*2.3. Data Analysis*

The population growth parameter was calculated by the following formula:

 $\sum\_{\mathbf{x}=\mathbf{0}}^{\infty} e^{-r\_m x} l\_x m\_x = 1, R\_0 = \sum l\_x m\_{x\prime}$  $\lambda = e^{r\_m}, T = \ln R\_0 \cdot r\_m^{-1}$ 

In the formula, *x* is the age period (d), *l*<sup>x</sup> is the survival rate at stage *x* (%), *mx* is the birth rate at stage *x*, *rm* is the intrinsic growth rate (d<sup>−</sup>1), *R*<sup>0</sup> is the net reproductive volume (individual), *T* is the generation period (d), and *λ* is the weekly growth rate (d<sup>−</sup>1).

Microsoft Excel 2010 was used to process test data, and IBM SPSS Statistics 23 (IBM Corporation, Armonk, NY, USA) was used for one-way ANOVA and Duncan's multiple comparison test to test for significance and variance homogeneity. The arithmetic mean of replicate groups was taken and expressed as mean ± standard deviation; *p* < 0.05 indicated significant difference; *p* < 0.01 indicated extremely significant difference.

The growth rate of body length was calculated as follows:

$$\text{LGR} = \frac{L\_1 - L\_0}{L\_0} \times 100\% \text{s}$$

In the formula, LGR is the growth rate body length (%), *L*<sup>0</sup> is the initial body length of the *D. tibetana* used in the experiment, and *L*<sup>1</sup> is the body length at the time of death in the experiment.

#### *2.4. Transcriptome Sequencing*

Approximately 400 *D. tibetana* were randomly selected from each treatment group as a biological replicate. Total RNA was extracted from *D. tibetana* using TRIzol (Invitrogen, Carlsbad, CA, USA), and DNase I (TaKara, Dalian, China) was used to remove gene DNA. Using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA), the concentration and purity of the extracted RNA were detected by ND-2000 microspectrophotometer (Thermo Scientific, Wilmington, DE, USA) to ensure the integrity of all RNA samples (OD260/OD280 = 1.8–2.2, OD260/OD230 ≥ 2.0, RIN ≥ 8.5, 28S/18S ≥ 1.0) and perform transcriptome sequencing. The mRNA sequencing was conducted using the HiSeq platform, and library construction was performed using the Illumina TruSeq™ RNA sample prep kit method as follows: total RNA was extracted (>1 μg), and mRNA was then enriched, fragmented, and inverted into cDNA; then, adapter ligation and illumina sequencing were performed.

#### *2.5. Differentially Expressed Genes (DEGs)*

To explore the differential gene expression of wild-type and domesticated *D. tibetana*, the expression levels of protein-coding genes were calculated by the FPKM method. DEGs were screened, and differential gene expression volcano plots were drawn. Quantitative analysis of gene expression levels was conducted using RSEM (https://deweylab.biostat. wisc.edu/resm/ accessed on 6 June 2019); after obtaining the number of read counts of gene transcripts, DEGseq (http://bioconductor.org/packages/stats/bioc/DESeq/ accessed on 6 June 2019) software was used to analyze gene expression differences between samples. The significance of differential expression was measured by FPKM (fragments per kilobases per million reads) using false discovery rate (FDR) and fold change (FC) as criteria. When a gene exhibited both FDR < 0.05 and |log2FC| > 1, it was considered differentially expressed.
