Wu Zhihua, Gui Songtao, Quan Zhiwu, Pan Lei, Wang Shuzhen, Ke Weidong, Liang Dequan, Ding Yi
BMC Plant Biol. 2014 Nov 19;14:289. doi: 10.1186/s12870-014-0289-0.
The chloroplast genome is important for plant development and plant evolution. Nelumbo nucifera is one member of relict plants surviving from the late Cretaceous. Recently, a new sequencing platform PacBio RS II, known as 'SMRT (Single Molecule, Real-Time) sequencing', has been developed. Using the SMRT sequencing to investigate the chloroplast genome of N. nucifera will help to elucidate the plastid evolution of basal eudicots.
The sizes of the de novo assembled complete chloroplast genome of N. nucifera were 163,307 bp, 163,747 bp and 163,600 bp with average depths of coverage of 7×, 712× and 105× sequenced by Sanger, Illumina MiSeq and PacBio RS II, respectively. The precise chloroplast genome of N. nucifera was obtained from PacBio RS II data proofread by Illumina MiSeq reads, with a quadripartite structure containing a large single copy region (91,846 bp) and a small single copy region (19,626 bp) separated by two inverted repeat regions (26,064 bp). The genome contains 113 different genes, including four distinct rRNAs, 30 distinct tRNAs and 79 distinct peptide-coding genes. A phylogenetic analysis of 133 taxa from 56 orders indicated that Nelumbo with an age of 177 million years is a sister clade to Platanus, which belongs to the basal eudicots. Basal eudicots began to emerge during the early Jurassic with estimated divergence times at 197 million years using MCMCTree. IR expansions/contractions within the basal eudicots seem to have occurred independently.
Because of long reads and lack of bias in coverage of AT-rich regions, PacBio RS II showed a great promise for highly accurate 'finished' genomes, especially for a de novo assembly of genomes. N. nucifera is one member of basal eudicots, however, evolutionary analyses of IR structural variations of N. nucifera and other basal eudicots suggested that IR expansions/contractions occurred independently in these basal eudicots or were caused by independent insertions and deletions. The precise chloroplast genome of N. nucifera will present new information for structural variation of chloroplast genomes and provide new insight into the evolution of basal eudicots at the primary sequence and structural level.
叶绿体基因组对植物发育和植物进化至关重要。莲是白垩纪晚期幸存的孑遗植物之一。最近，一种名为“单分子实时测序（SMRT）”的新测序平台PacBio RS II已被开发出来。利用SMRT测序研究莲的叶绿体基因组将有助于阐明基部真双子叶植物的质体进化。
通过桑格测序、Illumina MiSeq测序和PacBio RS II测序分别获得的从头组装的莲完整叶绿体基因组大小为163,307 bp、163,747 bp和163,600 bp，平均覆盖深度分别为7×、712×和105×。通过Illumina MiSeq reads校对PacBio RS II数据获得了莲精确的叶绿体基因组，其具有四分体结构，包含一个大单拷贝区域（91,846 bp）和一个小单拷贝区域（19,626 bp），由两个反向重复区域（26,064 bp）隔开。该基因组包含113个不同的基因，包括4种不同的rRNA、30种不同的tRNA和79种不同的肽编码基因。对来自56个目133个分类单元的系统发育分析表明，有1.77亿年历史的莲是悬铃木的姐妹分支，悬铃木属于基部真双子叶植物。使用MCMCTree估计基部真双子叶植物在侏罗纪早期开始出现，分歧时间约为1.97亿年。基部真双子叶植物内部的反向重复序列（IR）的扩增/收缩似乎是独立发生的。
由于PacBio RS II测序读长较长且对富含AT区域的覆盖没有偏差，它在生成高度准确的“完成”基因组方面具有很大潜力，特别是对于基因组的从头组装。莲是基部真双子叶植物之一，然而，对莲和其他基部真双子叶植物的IR结构变异的进化分析表明，这些基部真双子叶植物中的IR扩增/收缩是独立发生的，或者是由独立的插入和缺失引起的。莲精确的叶绿体基因组将为叶绿体基因组的结构变异提供新信息，并在一级序列和结构水平上为基部真双子叶植物的进化提供新见解。
