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Linkage Mapping and Quantitative Trait Loci (QTL) Analysis in Saltwater Crocodiles
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This report summarises efforts to construct the first genetic linkage map for the saltwater crocodile (Crocodylus porosus), and subsequent investigation into the presence and positioning of quantitative trait loci (QTL) for economically important traits in farmed saltwater crocodiles. Linkage and QTL mapping exercises will contribute significantly to elucidation and characterisation of the crocodile genome, and represent an important first step towards the development of genetic improvement tools for implementation in industry breeding programs. One of the major objectives of this research was to generate the requisite genomic resources to carry out genetic mapping studies in saltwater crocodiles. As such, herein we describe the development of a microsatellite marker resource, a DNA resource, a first generation genetic linkage map, a refined karyotype for C.porosus, and a proof of principle QTL study identifying the first QTL for crocodilian, or indeed any other non-avian reptile.
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... However, one major disadvantage of CrocPLAN is the large generation interval for the saltwater crocodile. Based on the recognition that the disadvantages associated with generation interval could be overcome by the implementation of MAS, Miles et al. (2009) developed a framework genetic linkage map and, using this resource, identified the first crocodilian quantitative trait loci (QTL; Miles et al., 2010a;Miles et al., 2010b). Based on this proof of concept, and leveraging off more recent advances in genetic technologies, including the recent release of the saltwater crocodile genome (Green et al., 2014), the development of molecular selection tools to further expedite selection decisions is achievable. ...
... The number of scale rows on a belly skin is not an important skin quality parameter but has so far been the only crocodile phenotype to be associated with a quantitative trait locus (QTL; Miles et al., 2010a;Miles et al., 2010b). To extend on this trait, it was of interest to investigate the symmetry of scale row number as well to predict the regularity of scale pattern. ...
... This project was highly successful and resulted in the construction of the first genetic linkage map for the saltwater crocodile (Miles et al., 2009). Furthermore, proof-of-principle QTL analyses undertaken for a limited number of traits identified the first QTL for a crocodilian demonstrating the use of these resources in identifying regions of the genome harbouring genes of interest (Miles et al., 2010a;Miles et al., 2010b). ...
The main objective of this project was to generate a large single nucleotide polymorphism (SNP) marker resource for later saturation of the genetic linkage map and fine mapping of quantitative trait loci (QTL). Another objective of this project was to learn more about basic crocodile biology, namely immune function and stress, and the underlying genetic function to evaluate their incorporation into CrocPLAN.
This report describes the development of new phenotypic trait panels for farmed saltwater crocodiles. Among these is the major crocodilian stress hormone, corticosterone (CORT), which should be useful for the development of animal welfare standards and the eventual selection of individuals in the future. Immune assays, some of which have never been previously used in crocodilians, were employed in this project to assess immune function. These immune assays, which are relatively easy to perform and cheap, could be employed in any farming setting to assess immune function in the future. Levels of estradiol (ESTR) and testosterone (TEST) are also detailed in this report, for the first time in the saltwater crocodile. At the same time as trying to expedite industry adoption of genetic improvement programs, it was necessary to expand on the current selection criteria available to gain a deeper insight into the breeding objectives already defined from RIRDC Project US-109A. The traits added were corticosterone (the main crocodilian stress hormone), two immune parameters, two sex hormones (testosterone and estradiol), two behaviour characters and four skin quality traits. Simultaneously, some of these traits could be used to gauge current industry practices which are set out in the “Code of Practice on the humane treatment of wild and farmed Australian crocodiles”. I am pleased to report that the lowest levels of corticosterone ever reported in saltwater crocodiles were found certifying the recommendations set out in the “code of practice”.
... Commercial crocodile farming industry in Australia was established in the 1970s, with a primary focus of meeting demands for the supply of crocodile skins to the luxury goods market. Both native crocodile species, namely the saltwater or estuarine crocodile Crocodylus porosus and the freshwater crocodile Crocodylus johnstoni, were originally farmed, but increased competition within the marketplace eventually resulted in production centred on C. porosus due to its higher skin quality and larger size (Miles and Isberg 2010). Compared with standard livestock enterprises (cattle, sheep and pigs), crocodile farming is still in its infancy and presents some unique challenges associated with the difficulty of trying to domesticate or, at best, captively manage this less understood and most dangerous of 'dinosaur-like' predators. ...
This review reports the current status of artificial breeding technology in the Crocodylia and the future requirements for the establishment of AI in the saltwater crocodile. Although there are challenges regarding safe restraint and immobilisation, semen collection of the saltwater crocodile by manual stimulation has proven effective in yielding sufficient volume and sperm concentrations for empirical and molecular analyses of sperm preservation and physiology. Nevertheless, there is still much to learn with respect to fundamental anatomy, physiology and behaviour in both sexes, but particularly in the female. Although lessons can be learned from successful AI in the alligator, the details of this research are not readily accessible. Future research needs to focus on the proximate factors of seasonality and the underlying control of the female’s annual reproductive cycle; this will require novel and innovative ways to collect blood samples without causing stress or injury, and ideally a dedicated crocodile research breeding colony. Because the saltwater crocodile is a farmed species, there is likely to be sufficient impetus for the application of assisted breeding technology to drive future productivity in the industry. These developments will also have benefits for the genetic and reproductive management of endangered captive populations.
