Detection of Chinook Salmon Environmental DNA with CRISPR Technology

Presented by: Tholen Blasko
Graduate Student
Phelps’ Lab

Abstract

Environmental DNA (eDNA), DNA released from an organism into the environment, is a non-intrusive tool for monitoring fish species such as the ESA-listed Chinook salmon (Oncorhynchus tshawytscha). Restoration projects aiming to restore Chinook require an accurate assessment of recolonization. Environmental DNA is used for monitoring Chinook recolonization and migrations, but current methods require a quantitative polymerase chain reaction (qPCR) in a lab to identify the presence or absence of a target species. While streamside eDNA collection takes only minutes, sample processing time may take weeks to months. Thus, a rapid and field-deployable eDNA processing system could save researchers resources and time. We developed a CRISPR Cas12a-based assay to detect the presence or absence of Chinook salmon mitochondrial DNA (mtDNA) using the unique sequence of that species.  An initial loop-mediated isothermal amplification method (LAMP) was developed to amplify a selective region of all salmonid mtDNA.  Chinook salmon samples are then confirmed by using specific CRISPR “guide” RNA sequences engineered to only identify the target species.  Field assays utilize a portable incubator to perform a one-tube LAMP amplification, followed by a CRISPR digest, with results analyzed on a lateral flow strip. The assay was piloted on eDNA samples collected from the Snake River and the South Fork Palouse River which have or do not have migratory Chinook salmon populations, respectively. The assay accurately identified expected Chinook presence or absence. Further work on this project will include optimization of specific assays for the salmon, trout and char species found in Washington State.

Prioritizing candidate genes for fertility in dairy cows using gene-based analysis, functional annotation and differential gene expression

Presented by: Victoria Kelson
Graduate Student
Neibergs’ Lab

Abstract

Dairy infertility results in economic losses and increased culling. A lack of selection for fertility has increased infertility in dairy cattle until recently (2006) when selection for fertility became a higher priority. Identifying additional fertility loci could further enhance the genetic gains made to improve reproductive performance in dairy cattle. This study aimed to identify loci associated with fertility traits by performing a genome-wide association analysis (GWAA). A two-step procedure was used to impute single nucleotide polymorphisms (SNP) from the Illumina (San Diego, CA) Bovine 50K BeadChip to a density of 778,000 SNP using IMPUTE2. The second imputation to whole genome sequence (WGS) level was performed using MiniMac2. The WGS GWAA was performed using genome wide complex trait analysis software with genotypes of 5,038 Nordic Holstein bulls. After filtering for minor allele frequencies < 0.5% and SNPs that failed Hardy-Weinberg equilibrium (P< 1×10^-6), associations with fertility were defined by SNPs that met Bonferroni’s multiple testing correction of (P≥ -log₁₀ 8.5). Seven loci were associated with female fertility traits and their positional candidate genes identified. Positional candidate genes identified in this study and found to be differentially expressed in a previous study of fertile, sub-fertile and infertile cattle were ADCY5, DIRC2, ITGBS, KARLRN, MYLK, and SEMA5B (BTA1), FRAS1 (BTA6), OTX2 (BTA10) and KLF6 (BTA13). This study validated fertility loci in Nordic Holstein cattle that were previously identified to be associated with fertility in cattle previously. Understanding genes associated with bovine reproduction could lead to more intense selection for fertility.

Authors: Zexi Cai, Bernt Guldbrandtsen, Mogens Sandø Lund, & Goutam Sahana