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Trifolium  L., which belongs to the IR lacking clade (IRLC), is one of the largest genera in the Leguminosae and contains several economically important fodder species. Here, we present whole chloroplast (cp) genome sequencing and annotation of two important annual grasses,  Trifolium alexandrinum  (Egyptian clover) and  T. resupinatum  (Persian clover). Abundant single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels) were discovered between those two species. Global alignment of  T. alexandrinum  and  T. resupinatum  to a further thirteen  Trifolium  species revealed a large amount of rearrangement and repetitive events in these fifteen species. As hypothetical cp open reading frame (ORF) and RNA polymerase subunits,  ycf1  and  rpoC2  in the cp genomes both contain vast repetitive sequences and observed high Pi values (0.7008, 0.3982) between  T. alexandrinum  and  T. resupinatum . Thus they could be considered as the candidate genes for phylogenetic analysis of  Trifolium  species. In addition, the divergence time of those IR lacking  Trifolium  species ranged from 84.8505 Mya to 4.7720 Mya. This study will provide insight into the evolution of  Trifolium  species.

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The Complete Chloroplast Genome of Two Important Annual Clover Species, Trifolium alexandrinum and T. resupinatum: Genome Structure, Comparative Analyses and Phylogenetic Relationships with Relatives in Leguminosae