Whole genome resequencing reveals diagnostic markers for investigating global migration and hybridization between minke whale species.

BACKGROUND: In the marine environment, where there are few absolute physical barriers, contemporary contact between previously isolated species can occur across great distances, and in some cases, may be inter-oceanic. An example of this can be seen in the minke whale species complex. Antarctic minke whales are genetically and morphologically distinct from the common minke found in the north Atlantic and Pacific oceans, and the two species are estimated to have been isolated from each other for 5 million years or more. Recent atypical migrations from the southern to the northern hemisphere have been documented and fertile hybrids and back-crossed individuals between both species have also been identified. However, it is not known whether this represents a contemporary event, potentially driven by ecosystem changes in the Antarctic, or a sporadic occurrence happening over an evolutionary time-scale. We successfully used whole genome resequencing to identify a panel of diagnostic SNPs which now enable us address this evolutionary question. RESULTS: A large number of SNPs displaying fixed or nearly fixed allele frequency differences among the minke whale species were identified from the sequence data. Five panels of putatively diagnostic markers were established on a genotyping platform for validation of allele frequencies; two panels (26 and 24 SNPs) separating the two species of minke whale, and three panels (22, 23, and 24 SNPs) differentiating the three subspecies of common minke whale. The panels were validated against a set of reference samples, demonstrating the ability to accurately identify back-crossed whales up to three generations. CONCLUSIONS: This work has resulted in the development of a panel of novel diagnostic genetic markers to address inter-oceanic and global contact among the genetically isolated minke whale species and sub-species. These markers, including a globally relevant genetic reference data set for this species complex, are now openly available for researchers interested in identifying other potential whale hybrids in the world's oceans. The approach used here, combining whole genome resequencing and high-throughput genotyping, represents a universal approach to develop similar tools for other species and population complexes.

Hybrids between common and Antarctic minke whales are fertile and can back-cross.

BACKGROUND: Minke whales are separated into two genetically distinct species: the Antarctic minke whale found in the southern hemisphere, and the common minke whale which is cosmopolitan. The common minke whale is further divided into three allopatric sub-species found in the North Pacific, southern hemisphere, and the North Atlantic. Here, we aimed to identify the genetic ancestry of a pregnant female minke whale captured in the North Atlantic in 2010, and her fetus, using data from the mtDNA control region, 11 microsatellite loci and a sex determining marker. RESULTS: All statistical parameters demonstrated that the mother was a hybrid displaying maternal and paternal contribution from North Atlantic common and Antarctic minke whales respectively. Her female fetus displayed greater genetic similarity to North Atlantic common minke whales than herself, strongly suggesting that the hybrid mother had paired with a North Atlantic common minke whale. CONCLUSION: This study clearly demonstrates, for the first time, that hybrids between minke whale species may be fertile, and that they can back-cross. Whether contact between these species represents a contemporary event linked with documented recent changes in the Antarctic ecosystem, or has occurred at a low frequency over many years, remains open.

Genotyping errors in a calibrated DNA register: implications for identification of individuals.

BACKGROUND: The use of DNA methods for the identification and management of natural resources is gaining importance. In the future, it is likely that DNA registers will play an increasing role in this development. Microsatellite markers have been the primary tool in ecological, medical and forensic genetics for the past two decades. However, these markers are characterized by genotyping errors, and display challenges with calibration between laboratories and genotyping platforms. The Norwegian minke whale DNA register (NMDR) contains individual genetic profiles at ten microsatellite loci for 6737 individuals captured in the period 1997-2008. These analyses have been conducted in four separate laboratories for nearly a decade, and offer a unique opportunity to examine genotyping errors and their consequences in an individual based DNA register. We re-genotyped 240 samples, and, for the first time, applied a mixed regression model to look at potentially confounding effects on genotyping errors. RESULTS: The average genotyping error rate for the whole dataset was 0.013 per locus and 0.008 per allele. Errors were, however, not evenly distributed. A decreasing trend across time was apparent, along with a strong within-sample correlation, suggesting that error rates heavily depend on sample quality. In addition, some loci were more error prone than others. False allele size constituted 18 of 31 observed errors, and the remaining errors were ten false homozygotes (i.e., the true genotype was a heterozygote) and three false heterozygotes (i.e., the true genotype was a homozygote). CONCLUSIONS: To our knowledge, this study represents the first investigation of genotyping error rates in a wildlife DNA register, and the first application of mixed models to examine multiple effects of different factors influencing the genotyping quality. It was demonstrated that DNA registers accumulating data over time have the ability to maintain calibration and genotyping consistency, despite analyses being conducted on different genotyping platforms and in different laboratories. Although errors were detected, it is demonstrated that if the re-genotyping of individual samples is possible, these will have a minimal effect on the database's primary purpose, i.e., to perform individual identification.

