Development of a non-lethal genetic-based method for aging Pacific halibut
Investigators
NOAA Fisheries
Overview
The issue
For many managed groundfish species, age has been traditionally estimated by manually counting the number of annuli in sagittal otoliths. For Pacific halibut, otolith-based methods for aging Pacific halibut are accurate and have been validated by using bomb radiocarbon dating. However, these have several shortcomings: (1) they cannot be used in fish that are not sacrificed, given that the otolith is a terminal (i.e. lethal) sample; (2) they require considerable manual processing and analysis of stored otoliths by trained personnel e.g., age readers) that could be difficult to retain and/or recruit in the future; and (3) they cannot be automated in a high-throughput manner to improve processing time and efficiency and to reduce potential human error and overall cost. DNA methylation-based aging is a well-established alternative aging method in fish species. This method is based on the known observation that the methylation patterns in DNA change predictably with age. Age-associated DNA methylation patterns can be modelled to generate molecular age predictors (known as “epigenetic clocks”) capable of estimating chronological age with high accuracy and can be developed from DNA isolated from any tissue, including non-lethal biological samples, such as a fin clip. The potential applicability for Pacific halibut is high given that non-otolith genetic samples (e.g., fin clips) that can be used for DNA methylation-based aging are also being used for other genetic applications, from genotyping for sex to investigating genetic variation in genomic population studies informing management and conservation strategies of Pacific halibut. Therefore, age information can be added to sex and other genetic information that is being collected for Pacific halibut to better understand population dynamics, support stock assessment, and evaluate possible responses to the Pacific halibut population in the face of climate change.
Objectives
The main goal of this proposal is to develop a genetic method for aging Pacific halibut using fin tissue, a sample that can be easily collected from either live or dead individuals. This method is based on the identification of DNA methylation patterns in fin tissue that are associated with age through the development of an age estimation model (i.e., an epigenetic clock) for Pacific halibut. The most informative DNA methylation patterns for aging will then be used to develop a targeted multiplex PCR assay (e.g., GT-seq), that is a cost-effective and high-throughput method. Although the proposed method represents an alternative to traditional aging methods (otolith or scale aging) and not a replacement, it will improve stock assessment by increasing the number of aged individuals used for estimating the age composition of the Pacific halibut population. Fin clips can be collected with little training and in areas not covered by IPHC field staff collecting otoliths. Since the proposed method is non-lethal it will provide age information to research projects and applications involving live Pacific halibut, including migration and discard survival studies using tags or image recognition, captive studies, etc., allowing for the first time to relate age to specific life history characteristics in live fish. Furthermore, the proposed method could provide the means to estimate the age composition of Pacific halibut discarded by non-directed fisheries (e.g., trawl).
How will researchers conduct their study?
DNA methylation patterns in Pacific halibut will be investigated by performing genome-wide DNA methylation at single base-pair resolution using reduced representation bisulfite sequencing (RRBS) by leveraging the high-quality genome assembly available for Pacific halibut. This is an efficient and cost-efficient method to identify methylation patterns (i.e., CpG sites) in DNA because it targets bisulfite sequencing to a well-defined set of genomic regions with high CpG density that can be sequenced at high read depth. Age-associated DNA methylation patterns will be modelled to generate an epigenetic age predictor (i.e., epigenetic clock) for Pacific halibut constructed using elastic net penalized regression models that select a group of CpG sites that have a monotonically increasing relationship with age in the selected training data set. By implementing these linear models that select and weight age-correlated CpG sites, chronological age of Pacific halibut will be estimated based on the percentage methylation at these key CpG sites in fin tissue samples. CpG sites that best correlated with age will be selected for the development of a cost-effective and high-throughput targeted multiplex PCR assay (GT-seq).
