7; probability of identity of
sibs P
id-sib
=2.23×10−3), 12 microsatellite
markers suitable for parentage testing when one parent is
known [G1A, G1D, G10B, G10L, G10P, UarMU5, UarMU23, UarMU50, UarMU51,
UarMU59, UarMU61, and UarMU64; probability of excluding one parent
(PE I=0.9991)], and 15 microsatellite markers for parentage
testing when neither parents are known [three markers added to
the above: G10M, G10X, and UarMU9;
probability of excluding both parents (PE II=0.9869)].
Genetic improvement of crocodilians is a novel concept. Here, breeding objectives for such a program are proposed and discussed in relation to the biological constraints inherent within the production system. The main source of income is the sale of skins, with meat as the major byproduct. Skins are sold according to a grading system. Objectives to increase the production efficiency of crocodile farms include: number of hatchlings per female per year, survival rate, food
conversion efficiency, and age to slaughter (growth rate). In the future, skin “quality” (scale row number and regularity, shape and thickness) could also become important.
ABSTRACT
Questions of population identification and gene flow have become more important in the conservation and management of crocodilians. Additionally there has long been an interest in understanding crocodilian mating systems. To address such questions we designed nuclear microsatellite markers from the DNA of C. acutus, C. porosus and C. johnstoni. We report on the development and testing of 26 new microsatellite loci for Crocodylus spp, which represent the first microsatellite loci found in Crocodylus.
Accurate pedigree information improves the reliability of breeding value estimates in Merino sheep improvement programs. Mismothering of Merino lambs results in incorrect pedigree information. DNA fingerprinting technology is now available to estimate the accuracy of on-farm pedigree recording methods. Dam pedigrees were erroneous for 10.5 % of all lambs genotyped and dam pedigree errors were as high as 15.5 % on one property. DNA pedigreeing could eliminate errors due to mismothering. INTRODUCTION Accurate pedigree information is necessary for reliable genetic evaluation of animals. The use of erroneous pedigree information in breeding programs reduces potential genetic gain (Long et al. 1990). The first Merino study that related mismothering to incorrect pedigree information was by Alexander et al (1983a), who observed that pedigree information was likely to be incorrect for 9 % of single born lambs when lambs were monitored constantly but identified once daily. This was due to ewes failing to keep their litters together, lamb desertion and lamb stealing. A survey established that Merino breeders generally record (dam pedigrees in one of two ways, either recording dam pedigrees at the time of, or close to, the time of birth, or else, 2-6 weeks after birth (Bamett 1998). Alexander (1983a) found that a significant proportion of Merino ewes desert their lambs soon after birth. Poindron and Le Neindre (1980) illustrated that the recognition of a lamb by a ewe develops within the first two hours after birth and Alexander et al (1986) established that the first hour post-partum was important for the development of exclusive maternal bonding. It has been shown that Merino lambs are more likely to be mismothered than other breeds of sheep (Stevens et al. 1984).
Paternity inference using highly polymorphic codominant markers is becoming common in the study of natural populations. However, multiple males are often found to be genetically compatible with each offspring tested, even when the probability of excluding an unrelated male is high. While various methods exist for evaluating the likelihood of paternity of each nonexcluded male, interpreting these likelihoods has hitherto been difficult, and no method takes account of the incomplete sampling and error-prone genetic data typical of large-scale studies of natural systems. We derive likelihood ratios for paternity inference with codominant markers taking account of typing error, and define a statistic delta for resolving paternity. Using allele frequencies from the study population in question, a simulation program generates criteria for delta that permit assignment of paternity to the most likely male with a known level of statistical confidence. The simulation takes account of the number of candidate males, the proportion of males that are sampled and gaps and errors in genetic data. We explore the potentially confounding effect of relatives and show that the method is robust to their presence under commonly encountered conditions. The method is demonstrated using genetic data from the intensively studied red deer (Cervus elaphus) population on the island of Rum, Scotland. The Windows-based computer program, CERVUS, described in this study is available from the authors. CERVUS can be used to calculate allele frequencies, run simulations and perform parentage analysis using data from all types of codominant markers.
The proportion of cows in the UK dairy herd whose sires were misidentified was estimated using DNA markers. Genetic marker genotypes were determined on 568 cows (from 168 milk samples and 400 hair samples) and 96 putative sires (from semen samples). The estimated pedigree error rate from the hair samples was 8.8%, and from the milk samples, 13.1%, giving an overall estimate of the error rate of 10%. This level of pedigree errors will have a relatively large impact on the efficiency of progeny testing and the accuracy of cow predicted breeding values. We predict a loss of response to selection of approximately 2 to 3% given this error rate.
Crocodylus siamensis, the Siamese crocodile, is a critically endangered species of freshwater crocodile previously distributed throughout much of SE Asia. Recovery plans call for reintroductions to the wild using founder individuals currently in captivity, mostly in commercial crocodile farms. On many farms C. siamensis has been intentionally hybridised with either Cuban crocodiles, C. rhombifer, or the estuarine crocodile, C. porosus, and hybrids may be difficult to distinguish morphologically. We report on the combined use of microsatellite and mtDNA genetic markers to determine the species status of potential founder individuals for reintroduction of C. siamensis. Genetic markers were used to characterise 103 captive and wild-caught individuals of C. siamensis, C. rhombifer and C. porosus in Vietnam and to distinguish purebred versus hybrid individuals. Although the microsatellite loci used had some overlap of allele sizes among species, assignment tests allowed differentiation. Four hybrids were identified, two of which had not been recognised morphologically as hybrids, and one of these was thought to be a C. siamensis suitable for reintroduction. Ten of the identified purebred C. siamensis were subsequently released into Cat Tien National Park in southern Vietnam.
The recent proliferation of hypervariable molecular markers has ushered in a surge of techniques for the analysis of parentage in natural and experimental populations. Consequently, the potential for meaningful studies of paternity and maternity is at an all-time high. However, the details and implementation of the multifarious techniques often differ in subtle ways that can influence the results of parentage analyses. Now is a good time to reflect on the available techniques and to consider their strengths and weaknesses. Here, we review the leading techniques in parentage analysis, with a particular emphasis on those that have been implemented in readily useable software packages. Our survey leads to some important insights with respect to the utility of the different approaches. This review should serve as a useful guide to anyone who wishes to embark on the study of parentage.
Crocodiles: Inside out
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Barnett NL, Purvis IW, van Hest B, and Franklin IR, 1999. The accuracy of
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