Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs
Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs
复制标题
通过九个新型通用引物对对被子植物叶绿体全基因组进行高效测序
DOI：
10.1111/1755-0998.12251
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发表时间：
2014-09-01
影响因子：
7.7
通讯作者：
Li, Hong-Tao
中科院分区：
文献类型：
--
作者：
Yang, Jun-Bo;Li, De-Zhu;Li, Hong-Tao
叶绿体基因组提供了不可缺少的信息，有助于提高系统发育分辨率，甚至作为细胞器尺度的条形码。新一代测序技术促进了叶绿体全基因组的测序，但与被子植物的数量相比，叶绿体基因组的测序相对较少。缺乏完整的叶绿体基因组测序主要有两个原因：(1)叶绿体测序需要大量的新鲜叶片；(2)由于难以分离高质量的叶绿体DNA，许多植物的叶绿体基因组测序存在相当大的空白，从而阻碍了完整的叶绿体基因组的组装。为了克服这些障碍，我们分析了迄今为止所有已知的被子植物叶绿体基因组，然后设计了9对与高度保守区域对应的通用引物。利用这些引物，被子植物的整个叶绿体基因组可以用远程PCR扩增，并使用下一代测序方法进行测序。引物具有较高的通用性，用24种被子植物的主要支系进行了测试。为了验证引物的功能，我们对被子植物主要类群——早分化被子植物、木兰科植物、单子科植物、沙花科植物、花叶科植物、花叶科植物和星形植物等8个物种进行了测序，并组装了它们的叶绿体基因组。在我们的试验中，只使用了100毫克的新鲜叶子。结果表明，该通用引物为被子植物叶绿体全基因组测序提供了一种简便、有效、可行的方法。设计的通用引物对提供了加速基因组规模数据采集的可能性，因此将扩大被子植物的系统发育分辨率和物种鉴定。
Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.