Investigating population genetic structure in a highly mobile marine organism: the minke whale Balaenoptera acutorostrata acutorostrata in the North East Atlantic.

Inferring the number of genetically distinct populations and their levels of connectivity is of key importance for the sustainable management and conservation of wildlife. This represents an extra challenge in the marine environment where there are few physical barriers to gene-flow, and populations may overlap in time and space. Several studies have investigated the population genetic structure within the North Atlantic minke whale with contrasting results. In order to address this issue, we analyzed ten microsatellite loci and 331 bp of the mitochondrial D-loop on 2990 whales sampled in the North East Atlantic in the period 2004 and 2007-2011. The primary findings were: (1) No spatial or temporal genetic differentiations were observed for either class of genetic marker. (2) mtDNA identified three distinct mitochondrial lineages without any underlying geographical pattern. (3) Nuclear markers showed evidence of a single panmictic population in the NE Atlantic according STRUCTURE's highest average likelihood found at K = 1. (4) When K = 2 was accepted, based on the Evanno's test, whales were divided into two more or less equally sized groups that showed significant genetic differentiation between them but without any sign of underlying geographic pattern. However, mtDNA for these individuals did not corroborate the differentiation. (5) In order to further evaluate the potential for cryptic structuring, a set of 100 in silico generated panmictic populations was examined using the same procedures as above showing genetic differentiation between two artificially divided groups, similar to the aforementioned observations. This demonstrates that clustering methods may spuriously reveal cryptic genetic structure. Based upon these data, we find no evidence to support the existence of spatial or cryptic population genetic structure of minke whales within the NE Atlantic. However, in order to conclusively evaluate population structure within this highly mobile species, more markers will be required.

Migration of Antarctic minke whales to the Arctic.

The Antarctic minke whale (Balaenoptera bonaerensis), and the common minke whale found in the North Atlantic (Balaenoptera acutorostrata acutorostrata), undertake synchronized seasonal migrations to feeding areas at their respective poles during spring, and to the tropics in the autumn where they overwinter. Differences in the timing of seasons between hemispheres prevent these species from mixing. Here, based upon analysis of mitochondrial and microsatellite DNA profiles, we report the observation of a single B. bonaerensis in 1996, and a hybrid with maternal contribution from B. bonaerensis in 2007, in the Arctic Northeast Atlantic. Paternal contribution was not conclusively resolved. This is the first documentation of B. bonaerensis north of the tropics, and, the first documentation of hybridization between minke whale species.

Forensic identification of severely degraded Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) tissues.

BACKGROUND: Aquaculture is a globally important and rapidly growing industry. It contributes positively to the economy and sustainability of coastal communities, but it is not without regulatory challenges. These challenges are diverse, and may include identification of fish discarded in an illegal manner, biological discharge from fish ensilage tanks, and partially destroyed or processed tissues. Robust genetic tools are required by management authorities to address these challenges. In this paper, we describe nine species-specific primer sets amplifying very short DNA fragments within the mitochondrial DNA cytochrome c oxidase (COI) gene, which were designed to permit diagnostic identification of degraded DNA from two of the most commonly farmed salmonids in Europe and North America. RESULTS: Of the nine designed primer sets, six were found to be species-specific (four Atlantic salmon, two rainbow trout), whereas the remaining three sets (two Atlantic salmon, one rainbow trout) also amplified a product from other, closely related, salmonid DNA templates. Screening of DNA templates from 11 other non-salmonid native fish species did not produce PCR products with any of the primer sets. Specific tests confirmed the ability of these markers to identify Atlantic salmon and rainbow trout tissues in treated food products, chemically treated ensilage waste and fillets left to degrade in saltwater for up to 31 days at 15°C. Importantly, these markers provided diagnostic identification in cases where other genetic methods failed because of degraded DNA quality. CONCLUSIONS: Results from this study demonstrate that amplification of very short DNA fragments using species-specific primers represents a robust and versatile method to create cheap and efficient genetic tests that can be implemented in a range of forensic applications. These markers will provide fishery, aquaculture and food regulatory authorities with a method to investigate and enforce regulations within these industries.