Resolving issues related to target-site resistance detection in Poa annua alpha-tubulin

Abstract

Advanced sequencing techniques have improved the ability to identify and understand target-site resistance in herbicide-resistant species. Despite innovations in sequencing, polyploid species can still face issues that are typically not seen in diploid species, often because of the presence of conflicting subgenomes. Further confounding the difficulties of polyploidy is the α-tubulin gene, which has subgenomic duplication of gene family copies. Poa annua L., an allotetraploid, is a persistent weed in turfgrass that has developed resistance to multiple herbicide modes of action, including mitotic-inhibiting herbicides. Sequencing α-tubulin cannot be performed by simple Sanger sequencing to identify target-site mutations because of frequent nucleotide conflictions between the numerous α-tubulin copies present on both subgenomes, which was illustrated by vector cloning in combination with Sanger. Improved sequencing is needed to understand resistance to mitotic-inhibiting herbicides. Amplicon sequencing via Illumina technology was used to identify target-site mutations. Eighty-two populations resistant to mitotic-inhibiting herbicides were sequenced via next-generation sequencing. Seventy-five populations presented with the known single nucleotide polymorphism, Thr239Ile. The ability to successfully sequence and analyze α-tubulin data provides a vehicle for further insight into herbicide resistance.