Testing Candidate Plant Barcode Regions In The True Citrus Fruit Trees Group (Aurantioideae, Rutaceae)

DNA barcoding is a technique for identifying and characterizing species of organisms using a short DNA region. It is becoming more and more attractive to biologists as a new method to aid in species identification. DNA barcoding can identify species rapidly and accurately based on the principle of inter-specific divergence is greater than intra-specific divergence. Since 2003, Paul Hebert have suggested that we should choose one standard gene fragment as new "taxonomy character" to help traditional taxonomy, the mitochondrial cytochrome c oxidase subunit 1 (COI or coxl) sequence as DNA barcode has been successfully employed for species identification and revealing cryptic species or new species in diverse groups of animals such as insects, fishes, birds, diatoms and prokaryotes. However, COI gene is not suitable candidate for plant DNA barcoding because of its slow substitution rate in higher plant. Plant DNA barcode technique is still in the stage of searching for a suitable DNA region. Although different candidate single region or combination of regions from chloroplast genome were reported in the existing literature. we still lack an obvious well-characterized plant locus that meets all the necessary criteria of a DNA barcoding region.The genus Citrus L. established in 1753 by Carl Linnaeus belongs to the subfamily Aurantioideae of the family Rutaceae. Swingle and Reece (1967) classified the genus Citrus and its five close relatives, Poncirus, Fortunella, Microcitrus, Eremocitrus and Clymenia into the true citrus fruit trees group based mainly on the number of stamen and structure of pulp-vesicles of the genera. A series of study from morphology to molecular markers on the taxonomy and evolution of the genus Citrus and its close relatives has been carried out both in China and abroad in the past more than 250 years. The taxonomic and evolutionary relationships between Citrus and its close relatives have remained unresolved. A better understanding of the phylogenetic relationship between Citrus and its closely related genera is needed for future variety breeding, germplasm conservation and utilization of the genus Citrus.In the present study, six candidate plant barcoding regions of chloroplast genome,rpoC1,trnG-trnS,psbH-petB and trnL-trnF, and nuclear ITS region of 59 biological types collected from the six genera of the true citrus fruit trees group were tested in an attempt to develop DNA barcoding makers for species identification and taxonomy of the genus Citrus and its closely related genera. To test the potential of the candidate makers being used as DNA barcoding of the true citrus fruit trees group, firstly, the inter-generic divergence of all genotypes and inter- and intra-specific divergence of Citrus L. were analyzed in present study based on genetic distances. The results of this study showed that none of the candidate markers proposed in the exsiting literature provided unique identifiers for all the species tested. The results of analysis of genetic distances show that ITS sequences have the potential as DNA barcoding of Citrus. And then, the maximum parsimony (MP), neighbor-joining (NJ) and unpaired group mean averages algorithm (UPGMA) trees of all genotypes were constructed with PAUP* version 4.10b software by using the DNA sequences obtained and Murraya paniculata (L.) Jack., Atalantia buxifolia Correa., and Severinia buxifolia (Poir.) Tenore as outgroups. In addition, the MP, NJ and UPGMA trees of 47 accessions of Citrus L. were constructed with PAUP* version 4.10b software by usingon the Citrus DNA sequences and P. trifoliata (L.) Raf., P. polyandra S. Q. Ding, X. N. Zhang, Z. R.Bao & M. Q. Liang, P. trifoliata var. monstrosa (T. Ito) Swingle as outgroups. Finally, the potential of using ITS, matK, or a combination of regions of cpDNA as the true citrus fruit trees group DNA barcoding was discussed in details.