目的 研究芍藥屬4種藥用植物葉綠體基因組特征、變異程度及芍藥屬和毛茛科植物的系統(tǒng)發(fā)育關(guān)系，為芍藥屬藥用植物的分類地位和系統(tǒng)發(fā)育提供參考。方法 應(yīng)用二代高通量測(cè)序技術(shù)測(cè)定芍藥Paeonia lactiflora葉綠體基因組，利用比較基因組學(xué)方法分析了芍藥與川赤芍P. Veitchii、牡丹P. suffruticosa及楊山牡丹P. ostii等芍藥屬藥用植物葉綠體基因組之間的結(jié)構(gòu)特征及變異程度，以山地虎耳草Saxifraga sinomontana等植物為外類群，分析了芍藥屬植物與毛茛科植物的系統(tǒng)發(fā)育關(guān)系。結(jié)果 芍藥屬4種藥用植物的葉綠體基因組均為典型的四分結(jié)構(gòu)，包含1個(gè)大單拷貝區(qū)（large single copy，LSC）、1個(gè)小單拷貝區(qū)（small single copy，SSC）和2個(gè)反向重復(fù)區(qū)（inverted repeats，IRa和IRb）。芍藥葉綠體基因組序列長(zhǎng)度為152 731 bp，GC含量為38.43%，共注釋到基因126個(gè)，其中82個(gè)蛋白質(zhì)編碼基因、36個(gè)tRNA基因和8個(gè)核糖體rRNA基因。重復(fù)序列分析發(fā)現(xiàn)芍藥葉綠體基因組重復(fù)序列數(shù)量最多為48個(gè)，楊山牡丹最少為39個(gè)；只有芍藥同時(shí)含有4種類型的重復(fù)序列。芍藥含有最多的SSR序列達(dá)52個(gè)，4種植物SSR序列中單核苷酸重復(fù)占比達(dá)81%～86%，絕大多數(shù)為A/T重復(fù)。密碼子偏好性分析表明芍藥屬4種藥用植物中氨基酸出現(xiàn)頻率均以亮氨酸最高，半胱氨酸最低；31種密碼子具偏好性，且密碼子偏好性與密碼子第3位堿基具顯著相關(guān)性。LSC/IRb、IRb/SSC、SSC/IRa和IRa/LSC邊界附近的基因類型相同，相對(duì)保守，但牡丹組和芍藥組之間仍具有差異。選擇壓力分析結(jié)果表明，絕大多數(shù)基因K_a/K_s值均小于1，受純化選擇，matK、ndhB和rpoA受到正選擇。葉綠體基因組比較分析發(fā)現(xiàn)芍藥屬內(nèi)4種藥用植物的葉綠體基因組中非編碼區(qū)比基因編碼區(qū)的變異程度大，篩選出了6個(gè)基因間區(qū)和2個(gè)基因編碼區(qū)的高變異區(qū)。系統(tǒng)發(fā)育分析結(jié)果顯示，芍藥與美麗芍藥親緣關(guān)系最近，所有芍藥屬植物被聚為一個(gè)分支，外類群把芍藥屬和毛茛科植物分隔開來(lái)，芍藥屬植物與毛茛科植物親緣關(guān)系較遠(yuǎn)。結(jié)論 芍藥屬4種植物在葉綠體基因組結(jié)構(gòu)特征方面較為保守，但其變異程度存在一定差異；篩選出的8個(gè)高變異區(qū)可為芍藥屬藥用植物的條形碼開發(fā)、物種鑒定及藥用植物質(zhì)量控制提供候選片段；系統(tǒng)發(fā)育分析表明芍藥屬植物與外類群植物親緣關(guān)系較近，而與毛茛科植物親緣關(guān)系較遠(yuǎn)，為芍藥屬植物系統(tǒng)進(jìn)化、芍藥屬藥用植物的物種鑒定、質(zhì)量控制及保護(hù)開發(fā)等研究奠定基礎(chǔ)。

Objective To understand the chloroplast genome characteristics, degree of variation of four medicinal plants in Paeonia and the phylogenetic relationship between Paeonia and Ranunculaceae. This study will provide a reference for classification status and phylogeny of medicinal plants in Paeonia. Methods High-throughput next-generation sequencing technology was applied to sequence and assemble chloroplast genome of Paeonia lactiflora. Comparative genomics methods were used to analyze the structural characteristics and degree of variation among the chloroplast genomes of four medicinal plants in Paeonia, including P. lactiflora, P. veitchii, P. suffruticosa, and P. ostii. The phylogenetic relationship between Paeonia and Ranunculaceae was analyzed, with Saxifraga sinomontana and other related plants as outgroups. Results The chloroplast genomes of four medicinal plants in Paeonia were all typical tetrad structures, including one large single copy region (LSC), one small single copy region (SSC), and two reverse repeat regions (IRa and IRb). The length of chloroplast genome of P. lactiflora was 152 731 bp, with GC content of 38.43%. A total of 126 genes were annotated, including 82 protein coding genes, 36 tRNA genes, and eight ribosomal rRNA genes. Repetitive sequence analysis revealed that the chloroplast genome of P. lactiflora had the highest number of repetitive sequences at 48, while P. ostii lowest at 39. Only P. lactiflora contains four types of repeat sequences simultaneously. P. lactiflora contained the largest number of SSR sequences with 52. Single nucleotide repeats accounted for 81%—86% of the SSR in the four medicinal plants chloroplast genome, with the vast majority being A/T repeats. Codon preference analysis showed that among the four medicinal plants of Paeonia, the highest frequency of amino acid was leucine, while lowest was cysteine. Thirty-one codons possessed preference, and codon preference was significantly correlated with the third base of the codon. The gene types near the boundaries of LSC/IRb, IRb/SSC, SSC/IRa and IRa/LSC were the same and relatively conserved, but there were still little differences between sect Moutan and Paeonia. The selection pressure analysis showed that the K_a/K_s values of the majority of genes were less than one. Only matK, ndhB, and rpoA were positively selected. Chloroplast genome comparative analysis revealed that the non-coding regions exhibited greater variability than the gene-coding regions. A total of six intergenic regions and two gene coding regions with high variability were screened out. The phylogenetic analysis showed that the relationship between P. lactiflora and P. maire was closest. All species in Paeonia were clustered into one branch, while outgroups can separate Paeonia from Ranunculaceae. The relationship between Paeonia and Ranunculaceae was relatively distant. Conclusion The chloroplast genome structural characteristics of the four medicinal plants in Paeonia are relatively conservative, with certain differences on chloroplast genome variation degree. The eight highly variable regions identified can be served as the candidate fragments for barcode development, species identification and quality control of medicinal plants. Phylogenetic analysis shows that species in Paeonia are more closely related to the outgroup plants, but far related to species in Ranunculaceae. This lays the foundation for phylogeny of Paeonia and research on species identification, quality control, protection and development of medicinal plants resources in Paeonia.

R286.12

國(guó)家自然科學(xué)基金資助項(xiàng)目（U1204323）