RNAseq and data analysis
For RNAseq analysis, RNA was extracted from three replicates of 25
mature seeds of wild type, Mthsfa9-1 and Mthsfa9-2 alleles
that were grown together.
RNAseq
was outsourced to BGI Genomics (Shenzhen, China). RNA quality was
checked with an Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit).
RNA was sequenced using the DNBseq platform, generating an average 24M
reads per sample. The sequencing reads which containing low-quality,
adaptor-polluted and high content of unknown base (N) reads were removed
and mapping of the clean reads was performed against the Mt4.0 genome.
Total mapping ratio was 90%, and unique mapping ratio 78%. DEGs
(differential expressed genes) between samples were determined using
DEseq2 algorithms. Genes were considered differential when the adjusted
P-value was <0.05 and log2FoldChange of 1. On the DEGs, GO
(http://www.geneontology.org/) and KEGG (https://www.kegg.jp/)
enrichment analysis of annotated different expression gene was performed
by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution)
based on Hypergeometric test. The significant levels of terms and
pathways were corrected by Q value with a rigorous threshold (Q value ≤
0.05) by Bonferroni. RNAseq data were deposited in the NCBI Gene
Expression Omnibus database (accession number GSE98199 and GSExxxx).