Cytoplasmic diversity, phylogenetic relationships and molecular evolution of Tunisian Citrus species as inferred from mutational events and pseudogene of chloroplast trnL-trnF spacer

• The trnL-trnF intergenic spacer was sequenced and analyzed for 27 citrus cultivars.
• The anticodon domain was identified as the most conserved element.
• Phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups.
• One copy of pseudogene of the original trnF gene in the 7 Citrus species at position 275 bp.
• A scenario of a neutral model of evolution and advocated a constant population size.

The genus Citrus L. is among the most important fruit trees in the world. In this report, cytoplasmic polymorphism of twenty seven Tunisian Citrus cultivars was explored using the chloroplast trnL-trnF intergenic spacer. Chloroplast sequences showed variation in length and nucleotide content. Haplotype and nucleotide diversity showed low variations. Molecular phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups distinguishing between mandarin and pummelo groups. The trnL-trnF intergenic spacer showed one copy of pseudogene of the original trnF gene in 27 Citrus species at position 275 bp with a size varying from 49 to 63 bp. The anticodon domain was identified as the most conserved element, but one transversion (T−>C) was found in the D-domain. Meanwhile, one transversion (T−>A) and one transition (T−>G) were found in the T-domain. Neutrality tests (Tajima, Fu & Li and Fu) which revealed positive and non-significant values and Pi and θW assume a neutral model of evolution and advocated a constant population size. The study demonstrates the resolving power of trnL-trnF sequence data to prove both pummelo and mandarin gene pool’s contribution in the development of Tunisian secondary species and inferring their genetic and phylogenetic relationships.