A High-Throughput Genotyping Workflow and an Optimized SSR Panel for High-Accuracy Parentage Analysis in Largemouth Black Bass

Abstract

Accurate pedigree information is paramount for genetic improvement programs in commercially important aquaculture species such as Largemouth black bass (Micropterus salmoides). Simple Sequence Repeats (SSRs) are highly polymorphic markers well-suited for parentage analysis, yet their application has been hampered by labor-intensive traditional genotyping methods. This study reports the development, validation, and optimization of a high-throughput SSR genotyping system for Largemouth black bass. We designed six 10-plex SSR panels, comprising 60 markers, by integrating MultiplexSSR for efficient primer design, Hi-TOM sequencing for targeted amplicon generation, and the SSRseq pipeline for automated and accurate genotyping. The developed SSRs exhibited moderate polymorphism (mean polymorphic information content, PIC = 0.396; mean number of alleles, Na = 2.48). Theoretical assignment power (P_u) analyses indicated that combinations of ≥ 30 markers achieved P_u values approaching 1.000 under ideal conditions. Empirical assignment accuracy mirrored this, exceeding 95% with ≥ 30 markers when no additional candidate parents were considered. While both P_u and accuracy were impacted by an increasing number of potential parents, a critical finding was that the targeted removal of eight problematic loci—identified via rigorous quality control thresholds for null allele frequencies and Mendelian inconsistencies—restored assignment accuracy from 90.77% to 100.00% in the in-sample validation. This retrospective optimization highlights the substantial impact of genotyping errors on parentage inference, although out-of-sample validation across independent cohorts is required. This resultant optimized 52-marker panel provides a robust tool, demonstrating that marker quality is a decisive factor for efficient, scalable, and precise SSR-based parentage assignment in Largemouth black bass.