Cloning of Toll3 and Toll4 and association analysis among their SNP haplotypes and disease resistance in red swamp crayfish (Procambarus clarkii)

With the expansion of the culture scale of red swamp crayfish (Procambarus clarkii), the high incidence of diseases has seriously threatened the development of its industry. In this study, PcToll3 and PcToll4 were respectively cloned and explored SNPs among the germplasm populations, which had been identified relating to disease resistance in crayfish based on our previous study. A total of 3036 bp and 2820 bp of the open reading frame of PcToll3 and PcToll4 encoded 1011 and 939 amino acids, respectively. They were specially expressed in haemolymph, and significantly up-regulated expression after stimulation by Vibrio parahaemolyticus, Aeromonas hydrophila and white spot syndrome virus. It was found that the expression of downstream genes PcALF, PcCru, PcIMD, PcMyD88, and PcNF-κB were repressed after interference of PcToll3 and/or PcToll4. Totally, 16 and 19 SNPs in the coding region of PcToll3 and PcToll4 were mined, and the favoured haplotypes and the combinations of them were classified according to the associated SNPs with the disease resistance in crayfish. The haplotypes of Toll3-Hap1, Toll4-Hap1 and the combination of Toll3+Toll4-Hap1 were further validated that they had the stronger disease resistance comparing to others haplotypes, and the related KASP markers were developed for further breeding application. This study will advance our understanding of the function of the two Toll genes in crayfish, and provide the markers for the molecular breeding.