Characteristics and Evolution of Panax Chloroplast Genomes
-
摘要:目的 对人参属（Panax L.）物种叶绿体基因组特征及其系统发育进行研究，为我国人参属资源的遗传学研究和开发利用提供理论依据。方法 基于14种人参属植物叶绿体基因组序列，利用生物信息学软件，对叶绿体基因组特征、序列重复、结构变异、基因进化和系统发育进行分析。结果 人参属物种叶绿体基因组均为典型的四分体结构，包含114个unique基因。长重复序列主要为回文重复和正向重复，30～39 bp的重复序列最多，SSR大多为A/T重复，单核苷酸重复是最丰富的类型。人参属叶绿体基因组未发生基因重排，反向重复区（IR）与单拷贝区边界高度保守，鉴定的12个核苷酸高度可变热点中7个位于大单拷贝（LSC）区，5个位于小单拷贝（SSC）区。根据dN/dS比率，发现功能未知基因clpP、ycf1和ycf2受正选择作用。系统发育分析显示，屏边三七和三叶参位于基部支系，四倍体人参和西洋参与其他二倍体物种聚在不同支系，三七、竹节参和越南参则亲缘关系较近。结论 人参属叶绿体基因组基因数目和顺序一致，基因组结构保守，重复序列数目和类型存在差异，单拷贝区核苷酸多态性高于IR区，正选择基因可能与物种的生态适应性有关。Abstract:Objective Characteristics and phylogeny of chloroplast genomes of the medicinally and economically valuable species in Panax genus were studied.Methods Using bioinformatics software, the properties, repeats, structural variation, evolution, and phylogeny of the genomes of chloroplasts from 14 ginseng species were analyzed.Results The genomes consisted of typical quadripartite structure with 114 unique genes. The long repeats in them were mainly of palindromic and forward types with a length between 30 bp and 39 bp. The simple sequence repeats were largely A/T type and most abundantly mononucleotides. No gene rearrangement occurred in the genomes was observed. The boundary between the inverted repeat region and the single copy region was highly conserved. Of the 12 regions with highly variable nucleotides, 7 were in the large and 5 in the small single copy region. Indicated by the dN/dS ratios, the positive selection could occur on clpP, ycf1, and ycf2 with unknown functions. The phylogenetic analysis showed that P. stipulenatus and P. trifolius were in the basal lineage, the tetraploid P. ginseng and P. quinquefolius separated from other diploid species, while P. notoginseng, P. japonicus, and P. vietnamensis closely related.Conclusion The chloroplasts of the ginseng species examined were basically same in number and order of genomes, conservative in structure, but divert in number and type of repeats. The nucleotide polymorphism of the chloroplasts was higher in single copy region than inverted repeat regions. The positive selection genes identified in the study might result from the ecological adaptation of these Panax species.
-
Keywords:
- Panax L. /
- chloroplast genome /
- repeat /
- variation /
- phylogeny
-
0. 引 言
