Plastome Annotation and Gene Identification

The plastomes were initially annotated by using GeSeq [68 (link)] with two reference genomes (Carnegiea gigantea, GenBank: NC_027618.1 and Lophocereus schottii, GenBank: NC_041727.1). Subsequently, annotations with problems were manually edited by using Apollo [69 ]. To further confirm the presence or absence of genes, we used the 80 unique protein-coding genes (PCGs) and the 30 unique tRNA genes annotated in Portulaca oleracea as query sequences to search for homologous sequences using the BLASTn program [70 (link)]. The parameters were as follows: -evalue 1e-5, -word_size 9, -gapopen 5, - gapextend 2, -reward 2, -penalty − 3, and -dust no. If only a partial sequence of the gene was identified in each genome, this gene was considered a pseudogene. However, for genes whose conserved functional domains still exist, such as accD, further experiments are still needed for confirmation. If a premature termination codon was encountered in the coding sequence, we also considered it to be a pseudogene, although we cannot rule out the possibility of an RNA editing event for correction.