| Summary: | There are 22 species in the <i>Phalaris</i> genera that distribute almost all over the temperate regions of the world. Among them, reed canary grass (<i>Phalaris arundinacea</i>, tetraploid and hexaploid) and hardinggrass (<i>P. aquatica</i>, tetraploid) have been long cultivated as forage grass and have received attention as bio-energy materials in recent years. We aimed to facilitate inter-species/ploidies comparisons, and to illuminate the degree of sequence variation within existing gene pools, chloroplast (cp) genomes of three <i>Phalaris</i> cytotypes (<i>P</i>. aquatica/4x, <i>P</i>. <i>arundinacea/</i>4x <i>and P.</i> <i>arundinacea</i>/6x) were sequenced and assembled. The result indicated that certain sequence variations existed between the cp genomes of <i>P. arundinacea</i> and <i>P. aquatica.</i> Several hotspot regions (<i>atpI</i>~<i>atpH</i>, <i>trnT-UGU</i>~<i>ndhJ</i>, <i>rbcL</i>~<i>psaI</i>, and <i>ndhF</i>~<i>rpl32</i>) were found, and variable genes (<i>infA</i>, <i>psaI</i>, <i>psbK</i>, etc.) were detected. SNPs (single nucleotide polymorphisms) and/or indels (insertions and deletions) were confirmed by the high Ka/Ks and Pi value. Furthermore, distribution and presence of cp simple sequence repeats (cpSSRs) were identified in the three <i>Phalaris</i> cp genomes, although little difference was found between hexaploid and tetraploid <i>P. arundinacea</i>, and no rearrangement was detected among the three <i>Phalaris</i> cp genomes. The evolutionary relationship and divergent time among these species were discussed. The RNA-seq revealed several differentially expressed genes (DEGs), among which <i>psaA</i>, <i>psaB</i>, and <i>psbB</i> related to leaf color were further verified by leaf color differences.
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