Demographic, environmental and genetic determinants of mating success in captive koalas (Phascolarctos cinereus).

Many factors have been shown to affect mating behavior. For instance, genes of the major histocompatibility complex (MHC) are known to influence mate choice in a wide variety of vertebrate species. The genetic management of captive populations can be confounded if intrinsic mate choice reduces or eliminates reproductive success between carefully chosen breeding pairs. For example, the San Diego Zoo koala colony only has a 45% copulation rate for matched individuals. Herein, we investigated determinants of koala mating success using breeding records (1984-2010) and genotypes for 52 individuals at four MHC markers. We quantified MHC diversity according to functional amino acids, heterozygosity, and the probability of producing a heterozygous offspring. We then used categorical analysis and logistic regression to investigate both copulation and parturition success. In addition, we also examined age, day length, and average pairwise kinship. Our post-hoc power analysis indicates that at a power level of 1-ß = 0.8, we should have been able to detect strong MHC preferences. However, we did not find a significant MHC effect on either copulation or parturition success with one exception: pairs with lower or no production of a joey had significantly lower MHC functional amino acid diversity in the categorical analysis. In contrast, day length and dam age (or age difference of the pair) consistently had an effect on mating success. These findings may be leveraged to improve the success of attempted pairs, conserve resources, and facilitate genetic management.

Immunomics of the koala (Phascolarctos cinereus).

The study of the koala transcriptome has the potential to advance our understanding of its immunome--immunological reaction of a given host to foreign antigens--and to help combat infectious diseases (e.g., chlamydiosis) that impede ongoing conservation efforts. We used Illumina sequencing of cDNA to characterize genes expressed in two different koala tissues of immunological importance, blood and spleen. We generated nearly 600 million raw sequence reads, and about 285 million of these were subsequently assembled and condensed into ~70,000 subcomponents that represent putative transcripts. We annotated ~16% of these subcomponents and identified those related to infection and the immune response, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), major histocompatibility complex (MHC) genes, and koala retrovirus (KoRV). Using phylogenetic analyses, we identified 29 koala genes in these target categories and report their concordance with currently accepted gene groups. By mapping multiple sequencing reads to transcripts, we identified 56 putative SNPs in genes of interest. The distribution of these SNPs indicates that MHC genes (34 SNPs) are more diverse than KoRV (12 SNPs), TLRs (8 SNPs), or RLRs (2 SNPs). Our sequence data also indicate that KoRV sequences are highly expressed in the transcriptome. Our efforts have produced full-length sequences for potentially important immune genes in koala, which should serve as targets for future investigations that aim to conserve koala populations.

Of contigs and quagmires: next-generation sequencing pitfalls associated with transcriptomic studies.

Molecular ecologists have good reasons to be excited about the newest DNA/RNA sequencing technologies. However, this exuberance should be tempered with a hefty dose of reality: new sequencing technologies come with significant new challenges. Herein, we offer a brief overview of some practical problems encountered during transcriptomics studies conducted in our laboratory, and of nontrivial issues that prospective practitioners should consider. These include template contamination (e.g. from xenobiotics) and the cutting-room floor problem, whereby most of the data are often unassembled, unannotated and unused. We also highlight computational requirements, including hardware, personnel time and associated skill sets. We are very optimistic about the future of molecular ecology, but we hope this cautionary overview will help neophytes better recognize some key challenges associated with new technologies.