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).