Forward primer

The ammonia-N-stress experiment adopted 400 healthy juvenile shrimps and followed the approach mentioned in Section 2.3. The first 40 shrimps that died belonged to the sensitive group, while the last 40 and those still alive were the resistant group. They were kept in ethanol for DNA extraction through MagPure Tissue/Blood DNA LQ Kit (Magen, Guangzhou, China).
The NCBI database was utilized to predict the exonic region of PmGrx2, upon which Premier 6.0 designed primers that spanned the exonic regions to amplify the samples’ DNA order. The PCR reaction solution included forward primer (1 µL), reverse primer (1 µL), DNA template (2 µL), and mix green (21 µL) (Tsingke, Beijing, China). The three PCR procedures included 98 °C for 2 min, 35 cycles of 98 °C (10 s), 55 °C (15 s), 72 °C (1 min), and 72 °C (10 min). Tsingke Biotechnology undertook the synthesis of primers and the sequencing of PCR products (Guangzhou, China). The details are given in Supplementary Table S5.
DNAMAN and Chromas were adopted to elucidate DNA sequences of each shrimp, thus acknowledging the SNPs of PmGrx2. WPS office was used to calculate genetic parameters. An χ2 test was conducted with SPSS 26.0 to explore how the SNPs of PmGrx2 are correlated to the ammonia-N-stress-tolerance trait, and p < 0.05 indicated a significant difference.

A pair of universal primers (forward primer: 5′-TATTCTCAACTAACCACAA AGA-3′; reverse primer: 5′-ACTTCTGGTTGACCAAAGAATCA-3′) was screened to amplify the DNA fragments from the five species. PCR amplification was performed in a total volume of 25 µl containing 21 µl of PCR Mix (TsingKe, Beijing, China), 1 µl of forward primer (10 µM, Sangon), 1 µl of reverse primer (10 µM, Sangon), 2 µl of total DNA. Thermal cycling was performed in a Bio-Rad thermocycler (Bio-Rad, California, CA, USA) using a PCR process: 94°C for 3 min; 40 cycles of 94°C for 40 s, 48.5°C for 30 s, and 72°C for 60 s; 72°C for 7 min, and subsequent storage at 4°C. The amplified fragments were sequenced by the ABI3730XL sequencer (Applied Biosystems Co., Shanghai, China). Sequencing data were processed by the CodonCode Aligner V3.7.1 (CodonCode Co., Centerville, MA, USA). Phylogenetic relationship was analyzed by the neighbor-joining (NJ) method in the MEGA7 program (National Institutes of Health, Bethesda, MD, USA).