... poxvirus-like lesions on their belly region. Throughout the duration of the study the animals, were housed in accordance with the Code of Practice on the Humane Treatment of Wild and Farmed Australian Crocodiles (NRMMC, 2009) in facilities previously described by Miles et al. (2010). ...
... So far, these efforts include the construction of a Bacteria Artificial Chromosome (BAC) library (Shan et al. 2009), a genetic linkage map ), and a genome-sequencing and annotation project (St John et al. 2012). Faster and more substantial genetic gains could be achieved if the genes affecting a trait of interest was identified and incorporated into the breeding program through marker assisted selection or selection based on a causative gene-mutation responsible for the variation in a particular trait (Miles 2009). One such point of interest is the assessment of traits involved in disease prevalence or immunocompetence as mortalities caused by disease incur substantial economic losses annually. ...
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 commercial wildlife trade involves billions of animals each year, consumed for various purposes, including food, fashion, entertainment, traditional medicine, and pets. The experiences of the animals involved vary widely, with negative welfare states being commonplace. To highlight the broad scope of animal welfare impacts across the commercial wildlife trade, we present ten case studies featuring a range of species traded globally for different purposes: (1) Ball pythons captured and farmed to serve as pets; (2) Zebrafish captive bred to serve as pets; (3) African Grey Parrots taken from the wild for the pet industry; (4) Sharks de-finned for traditional medicine; (5) Pangolins hunted for traditional medicine; (6) Crickets farmed for food and feed; (7) Frogs wild-caught for the frog-leg trade; (8) Crocodilians killed for their skins; (9) Lions farmed and killed for tourism; and (10) Elephants held captive for tourism. The case studies demonstrate that wild animals commercially traded can suffer from negative welfare states ranging from chronic stress and depression to frustration and extreme hunger. The individuals involved range from hundreds to billions, and their suffering can last a lifetime. Given the welfare issues identified and the growing recognition and scientific evidence for animal sentience, we propose reducing and redirecting consumer demand for these consumptive wildlife practices that negatively impact animals.
Profit from farmed crocodile is essentially a function of the returns and costs from the average lifetime productivity of the herd. The aim of a genetic improvement program is to improve the total economic value of the herd, and consequently maximise profit. To date, no research has been conducted to evaluate the potential of a genetic improvement program in the Australian crocodile industry. By implementing a selection program based on reproductive performance, juvenile growth rates and juvenile survival rates, the resultant superior breeding animals will increase the profitability of crocodile farms. The major benefits to the industry will be decreasing overhead costs by growing animals to marketable size in a quicker time, increasing profitability by offsetting some of the production costs per animal and increasing the number of animals obtained from the farm each year. The major aim of this project was to create a practical genetic improvement program for immediate adoption by the Australian crocodile industry, to be called CrocPLAN.
Comparative mapping studies of X-linked genes in mammals have provided insights into the evolution of the X chromosome. Many reptiles including the American alligator, Alligator mississippiensis, do not appear to possess heteromorphic sex chromosomes, and sex is determined by the incubation temperature of the egg during embryonic development. Mapping of homologues of mammalian X-linked genes in reptiles could lead to a greater understanding of the evolution of vertebrate sex chromosomes. One of the genes used in the mammalian mapping studies was ZFX, an X-linked copy of the human ZFY gene which was originally isolated as a candidate for the mammalian testis-determining factor (TDF). ZFX is X-linked in eutherians, but maps to two autosomal locations in marsupials and monotremes, close to other genes associated with the eutherian X. The alligator homologue of the ZFY/ZFX genes, Zfc, has been isolated and described previously. A detailed karyotype of A. mississippiensis is presented, together with chromosomal in situ hybridisation data localising the Zfc gene to chromosome 3. Further chromosomal mapping studies using eutherian X-linked genes may reveal conserved chromosomal regions in the alligator that have become part of the eutherian X chromosome during evolution.
Reptiles occupy a crucial position with respect to vertebrate phylogeny, having roamed the earth for more than 300 million years and given rise to both birds and mammals. To date, this group has been largely ignored by contemporary genomics technologies, although the green anole lizard was recently recommended for whole genome sequencing. Future experiments using flow-sorted chromosome libraries and high-throughout genomic sequencing will help to discover important findings regarding sex chromosome evolution, early events in sex determination, and dosage compensation. This information should contribute extensively toward a general understanding of the genetic control of development in amniotes.
In this paper we consider the detection of individual loci controlling quantitative traits of interest (quantitative trait loci or QTLs) in the large half-sib family structure found in some species. Two simple approaches using multiple markers are proposed, one using least squares and the other maximum likelihood. These methods are intended to provide a relatively fast screening of the entire genome to pinpoint regions of interest for further investigation. They are compared with a more traditional single-marker least-squares approach. The use of multiple markers is shown to increase power and has the advantage of providing an estimate for the location of the QTL. The maximum-likelihood and the least-squares approaches using multiple markers give similar power and estimates for the QTL location, although the likelihood approach also provides estimates of the QTL effect and sire heterozygote frequency. A number of assumptions have been made in order to make the likelihood calculations feasible, however, and computationally it is still more demanding than the least-squares approach. The least-squares approach using multiple markers provides a fast method that can easily be extended to include additional effects.
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