Characterization of the complete chloroplast genome of the ‘Huishui Jin Ju' (Citrus erythrosa Hort. ex Tan.)

Abstract: 【Objective】In this study, we reported a complete chloroplast genome of‘Huishui Jin Ju' (Citrus erythrosa Hort. ex Tan.) with assistance from the next-generation sequencing, and the objective is to ascertain the chloroplast genetic difference between it and other citrus species, and to develop molecular markers for citrus varieties identification.【Methods】The total genomic DNA was extracted from the leaves of‘Huishui Jin Ju', and then the sequencing library was constructed with fragmented and qualified DNA samples. The Illumina HiSeq 4000 PE150 was used to sequence the library. The high quality short reads of sequencing were assembled with the assistance from Citrus sinensis chloroplast reference genome (NCBI Reference Sequence: NC＿008334.1) . The coding genes were annotated by DOGMA (http://dogma.ccbb.utexas.edu/) . Using MISA (http://pgrc.ipk-gatersleben.de/misa/misa.html) , it was undertaken to conduct SSRs chloroplast genome analysis. The genetic variation analysis was performed by MUMmer (version 3.23) nucmer. The neighbor-joining (NJ) methods from the MEGA (version 5.2) program were used to create phylogenetic trees.【Results】The complete chloroplast genome of‘Huishui Jin Ju'was a double-stranded circular DNA of 160 698 bp in length, consisting of a pair of inverted repeats (IR) regions of 26 990 bp, large single copy (LSC) region of 87 919 bp and a small single copy (SSC) region of 18 799 bp. The overall GC content accounted for 38.42% of the whole chloroplast genome. A total of 117 genes were predicated, including 83 protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes. In addition, 92 simple sequence repeats (SSRs) loci (≥5 bp) were identified including 90 single nucleotide repeats (97.8%) , one trinucleotide repeat and one tetranucleotide repeat. In these chloroplast SSRs (cpSSR) , the most of single nucleotide repeat sequences were A and T. The pairwise sequence alignment between the sweet orange and‘Huishui Jin Ju'chloroplasts genomes revealed approximately 0.71% (relative to the length of‘Huishui Jin Ju') sequence divergence, including 417 SNPs (0.45%) and 149 InDels (0.26%) . The labeled density of InDel was 0.927 kb. The analysis of phylogenetic tree showed that‘Huishui Jin Ju'and some mandarin species were grouped into one branch in this study.【Conclusion】In this study, the high-throughput sequencing technology was used to sequence the complete chloroplast genome of‘Huishui Jin Ju', and we obtained the complete chloroplast sequence of 160 698 bp in length, and a total of 117 genes were predicated and annotated. The analysis of phylogenetic tree showed that‘Huishui Jin Ju'was closely related to mandarins. As for the highly conserved feature of chloroplast genome, we can develop molecular markers with chloroplast genomic to identify citrus varieties and genetic breeding materials.