DNA Barcoding to Identify Members of Aurantioideae

DNA barcoding facilitates proper and easy identification of species. Two chloroplast genes, rbcL and matK have been recognized as potentially ideal candidates for barcoding of plants. The sub-family Aurantioideae, including citrus, is known to have a high level of hybridization and incomplete lineage sorting resulting in maintenance of ancestral polymorphisms. Use of uniparentally inherited markers for barcoding will not be adequate to identify this biologically complex taxonomic group. We have used a low copy nuclear gene, malate dehydrogenase (MDH), as a barcoding gene to identify members of Aurantioideae. Sequences of a 1600 bp fragment of MDH were generated using vouchered specimens from 29 genera and 120 cultivars of Aurantioideae. The region selected has several introns providing the variability needed to distinguish closely related cultivars. We have identified regions that are short enough for easy sequencing and variable enough to provide discrimination. A two tiered approach was used for barcoding. Primers designed to amplify short exon regions are useful in identification of the genera while a second PCR using primers designed to amplify variable intron regions is useful in identification at the lower taxonomic levels. A distance matrix was constructed to analyze the sequence differences between different groups. The information generated serves as a guide for proper and reliable identification of citrus and its relatives and is useful in monitoring plant movement essential for preventing disease spread.