Linder C R, Goertzen L R, Heuvel B V, Francisco-Ortega J, Jansen R K
Section of Integrative Biology-C0930, The University of Texas, Austin, Texas 78712, USA.
Mol Phylogenet Evol. 2000 Feb;14(2):285-303. doi: 10.1006/mpev.1999.0706.
For molecular phylogenetic reconstruction of some intrageneric groups of plants, a DNA region is needed that evolves more rapidly than the internal transcribed spacer (ITS) of the 18S-26S nuclear ribosomal DNA (nrDNA) repeat. If the region identified is nuclear, it would also be desirable for it to undergo rapid concerted evolution to eliminate problems with coalescence. The external transcribed spacer (ETS) of the nrDNA repeat has shown promise for intrageneric phylogenetic reconstruction, but only the 3' end of the region has been utilized for phylogenetic reconstruction and "universal" primers for PCR amplification have been elusive. We present a method for reliably amplifying and sequencing the entire ETS throughout Asteraceae and some closely allied families. We also show that the ETS is more variable and phylogenetically informative than the ITS in three disparate genera of Asteraceae-Argyranthemum (tribe Anthemideae), Asteriscus (tribe Inuleae), and Helianthus (tribe Heliantheae). The full ETS was amplified using a primer (ETS1f) within the intergenic spacer in combination with a primer (18S-2L) in the 5' end of the highly conserved 18S gene. ETS1f was designed to correspond to a highly conserved region found in Helianthus and Crepis, which are in separate subfamilies of Asteraceae. ETS1f/18S-2L primed in all of the tribes of Asteraceae as well as exemplar taxa from Campanulaceae, Goodeniaceae, and Calyceraceae. For both Argyranthemum and Asteriscus, we were able to directly sequence the ETS PCR products when a single band was produced. When multiple bands were produced, we gel-purified and occasionally cloned the band of interest before sequencing. Although PCR produced single bands for Helianthus species, it was necessary to clone Helianthus amplifications prior to sequencing due to multiple intragenomic ETS repeat types. Alignment of ETS sequences for Argyranthemum and Asteriscus was straightforward and unambiguous despite some subrepeat structure in the 5' end. For Helianthus, different numbers of large tandem subrepeats in different species required analysis of the orthology of the subrepeats prior to alignment. In all three genera, the ETS provided more informative variation for phylogenetic reconstruction and allowed better resolution of relationships than the ITS. Although cloned sequences from Helianthus differed, intragenomic clones consistently formed clades. This result indicated that concerted evolution was proceeding rapidly enough in ETS that species-specific phylogenetic signal was retained. It should be now be possible to use the entire ETS for phylogenetic reconstruction of recently diverged lineages in Asteraceae and at least three other families (approximately 26,000 species or about 8% of all angiosperms).
对于一些植物属内类群的分子系统发育重建，需要一个比18S - 26S核糖体DNA（nrDNA）重复序列的内部转录间隔区（ITS）进化更快的DNA区域。如果鉴定出的区域是核基因的，那么它还需要经历快速的协同进化，以消除合并问题。nrDNA重复序列的外部转录间隔区（ETS）已显示出在属内系统发育重建方面的潜力，但该区域仅3'端已被用于系统发育重建，且用于PCR扩增的“通用”引物一直难以获得。我们提出了一种可靠地扩增和测序整个菊科及一些近缘科ETS的方法。我们还表明，在菊科的三个不同属——滨菊属（春黄菊族）、小滨菊属（旋覆花族）和向日葵属（向日葵族）中，ETS比ITS更具变异性且在系统发育上更具信息性。使用基因间隔区内的一个引物（ETS1f）与高度保守的18S基因5'端的一个引物（18S - 2L）组合来扩增完整的ETS。ETS1f被设计成对应于向日葵属和还阳参属中发现的一个高度保守区域，这两个属属于菊科的不同亚科。ETS1f/18S - 2L引物在菊科的所有族以及桔梗科、草海桐科和卡利塞拉科的代表性分类群中均能引发扩增。对于滨菊属和小滨菊属，当产生单一条带时，我们能够直接对ETS PCR产物进行测序。当产生多条带时，我们在测序前对感兴趣的条带进行凝胶纯化，偶尔还进行克隆。虽然PCR对向日葵属物种产生单一条带，但由于基因组内ETS重复类型多样，在测序前有必要对向日葵属的扩增产物进行克隆。尽管滨菊属和小滨菊属的ETS序列比对直接且明确，尽管5'端存在一些亚重复结构。对于向日葵属，不同物种中不同数量的大串联亚重复需要在比对前分析亚重复的直系同源性。在所有三个属中，ETS为系统发育重建提供了更多信息性变异，并且比ITS能更好地解析亲缘关系。尽管向日葵属的克隆序列存在差异，但基因组内克隆始终形成分支。这一结果表明，ETS中的协同进化进行得足够快，以至于保留了物种特异性的系统发育信号。现在应该可以使用整个ETS对菊科以及至少其他三个科（约26,000种，约占所有被子植物的8%）中最近分化的谱系进行系统发育重建。
