Genetic markers (SNPs) for sex identification

Trends in the average size of Pacific halibut over time - in concert with changes in sex ratio and age distributions as cohorts move through the fishery - can have substantial effects on the nature of fishery landings and our ability to properly characterize the stock. Through approximately 1985, male and female Pacific halibut entered the commercial halibut fishery on average roughly at the same age: in the central Gulf of Alaska (GOA), males recruited to the fishery at approximately age-7 and females at age-8. As a result, commercial catches were composed of sex ratios relatively similar to underlying abundance (Clark 2004). However, between the mid-1980s and mid-1990s, mean size-at-age declined across the population (Clark et al. 1999) and led to the concern that commercial catches were becoming increasingly sex-biased. By 2009, the average age for exploitation of the female stock component in most areas of the fishery was estimated to be three years younger than that of males (Hare and Clark 2009), and it was further suggested the male stock component would not reach full fishery selectivity (Clark and Hare 2006). Understanding the annual contribution of both sexes to the commercial harvest is important for predicting population dynamics and setting catch limits. However, halibut are eviscerated at sea and therefore the collection of sex data has not historically been possible within the context of the IPHC’s commercial port sampling program. As such, in 2007 the IPHC began investigating the use of genetic methods for determining the sex of Pacific halibut.

Originally envisioned as an independent method for sexing commercial catch, the IPHC’s genetic sex-identification research evolved through three phases to currently represent a component of an integrated commercial sampling program that may include physical marking of the landed catch in conjunction with genetically-based validation. The work began with the discovery in 2006 – quite by accident – of sex-linked microsatellite markers during the course of population genetics research; it advanced in 2010 to include a formal test of the relative accuracy of genetic versus statistical methods for determining sex ratios in commercial catches; and in 2017 saw the completion of the development of an assay for sex that is based on single-nucleotide polymorphisms (SNPs).

The initial discovery of sex-associated alleles 

In the course of conducting population genetic analyses, a chance discovery occurred: out of sixteen presumably neutral microsatellites being examined for geographic variation in their occurrence in Pacific halibut, three exhibited strong association with sex. Microsatellites are regions within the genetic code that lie between “coding regions” and are composed of repetitive sequences of DNA. Because they typically do not code for protein production, they are free to occur in nearly any size (although, once large enough they are referred to as either “minisatellites” or “macrosatellites”, depending upon repeat-length) without negatively impacting the organism’s function, and are therefore often subject to high mutation rates. This can make them highly variable within a population and ideal candidates for a variety of genetic applications, including population genetics. The loci discovered in Pacific halibut were originally identified in Atlantic halibut (Hippoglossus hippoglossus) but apparently showed no such correlation to sex in that species, presumably because their variability is very low in that species (Reid et al. 2007). Although the discovery was completely unexpected and presumably highly unlikely in the course of such a small genetic survey, the discovery of these markers opened the door for development of genetic tests for sex in Pacific halibut. Although familiarity with sex-determination in humans and other mammals would suggest that this was no great breakthrough, this was an exciting discovery because sex is a far more ambiguous attribute in fishes. In fishes, sex is often under the control of external environmental factors, can be influenced by complex interactions among multiple genetic loci, and may change during the course of an individual’s lifetime (see review in Devlin and Nagahama 2002).

To further investigate the nature of these loci and evaluate the likelihood that sex is determined by a simple genetic system in Pacific halibut – and therefore amenable to genetic testing – the three sex-linked loci were screened in 550 individuals spanning the geographic range from British Columbia to the central Aleutian Islands, and across nine year classes (Galindo et al. 2011). The genetic sex assignments were 92% accurate, with all but two females containing at least one copy of a characteristic allele. The pattern led to the hypothesis that female Pacific halibut are the heterogametic sex; that is, they possess two different sex chromosomes, much in the same way that human males contain an X and a Y chromosome. The result was very promising in suggesting that accurate genetic sexing may be possible in Pacific halibut; but, at the same time, less accurate than would be ideal, because a fair number of males contained a copy of an allele that was otherwise considered diagnostic for females.

Comparison of genetic and statistical methods

As an initial step in the attempt to accurately partition commercial catch in a sex-specific manner, in 2004 the IPHC developed a statistical method for estimating sex ratios at size and length, based on sex ratios observed in longline survey data (Clark 2004). However, concerned that survey and commercial catches may not perfectly reflect one another, the IPHC sought to compare the sex ratios that would be estimated using the statistical technique (Clark 2004) to genetic testing (Galindo et al. 2011), primarily as a means to evaluate the need for further development of genetic techniques. In 2010, the IPHC’s summer intern – Monica Woods – was placed aboard longline vessels in IPHC Regulatory Areas 2B and 3A in order to obtain length, sex, tissue samples, and otoliths from commercial catches prior to landing. Sampling was continued in 2011, again collecting samples from Area 2B, as well as from Areas 3B, 4A, 4C, and 4D. Ultimately, it was found that statistically-derived sex ratios were more variable than genetically-based estimates, relative to the true ratios within those catches. Furthermore, the results provided evidence that the statistical method is more accurate in characterizing commercial catches during the summer than during spring and fall. This is presumably due to seasonal migration causing the population to shift and underlying dynamics to change in spring and fall, relative to survey data that are collected in summer. In summary, the work (detailed in Loher et al. 2016) indicated that continuing to seek direct observations of sex in the commercial fishery and refining genetic-sexing techniques is warranted.

