Development of a multiplex PCR assay for parentage assignment of the redclaw crayfish (Cherax quadricarinatus)

Abstract

Cherax quadricarinatus, commonly known as the redclaw crayfish, possesses enormous commercial benefits. To prevent germplasm degradation of C. quadricarinatus populations, there is an urgent need to protect its germplasm resources. One of the important links in the breeding of improved varieties is family selection. In this study, we screened for microsatellite markers based on the whole genome sequence of C. quadricarinatus. The obtained polymorphic tetranucleotide microsatellite genetic markers were combined with capillary electrophoresis to establish an experimental system suitable for parentage assignment of C. quadricarinatus. Sixty polymorphic tetrameric microsatellite markers with stable amplification were screened from the reference genome sequence of C. quadricarinatus. By continuously optimizing the annealing temperature, primer concentration, and cycle numbers of the combination, 20 tetranucleotide microsatellite loci with good amplification effect and a high polymorphism information content (PIC) were obtained. Multiplex PCR systems were then constructed using five groups of microsatellites. Parentage assignment in 12 families of C. quadricarinatus with known pedigree information was carried out using CERVUS 3.0 software. The results showed that 112 alleles were amplified from the 20 loci. The observed heterozygosity (Ho) ranged from 0.458 to 0.917. The expected heterozygosity (He) ranged from 0.435 to 0.832 The PIC ranged from 0.375 to 0.788. Most of the microsatellite markers were highly polymorphic and could meet the requirements for parentage assignment. Using any four combinations, the actual successful assignment rates ranged from 97.20% to 99.30%, and when using all five combinations, the assignment rate reached 100%. Therefore, selecting these sets could not only obtain accurate pedigree information but also would reduce the workload and cost. The microsatellite multiplex PCR system constructed in this study provides a convenient and useful method for molecular pedigree construction, population breeding, and family management of C. quadricarinatus, thus forming a basis to ensure the normal development of this important freshwater crustacean.