Complete genome sequencing of cytoplasmically inherited chloroplast DNA provides novel insights into the origins of clonally propagated crops such as banana and plantain (Musa spp.). This study describes the structural organization of the chloroplast genome of M. balbisiana Colla and its phylogenetic relationship with other wild progenitors of the domesticated banana cultivars. The M. balbisiana chloroplast genome was sequenced using Illumina HiSeq 2000 platform, followed by a combination of de novo short-read assembly and reference-guided mapping of contigs to generate complete plastome sequence. The M. balbisiana chloroplast genome is 169,503 bp in length, exhibits a typical quadripartite structural organization with a large single-copy (LSC; 87,828 bp) region and a small single-copy (SSC; 11,547 bp) region interspersed between inverted repeat (IRa/b; 35,064 bp) regions. Overall, its gene content, size, and gene order were identical to that of M. acuminata Colla with extensive expansion of the inverted repeat-small single-copy (IR-SSC) junctions. Comparative analyses revealed the conserved IRa-SSC expansion in three wild Musa species and members of the order Zingiberales. In contrast, IRb-SSC expansion was conspicuously absent in the sister taxon M. textilis Nee and related species of Zingiberales. Interestingly, phylogenomic assessment based on whole-plastome and protein-coding gene sets have provided robust support for the association of M. balbisiana and M. textilis as a sister group, despite the variation in IRb-SSC expansion. Although the current study substantiates the infrageneric IRb-SSC fluctuations in Musaceae, extensive taxon sampling is necessary to confirm whether the accessions of section Musa have undergone independent IRb-SSC expansion relative to section Callimusa. Chloropl asts are a quintessential feature of land plants and algae, harboring oxygenic photosynthetic machinery and an array of metabolic pathways. Believed to have originated from endosymbiotic associations between cyanobacteria and eukaryotic cells, chloroplasts have evolved complex mechanisms of intracellular gene
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