Development of SNP-based sex identification

From 2015-2017 laboratory work was conducted to identify genetic markers that would be highly diagnostic for sex and to develop a simple laboratory assay that will allow for large numbers of samples to be screened at relatively low cost. Although microsatellites proved promising, they were not 100% diagnostic for sex and in general microsatellites can be difficult and relatively expensive to isolate. To overcome these issues, a new technique was used that utilizes genetic markers known as “single nucleotide polymorphisms” (SNPs): one of the most common forms of natural genetic variation. 

In brief, genetic code is written in a very simple “language” that is composed of four unique DNA building-blocks known as nucleotides: adenine and thymine; guanine and cytosine. Every protein code that is written into the DNA sequence is represented by a series of these nucleotides, in which adenine and guanine are essentially synonyms, as are guanine and cytosine. As such, whether a specific position in the code is occupied by one of the synonyms or its counterpart makes no difference; either way, the code will be fully functional and specify the same protein. The result is that different individuals can have codes that are functionally identical (i.e., make the same proteins) while being written somewhat differently. At each position in which some individuals possess one of the synonym nucleotides and other individuals the other nucleotide, a SNP is said to have occurred. Given the extremely large size of most genomes (i.e., the full genetic code; on the order of hundreds of millions to hundreds of billions of base-pairs in fishes), the potential for SNP variation is extensive.

For Pacific halibut, SNPs were identified using restriction-site associated DNA sequencing (RADseq) techniques (Baird et al. 2008) resulting in the identification of more than 40,000 genetic loci. Examination of the loci demonstrated considerable similarity between Pacific halibut and two other flatfish: the closely-related Atlantic halibut (Hippoglossus hippoglossus) and the more distantly-related half-smooth tongue sole (Cynoglossus semilaevis). Within the Pacific halibut genome, 70 SNPs were identified that are linked to sex and the three most-diagnostic were chosen for further assay development. These three SNPs are present in females and all occur on a single chromosome, consistent with the ZW sex-determination system that was first hypothesized for the Pacific halibut on the basis of nuclear microsatellites and suggest that the sex-determining chromosome has likely been identified.

For two selected SNPs, TaqMan® assays (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were developed, and their suitability for use with both ethanol-preserved tissue and samples that are affixed to chromatography paper and stored dry was verified. The latter is highly desirable as dry collection is often easier in field settings, especially when samples need to be shipped across state and international borders, as occurs routinely for port-collected biological samples.

For additional information on this component, please see Drinan et al. (2017); note also that a manuscript fully describing the work is currently in peer review.

Linkage to the IPHC’s commercial sex-marking project

In 2014, the IPHC initiated a project intended to lead to direct observation of sex as a routine element of its ongoing Port Sampling program. The project includes at-sea sex-marking in the directed longline fishery, to be accompanied by genetic methods to validate the sex-marking. For an overview of the at-sea sex-marking project, please see this page: Commercial sex marking and genetic sex identification.

References

Clark, W.G.  2004.  A method of estimating the sex composition of commercial landings from setline survey data.  Int. Pac. Halibut Comm. Rep. Assessment Res. Activ. 2003: 111-162.

Clark, W.G., Hare, S.R.  2006.  Assessment and management of Pacific halibut: data, methods, and policy. Int. Pac. Halibut Comm. Rep. Sci. Rep. 83.

Clark, W.G., Hare, S.R., Parma, A.M., Sullivan, P.J., Trumble, R.J.  1999.  Decadal changes in growth and recruitment of Pacific halibut (Hippoglossus stenolepis). Can. J. Fish. Aquat. Sci. 56: 242-252.

Devlin, R.H., Nagahama, Y.  2002.  Sex determination and sex differentiation in fish: an overview of genetic, physiological and environmental factors.  Aquaculture 208: 191-364.

Drinan, D.P., Hauser, L., Loher, T.  2017.  Development of production-scale genetic sexing techniques for routine catch sampling of Pacific halibut.  Int. Pac. Halibut Comm. Rep. Assessment Res. Activ. 2016: 511-526.

Galindo, H.M., Loher, T., Hauser, L.  2011.  Genetic sex identification and the potential evolution of sex determination in Pacific halibut (Hippoglossus stenolepis).  Mar. Biotechnol. 13: 1027-1037.  doi: 10.1007/s10126-011-9366-7

Hare, S.R., Clark, W.G.  2009.  Assessment of the Pacific halibut stock at the end of 2008. Int. Pac. Halibut Comm. Rep. Assessment Res. Activ. 2008: 137-201.

Loher, T., Woods, M.A., Jimenez-Hidalgo, I., and Hauser, L.   2016.  Variance in age-specific sex ratios of Pacific halibut catches and comparison of statistical and genetic methods for reconstructing sex ratios.  J. Sea Res. 107: 90-99.  doi: 10.1016/j.seares.2015.06.004