基于454FLX高通量技术的厚朴叶绿体全基因组测序及应用研究

High-throughput pyrosequencing of the complete chloroplast genome of Magnolia officinalis and its application in species identification

叶绿体基因组序列在中药DNA条形码鉴定及基因工程生物制药方面具有广泛的应用前景。本文应用454高通量测序技术对厚朴进行了叶绿体全基因组测序,建立了标准测序流程。生物信息学分析共获得有效序列重叠群(contig)14个,测序覆盖度达到99.99%。厚朴叶绿体基因组大小为160 183 bp,大(LSC)、小(SSC)单拷贝区大小分别为88 210 bp和18 843 bp,反向互补重复区(IR)大小为26 565 bp,共注释叶绿体基因126个,其中每个IR区17个。厚朴与木兰亚纲其他物种的叶绿体编码基因在种类、排列顺序及结构大小上基本一致。采用叶绿体81个蛋白编码基因对厚朴及同属5个种9个样本进行了分子鉴定,鉴定效率100%,并且可以成功区分不同产地的凹叶厚朴。以上结果表明,本研究建立的标准测序流程适用于叶绿体基因组测序,叶绿体基因组序列可有效区分厚朴及近缘物种。

Chloroplast genome sequences have comprehensive application prospects in DNA barcoding and chloroplast engineering in traditional Chinese medicine. The complete chloroplast genome of Magnolia officinalis sequenced by high-throughput pyrosequencing and a sequencing procedure was established. Fourteen contigs were obtained after de nove assembly. The sequencing percent of coverage was 99.99%. The chloroplast genome is 160 183 bp in size, and has a typical quadripartite structure with the large （LSC, 88 210 bp） and small copy （SSC, 18 843 bp） regions separated by two copies of an inverted repeat ORs, 26 565 bp each）. 126 chloroplast genes were successfully annotated, of which 17 genes located in each IR region. The chloroplast genome features in Magnolia officinalis are nearly identical to those from other Magnoliid chloroplast genomes. Phylogenetic analyses were performed based on 81 shared coding-genes for a total of 9 Magnolia samples of 5 closely related species. Results showed that distinguishing among species was generally straightforward at the species and population level. This study confirmed the effectiveness of our chloroplast genome sequencing procedure. The chloroplast genome can provide distinguishing differences to help identify Magnolia officinalis and its closely related plants.