Bausher Michael G, Singh Nameirakpam D, Lee Seung-Bum, Jansen Robert K, Daniell Henry
USDA-ARS, Horticultural Research Laboratory, Fort Pierce, FL 34945-3030, USA.
BMC Plant Biol. 2006 Sep 30;6:21. doi: 10.1186/1471-2229-6-21.
The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over nuclear transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms.
The complete chloroplast genome sequence of Citrus sinensis is 160,129 bp in length, and contains 133 genes (89 protein-coding, 4 rRNAs and 30 distinct tRNAs). Genome organization is very similar to the inferred ancestral angiosperm chloroplast genome. However, in Citrus the infA gene is absent. The inverted repeat region has expanded to duplicate rps19 and the first 84 amino acids of rpl22. The rpl22 gene in the IRb region has a nonsense mutation resulting in 9 stop codons. This was confirmed by PCR amplification and sequencing using primers that flank the IR/LSC boundaries. Repeat analysis identified 29 direct and inverted repeats 30 bp or longer with a sequence identity > or = 90%. Comparison of protein-coding sequences with expressed sequence tags revealed six putative RNA edits, five of which resulted in non-synonymous modifications in petL, psbH, ycf2 and ndhA. Phylogenetic analyses using maximum parsimony (MP) and maximum likelihood (ML) methods of a dataset composed of 61 protein-coding genes for 30 taxa provide strong support for the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids and asterids. The MP and ML trees are incongruent in three areas: the position of Amborella and Nymphaeales, relationship of the magnoliid genus Calycanthus, and the monophyly of the eurosid I clade. Both MP and ML trees provide strong support for the monophyly of eurosids II and for the placement of Citrus (Sapindales) sister to a clade including the Malvales/Brassicales.
This is the first complete chloroplast genome sequence for a member of the Rutaceae and Sapindales. Expansion of the inverted repeat region to include rps19 and part of rpl22 and presence of two truncated copies of rpl22 is unusual among sequenced chloroplast genomes. Availability of a complete Citrus chloroplast genome sequence provides valuable information on intergenic spacer regions and endogenous regulatory sequences for chloroplast genetic engineering. Phylogenetic analyses resolve relationships among several major clades of angiosperms and provide strong support for the monophyly of the eurosid II clade and the position of the Sapindales sister to the Brassicales/Malvales.
柑橘是具有国际经济价值的最大水果作物，由于细菌性柑橘溃疡病的传入，其产量最近受到威胁。通过植物育种和核转基因方法对抗这种疾病尚未取得显著进展。叶绿体基因工程相对于核转化具有许多优势；它不仅增加了转基因表达，还便于转基因的限制，这是转基因树木发展的主要障碍之一。我们对柑橘叶绿体基因组进行了测序，以促进这种作物的遗传改良，并评估被子植物主要谱系之间的系统发育关系。
甜橙完整的叶绿体基因组序列长度为160,129 bp，包含133个基因（89个蛋白质编码基因、4个rRNA基因和30个不同的tRNA基因）。基因组结构与推测的被子植物祖先叶绿体基因组非常相似。然而，柑橘中不存在infA基因。反向重复区域已经扩展，重复了rps19和rpl22的前84个氨基酸。IRb区域中的rpl22基因存在无义突变，导致9个终止密码子。通过使用位于IR/LSC边界两侧的引物进行PCR扩增和测序证实了这一点。重复序列分析鉴定出29个30 bp或更长的正向和反向重复序列，其序列同一性≥90%。将蛋白质编码序列与表达序列标签进行比较，发现6个推定的RNA编辑，其中5个导致petL、psbH、ycf2和ndhA中的非同义修饰。使用最大简约法（MP）和最大似然法（ML）对由30个分类群的61个蛋白质编码基因组成的数据集进行系统发育分析，为包括单子叶植物、真双子叶植物、蔷薇类植物和菊类植物在内的几个被子植物主要分支的单系性提供了有力支持。MP树和ML树在三个区域不一致：无油樟属和睡莲目的位置、木兰类植物腊梅属的关系以及真蔷薇类I分支的单系性。MP树和ML树都为真蔷薇类II的单系性以及柑橘（无患子目）作为包括锦葵目/十字花目在内的一个分支的姐妹分支的位置提供了有力支持。
这是芸香科和无患子目成员的首个完整叶绿体基因组序列。反向重复区域扩展以包括rps19和rpl22的一部分以及存在两个截短的rpl22拷贝在已测序的叶绿体基因组中是不寻常的。完整的柑橘叶绿体基因组序列的可用性为叶绿体基因工程的基因间隔区和内源调控序列提供了有价值的信息。系统发育分析解决了被子植物几个主要分支之间的关系，并为真蔷薇类II分支的单系性以及无患子目作为十字花目/锦葵目姐妹分支的位置提供了有力支持。
