Nanopore Technology for Amplicon Sequencing to Investigate DNA Polymorphism of Glutamic-Oxaloacetic Transaminase 1 (GOT1), a Candidate Gene for Wooden Breast Disease in Commercial Broiler Chickens

• Madelyn Winzig, Agriculture and Natural Resources, University of Delaware

• Behnam Abasht, Plant and Animal Genetics, University of Delaware

Wooden Breast Disease (WBD) mainly impacts commercial broiler chickens. This myopathy results in reduced animal welfare and meat quality. Leading to major economic loss in the poultry industry. Genetic selection for fast growth rates and high muscle yield has been the driving force behind the disease developing. WBD is characterized by the hardening of breast muscle tissue and white striations running through the muscle tissue upon necroscopy. Metabolic imbalances caused by suspected genes result in these irregularities. To identify genes linked with WBD, DNA was isolated then amplified through a polymerase chain reaction. Our research focused on DNA that was isolated from eight hatches of mainly broiler chickens, one hatch containing samples from hens and roosters. Each hatch contained 96 blood samples that were broken down and cleaned with buffers and DNeasy filtration plates. Primers were designed through Ensembl, Primer 3 and Primer Design to specifically target our gene of focus: GOT1. Glutamic- Oxaloacetic transaminase 1 (GOT1) is a target gene for WBD. This gene provides balance between glutamate and aspartate between the cytosol and mitochondria. Which is vital to production of amino acids. The primers cut sequences of 5000 base pairs to amplify the presence of the gene through pairs of reverse and forward primers. A thermocycler performs a polymerase chain reaction (PCR) through rapidly heating and cooling the samples. DNA segments specific to the primers were amplified for subsequent use creating PCR products. PCR products are loaded into gel electrophoresis, which allows for specific DNA band visualization through migration of the negatively charged DNA sequences. Based on the location of the sequence, its length can be verified, providing additional information on the gene’s position. Further data analysis and DNA sequencing can be performed to pinpoint the exact location of the gene. Giving insight on where to focus gene editing and selection.