Curci Pasquale L, De Paola Domenico, Sonnante Gabriella
Institute of Biosciences and Bioresources, National Research Council, Via Amendola, 165/A, 70126, Bari, Italy.
Mol Ecol Resour. 2016 Mar;16(2):562-73. doi: 10.1111/1755-0998.12457. Epub 2015 Sep 10.
In this study, new chloroplast (cp) resources were developed for the genus Cynara, using whole cp genomes from 20 genotypes, by means of high-throughput sequencing technologies. Our target species included seven globe artichokes, two cultivated cardoons, eight wild artichokes, and three other wild Cynara species (C. baetica, C. cornigera and C. syriaca). One complete cp genome was isolated using short reads from a whole-genome sequencing project, while the others were obtained by means of long-range PCR, for which primer pairs are provided here. A de novo assembly strategy combined with a reference-based assembly allowed us to reconstruct each cp genome. Comparative analyses among the newly sequenced genotypes and two additional Cynara cp genomes ('Brindisino' artichoke and C. humilis) retrieved from public databases revealed 126 parsimony informative characters and 258 singletons in Cynara, for a total of 384 variable characters. Thirty-nine SSR loci and 34 other INDEL events were detected. After data analysis, 37 primer pairs for SSR amplification were designed, and these molecular markers were subsequently validated in our Cynara genotypes. Phylogenetic analysis based on all cp variable characters provided the best resolution when compared to what was observed using only parsimony informative characters, or only short 'variable' cp regions. The evaluation of the molecular resources obtained from this study led us to support the 'super-barcode' theory and consider the total cp sequence of Cynara as a reliable and valuable molecular marker for exploring species diversity and examining variation below the species level.
在本研究中，利用高通量测序技术，基于20个基因型的完整叶绿体基因组，为刺苞菜属开发了新的叶绿体（cp）资源。我们的目标物种包括7个球茎甘蓝、2个栽培刺芹、8个野生菊芋，以及3个其他野生刺苞菜属物种（巴氏刺苞菜、角状刺苞菜和叙利亚刺苞菜）。其中一个完整的cp基因组是从全基因组测序项目的短读长中分离出来的，而其他基因组则通过长距离PCR获得，本文提供了相应的引物对。从头组装策略与基于参考的组装相结合，使我们能够重建每个cp基因组。对新测序的基因型与从公共数据库中获取的另外两个刺苞菜属cp基因组（“布林迪西诺”菊芋和矮刺苞菜）进行比较分析，结果显示刺苞菜属中有126个简约信息位点和258个单态位点，共计384个可变位点。检测到39个SSR位点和34个其他插入缺失事件。数据分析后，设计了37对用于SSR扩增的引物对，这些分子标记随后在我们的刺苞菜属基因型中得到验证。与仅使用简约信息位点或仅使用短的“可变”cp区域进行系统发育分析相比，基于所有cp可变位点的系统发育分析提供了最佳分辨率。对本研究获得的分子资源进行评估后，我们支持“超级条形码”理论，并认为刺苞菜属的完整cp序列是探索物种多样性和研究种内变异的可靠且有价值的分子标记。
