‘惠水金橘’的叶绿体基因组特征分析

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

【目的】分析贵州地方柑橘品种‘惠水金橘’的叶绿体基因组特征及分子标记,为研究该品种的系统进化关系、丰富柑橘类果树叶绿体分子标记奠定基础。【方法】提取‘惠水金橘’成熟叶片总DNA,构建小片段插入文库并测序,以甜橙叶绿体基因组为参考,筛选叶绿体基因组相关序列组装,开展SSR(simple sequence repeat,简单重复序列)预测、SNP(single nucleotide polymorphisms,单核苷酸多态性)/InDel(insertion-deletion,插入缺失)分析以及聚类分析。【结果】组装后获得叶绿体基因组全长为160 698 bp,GC含量为38.42%,共有117种基因被注释;检测到92个SSR位点,大部分的SSR位点都偏向于A/T组成;检测到417个SNPs位点、149个InDels位点、InDel的标记密度约为每kb 0.927个。进化树显示该品种与其他宽皮柑橘聚为一大类。【结论】利用高通量测序技术挖掘柑橘叶绿体基因组DNA分子标记是可行的。

【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: NC008334.1). 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.