2.13. Identification and analysis of conserved noncoding sequences

We identified conserved noncoding sequences (CNSs) using GERP++ software (Cooper et al., 2005). The nucleotide substitution frequency of a CNS was estimated by nucleotide_substitution_frequency=2∑i<jdijnn-1, where d_ij is an estimate of the number of nucleotide substitutions per site between DNA sequence i and j, and n is the number of the examined sequences. For each rapidly evolving region in CNSs, nucleotide substitution frequency was subsequently calculated for each species using an average of nucleotide substitution frequency of a species within this genomic region compared to other seven Oryza AA‐genome species. We then used the nucleotide substitution frequency of this rapidly evolving region to compare with average nucleotide substitution frequency of total CNSs by Chi‐squared test.