Microsatellites are an excellent choice of genetic marker for genome mapping due to their hyper-variability and abundance throughout most vertebrate genomes [1
]. Typing of microsatellite DNA loci by routine polymerase chain reaction (PCR) was developed almost 20 years ago [2
], and has since facilitated the construction of dense genetic maps in many species. Genetic maps are valuable tools in numerous areas of genetic research, particularly for the localization and dissection of quantitative trait loci (QTL), and for comparative mapping between species. Comprehensive linkage maps have been developed using microsatellite markers in humans, many biomedical models, livestock, fish, birds, invertebrates, plants and other organisms [5
]. Although genetic linkage maps have been developed for a broad array of species, no such maps exist for any crocodilians, or indeed any other non-avian member of the Class Reptilia.
Crocodilians are the sole surviving reptilian archosaur, a group of diapsids that include dinosaurs and other ancient reptiles that gave rise to birds [14
]. Diverging from the evolutionary lineage that gave rise to mammals more than 300 million years ago [15
], Reptilia represent a valuable intermediate evolutionary group placed between mammals and more distantly related vertebrate species such as fish [7
]. Recent progress in the development of genomic resources for studies in reptiles has given impetus to comparative genomics aimed at understanding the evolution and structure of the reptilian genome [17
]. Genome sequences are now available for two avian species [chicken [20
], and zebra finch http://www.songbirdgenome.org/index.html
], as well as for one non-avian reptile, the green anole (Order Squamata, http://www.broad.mit.edu/models/anole/
). Work is also currently underway to sequence the genome of the painted turtle (Order Chelonia, http://www.genome.gov/10002154
). Although the Order Crocodylia remains unrepresented, the generation of a comprehensive genetic map for a crocodilian will provide a significant step towards the elucidation of the crocodilian genome, providing a valuable scaffold for genome sequence assembly, and will be of intrinsic value to comparative mapping efforts aimed at understanding the molecular evolution of reptilian, as well as other amniote genomes.
From an economic perspective, crocodilians play an important role in modern agriculture, as well as forming a basis for tourism, with management programs in more than 40 nations worldwide [21
]. The Australian crocodile industry produces farmed saltwater crocodiles (Crocodylus porosus
) for the international skin trade. Although still an emerging livestock industry, the Australian crocodile industry, following the lead of other livestock industries, has recently developed a comprehensive genetic improvement program [22
]. Research efforts have thus far focused on genetic and phenotypic parameter estimation for selection objectives and selection criteria required for multi-trait index selection [22
]. However, this type of animal selection occurs with little or no knowledge of what is occurring at the DNA level. One of the major limitations to performance-based selection in crocodiles is the large generation interval, which is estimated to be 13 years [22
]. To improve the rate of genetic gain currently achieved in the industry, particularly for traits that are difficult to measure such as disease resistance and sex limited traits, as well as other complex traits such as growth rate, animal survival and skin quality, trait-linked DNA markers will be necessary. Animal selection employing marker information will increase the rate of genetic gain by permitting early selection decisions to be made on large animal resources, thereby both increasing selection intensity and reducing the generation interval. The availability of a comprehensive linkage map, with markers evenly spaced across the genome, will facilitate the systematic searches necessary to identify genes affecting traits of economic importance, with the potential to incorporate marker information into the animal selection process using marker assisted selection (MAS) [27
]. Accordingly, a genetic linkage map for the saltwater crocodile will have both scientific and commercial benefits.
Evidence of genetic linkage between ten microsatellites was previously reported for the saltwater crocodile by Isberg et al. [29
] based on a limited number of genetic markers developed by Fitzsimmons et al. [30
]. However, Miles et al. (2009a) [31
] have since developed 253 novel polymorphic microsatellite markers for saltwater crocodiles, thus providing a sufficiently large marker resource for genome mapping. Besides the previous lack of suitable markers, another major reason why there are no genome maps for the Order Crocodylia is the difficulty in breeding informative predigrees from which DNA resources can be developed. Indeed, the lack of pedigreed animals has thus far thwarted efforts to generate a linkage map for other model reptiles, such as Anolis
. Fortunately, the use of unitized breeding pens, coupled with detailed pedigree records in the Australian crocodile industry, has provided the pedigrees and complementary DNA resources necessary for the construction of the first crocodilian genetic map.