Parabotia fasciatus unigene sequences for SSR primers
Genomic RNA was extracted from the P. fasciata intestine collected from Wuhan (Hubei, China), and was sent to Biomarker Technologies Co. Ltd (Beijing, China) for transcriptomic sequencing using Illumina platform. Simple repeat motifs in the DNA sequence were detected and a total of 132 pairs of SSR primers were then designed by MISA software (Beier et al. 2017) according to the following criteria: the size of the PCR product ranges from 130 to 300 bp; the Tm value is greater than 55 C and the Tm difference between the two primers is less than 6 C, GC content is in the range of 4060%. The designed primers were synthesized by the Sangon Biotech (Shanghai) Co., Ltd and were then tested for polymorphism using 36 P. fasciata individuals collected from Dexing (Jiangxi, China). The PCR system consists of 2 L 10. PCR Buffer, 2 L dNTPs, 1 L forward primer, 1 L reverse primer, 0.2 L Taq polymerase, 12.8 L ddH2O and 1 L DNA template. The PCR amplification conditions were as follows: initial denaturation at 94 C for 5 min, 35 cycles of 94 C for 30 s, 45 s at the optimal annealing temperature and 72 C for 60 s, final extension at 72 C for 10 min. The amplification products were then separated on 10% denaturing polyacrylamide gel using a 20-bp DNA marker to tested the polymorphism and further determine allele sizes.