基于叶绿体DNA序列trnL-F分析李亚属植物的系统发育关系

Phylogenetic relationship in the plants of subgenus Prunophora (Rosaceae) inferred from the chloroplast DNA region,trnL-F

【目的】探索李亚属内物种间的系统发育关系,明确最近新命名物种的起源与植物学分类地位。【方法】在克隆测序trnL-F序列的基础上,分析李亚属各种级分类群的分子特征,并进行李亚属的分子系统发育学分析。【结果】在李亚属供试材料中检测到的叶绿体DNA trnL-F序列长度变化为945~956 bp,所有序列的AT含量明显高于GC含量。李亚属内各种间的序列存在着不同的差异,部分种具有显著的分子序列特征,可用于物种鉴定。共检测到21个变异位点和20个单倍型。根据邻接法构建系统发育树,将所有种共分为6个不同的组群。【结论】仅通过trnL-F序列片段无法完全将李亚属的所有种级分类群分开,但是部分种间具有显著的分子特征;中国李(P.salicina)和樱桃李(P.cerasifera)分别是种间杂交种李梅杏(P.limeixing)和紫杏(P.dasycarpa)的母系亲本提供者。

[Objective] The phylogenetic classification of the economically important genus Prunus L. is controversial and the taxonomy of plum and apricots has changed with time, due to the high levels of convergent or the parallel evolution of morphological traits. Most pomologists considered that Prunus should include plums along with peach, cherry, apricot and almond. Integrated Taxonomic Information System （ITIS） divided the Prunophora subgenus into three sections： Euprunus section, Armeniaca section and Prunocerasus section. This study aimed to assess the phylogenetic relationships of new species in the sub- genus Prunophora （Rosaceae） and elucidate the possible origin and its botanical classification. [Methods] Chloroplast DNA fragments （trnL-F） of 45 accessions representing 17 species （including apricots and plums） were amplified by polymerase chain reaction （PCR） method and sequenced in this investigation. Moreover, the additional 22 sequences in the subgenus Prunophora were downloaded from GenBank, and 1 accession of P. tomentosa from GenBank （AM282663） was chosen as out-group. The alignment of DNA sequences was initially performed with DNAMAN and ClustalX, then with subsequent manual adjustment by eye. Variable positions in the data matrices were double checked against the original chromatogram files to make sure that all base calls were true at all variable positions. The SNPs and phylogenetic tree were analyzed using the neighbor-joining method in MEGA 6.0 software. [ Results] Sequencing of trnL-F region was successful in all of the species. Aligned nucleotides ranged from 945 to 956 bp, and the align- ment matrix was 962 bp. The aligned matrix consisted of 941 invariable sites, 9 variable sites that were parsimony un-informative and 12 parsimony-informative sites, in addition, a total of 3 phylogenetically informative indels were coded for inclusion in the analysis. Based on the trnL-F spacer sequences, signifi- cant molecular characteristics were found in some species, which could be used for species identification. For instance, two specific sites of deletion fragments （AAACG） in 554 bp and mutation （T ←→C） in 165 bp only existed in Armeniaca section, such as P. armeniaca, P. holosericea, P. sibirica and P. mandshurica; the mutation site （A←→G） in 660 bp were only detected in P. cerasifera and P. dasycarpa; the mutation site （G←→A） in 288 bp occurred only in P. brigantlna, P. spinosa and wild population of P. domestica; the site- specific mutation, a single-base repeat sequences （T12）, of P. ussuriensis was located in 798 bp; the specific site （A←→G） of P. brigantina was only located in 931 bp. Compared with P. salicina, P. mume, P. limeixing, P. zhenghensis and P. simonii were not any of specific variation sites. We identified 20 haplotypes, which were tentatively divided into six clades. The results of phylogenetic tree showed that taxa of the subgenus Prunophora formed a well-supported clade, and within these remaining species the data were distinguished into six major clades： 1） the apricot group, including the majority of members of the Armeniaca sections, P. armeniaca, P. holosericea, P. sibirica, P. mandshurica; 2） the Chinese plum group I, comprising of the majority of the P. salicina and P. simonii, and P. mume （as Armeniaca section）, P. limeixing, P. zhenghensis; 3） the Chinese plum group II, including one accession of the P. salicina and all of P. ussuriensis; 4） the Asian/Eur plum group, including all the European species, and which were divided into the two groups distributed in the wild populations （P. spinosa, P. brigantina, and wild population of P. domestica） and cultivation populations （P. cerasifera, P. dasycarpa, P. insititia and cultivation population of P. domestica）; 5） only one accession of P. salicina （Fuzhou Qingnai）; 6） two accessions of P. siblrica （Dashanxing and B 1-10-2-1）. [ Conclusion] As a consequence, single trnL-F sequence could not solely used for distinguishing species in the subgenus Prunophora, and the sequences were similar in closely related spe- cies. The results were in accord with the points that P. brigantina belonged to the Euprunus section; P. li- meixing and P. dasycarpa were derived from a natural interspecific hybrid with P. salicina or P. cerasifera as its maternal progenitor and P. armeniaca as the female parent; the Euprunus section was divided into two non-sister sections, and all polyploidy species were closely related to the diploid species of P. cera- sifera, while the P. salicina species was scattered; wild populations of domestica, P. spinosa and P. brigan- tina were close relatives, while cultivation populations of domestica and P. insititia were close relatives.