Species discrimination of the genus Citrus and the hybridization analysis

The genus Citrus (Aurantioideae, Rutaceae) is the most important fruit crops all around the world. As many cultivated and weedy species/varieties exist in this genus, botanists hold different opinions on the taxonomy and phylogeny of the genus Citrus all the time. To evaluate the taxonomy and phylogenetic relationship of Citrus species, we collected 22 Citrus species and evaluated the genetic diversity and phylogeny based on the genetic variation of the ribosomal RNA (rRNA) 5S gene sequences. The 5S DNA alignment showed high nucleotide variations and sequence length difference among investigated species. In the rRNA 5S gene, four nucleotide sites were found to be easily nucleotide-substituted and hybridization-reflective. Some Citrus species, including C. kinokuni, C. unshiu, C. sinensis, C. leiocarpa, C. tagerina andC. hybrid are found to be hybridization-susceptible species. The analysis result of genetic distance suggested that C. clemntina, C. hybrid, C. leiocarpa, C. nippokoreana, C. kinokuni and C. tagerina showed low values with each other and divided into one group in the rRNA 5S phylogenetic tree. Others were grouped together in the 77% similarity criterion. According to six-tribe discrimination method, the clading seen from the 5S phylogenetic tree suggested that the species belonging to Sinocitrus tribe showed higher sequence variation than other tribes. This work provides more sequence evidences for Citrus species identification and helps us further understand the phylogenetic relationships and species identification of the genus Citrus. The results would help citrus breeding and germplasm conservation programs.