Genomic basis of divergence between O. kokonorica andC. songorica
In order to investigate the genomic basis of divergence between O. kokonorica and C. songorica , we compared the two species at the whole-genome level and the differentiation in gene expression under diverse abiotic stresses. To detect genomic variation between the two species, we first used NUCDIFF (Khelik et al., 2017) to locate and categorize differences between the two species. The results were uploaded to RectChr (https://github.com/hewm2008/RectChr) for visualization. We also performed whole-genome comparisons using the nucmer (nucmer -maxmatch -l 100 -c 500) function from the MUMmer package. Structural variations (SVs) were called using Assemblytics (Nattestad & Schatz, 2016) based on the output of nucmer. A custom Python script (Xie et al., 2019) was used to reformat the results from Assemblytics, and the effects of SVs (including high-, moderate-, low-, and modifier-impacted SVs) were annotated using SnpEff (Cingolani et al., 2012). The genes categorized as highly-impacted-by-SV were subjected to GO enrichment and KEGG analyses.
To evaluate the differentiation in gene expression under drought, heat and cold treatments between the two species, seeds of O. kokonorica were collected from the same place where plants were collected for sequencing. Bleach-sterilized seeds were germinated and grown in a glasshouse under the same controlled conditions as forC. songorica used in Zhang et al. (2021a). Four-week-old seedlings were transplanted into individual pots with the same growth medium. Each pot was irrigated with 100 mL Hoagland nutrient solution every three days. We used the same conditions described in Zhang et al. (2021a) for the heat and cold treatment. Nine-week-old plants were treated under heat and cold (40°C and 4°C). For drought treatment, the soil water content was decreased to 10-20%, corresponding to light drought stress in Zhang et al. (2021a). Shoots and roots of each plant were collected 24h after treatment, immediately immersed in liquid nitrogen, then stored at -80°C. Four biological replicates were used for each sample for transcriptomic analysis.
Total RNA extraction, library construction and sequencing were performed by BGI-Shenzhen Company (Wuhan, China) on the MGI2000 platform by 2 × 150 bp pair-end mode. Adapters, reads containing poly-N and lower-quality reads (< Q30) in the raw sequencing outputs were removed to obtain clean reads, which were then mapped to the O. kokonorica genome using HISAT2 v. 2.0.4 (Kim et al., 2015). We used StringTie v. 1.3.1 (Pertea et al., 2015) to calculate transcripts per million (TPM) of mRNA in each sample. TPM of genes was computed by summing the TPMs of transcripts in each gene group, and the DESeq2 R package (Love et al., 2014) was used to perform differential expression analysis. Differentially expressed genes (DEGs) were defined as those having at least a twofold change in expression compared with those in controlled samples (false discovery rate, FDR < 0.05).