【研究意义】百合叶烧病是一种生理性病害，国内外百合种植过程中普遍发生，严重影响切花百合和盆栽百合生产质量。其发病症状表现为百合生长至30～40 cm，花蕾出现前，顶部6～10片新叶离叶尖约2 cm处出现灰白色病斑，后期逐渐向叶尖蔓延，最后变成焦枯状。百合叶烧病具有显著的品种特异性，但目前尚未发现与叶烧病存在紧密联系的品种特征[1]，因此探索百合叶烧病发病机理，对百合新种质创制和栽培技术创新具有重要意义。【前人研究进展】前人研究发现，通过剪掉一半刚展开的下部叶片[2]、在发病之前喷施氯化钙和硝酸钙溶液[3]、通过手动展开尚未展开的新叶和降低栽培环境的湿度[4]等方法能够降低叶烧病的发生率，同时施用生根剂、微生物菌肥和使用合适的栽培基质均能有效地降低叶烧病的发生[5-7]。【本研究切入点】目前，百合叶烧病发病原因及防治方法等相关研究已较为深入，其中由缺钙导致叶烧病发生已被确认，但由于钙参与多项植物生理调节过程，如何确定叶烧病发生分子机理，以及缺钙导致哪些代谢途径受阻从而产生叶烧病，是今后选育抗叶烧病品种的重要研究基础。【拟解决的关键问题】通过扫描电镜和投射电镜观察东方百合Tarrango正常叶片、轻度叶烧叶片和重度叶烧叶片超微结构的差异，并通过比较东方百合Tarrango正常叶片、叶烧叶片、正常叶片喷钙和叶烧叶片喷钙4种处理的转录组测序数据，以期探索与叶烧病发病相关的分子机理和关键调控基因。
1. 材料与方法
1.1 试验材料
以荷兰进口的Tarrango百合种球为试验材料，种植于连云港市农业科学院东辛农场实验基地日光温室，Tarrango百合生长至现蕾期，对部分百合叶烧叶片和正常叶片喷施30 mmol·L−1 硝酸钙叶面肥一次，其余百合喷施等量清水。24 h后分别取正常叶片（TarCK）、叶烧叶片（TNCK）、叶面喷钙正常叶片（TarCa）和叶面喷钙叶烧叶片（TNCa）4个处理，干冰冷藏送样检测，每个处理生物学重复3次。
1.2 转录组测序及分析试验方法
1.2.1 RNA提取及处理
上述4个处理中，取每个处理叶片混合样品1 g，参照TaKaRA全RNA提取试剂操作说明进行叶片总RNA提取。
1.2.2 文库构建及测序
百合叶片cDNA文库构建及测序均委托苏州金唯智生物科技有限公司完成。
1.2.3 转录组数据组装及基因功能注释
百合叶片转录组测序及对获得的数据库 Unigene 的全面分析和注释，均委托苏州金唯智生物科技有限公司完成。
1.3 百合叶片扫面电镜及透射电镜观察
采集Tarrango正常叶片、轻度发病叶烧叶片和重度发病叶烧叶片（图1），无菌水漂洗干净后，手术刀裁成3 mm×3 mm大小，置于电镜固定液中固定2 h，再转移至4 ℃保存，4 ℃冰袋运输送样至武汉赛维尔生物科技有限公司。
![]()
图 1 不同叶烧程度百合叶片注：A，百合Tarrango正常叶片；B，百合Tarrango轻度叶烧叶片；C，百合Tarrango重度叶烧叶片。图2同。Figure 1. Leaves from different degrees of ULN-infectionNote: A: Leaf from normal lily plant; B: Leaf from lily plant mildly infected by ULN; C: Leaf from lily plant severely infected by ULN. Same for Fig. 2.2. 结果与分析
2.1 百合叶片叶烧病扫描电镜分析
如图2所示，近轴面表皮层均无气孔分布，其中Tarrango正常叶片和Tarrango轻微叶烧叶片的上表皮细胞横直径为500 μm。而Tarrango重度叶烧叶片上表皮细胞发生严重皱缩，其上皮细胞的横直径只有312.5 μm。轻度叶烧时，百合叶片上表皮细胞出现轻微的收缩，重度叶烧时，百合叶片表皮细胞表面出现褶皱痕迹。
如图3所示，百合叶片远轴面表皮层分布了大量气孔，气孔数量最多的是Tarrango重度叶烧叶片（图3-A），其次为Tarrango轻度叶烧叶片（图3-B）和Tarrango正常叶烧叶片（图3-C）叶片。Tarrango正常叶片、Tarrango轻微叶烧叶片和Tarrango重度叶烧叶片气孔直径大小基本一致，均在400 μm左右。Tarrango轻度叶烧叶片下表皮层便发生皱缩现象，重度叶烧叶片皱缩程度更加严重。
![]()
图 3 百合叶片远轴面超微结构注：A，百合Tarrango重度叶烧叶片；B，百合Tarrango轻度叶烧叶片；C，百合Tarrango正常叶片。Figure 3. Ultrastructure of lily leaves in abaxial viewNote: A, Leaf from lily plant severely infected by ULN; B, Leaf from lily plant mildly infected by ULN; C, Leaf from normal lily plant.2.2 百合叶片透射电镜分析
正常叶片叶肉细胞（图4-A、B）和轻度叶烧叶肉细胞（图4-C、D）液泡较大，叶绿体分布在边缘。重度叶烧叶片的叶肉细胞由于液泡失水，叶绿体分布在整个细胞，细胞较为规则；重度叶烧细胞由于失水，整个细胞皱缩，形状不规则（图4-E、F）。在叶烧病发展过程中，叶绿体结构未发生太大变化，基粒垛叠层数较多且排列规律，3个处理中叶绿体均含有数量不等的淀粉粒。但重度叶烧叶片叶肉细胞中，线粒体数量明显较少。
![]()
图 4 百合叶片透射电镜下超微结构注：A和B：百合Tarrango正常叶片；C和D：百合Tarrango轻度叶烧叶片；E和F：百合Tarrango重度叶烧叶片。Figure 4. Ultrastructure of lily leaves shown by TEMNote: A and B: normal leaf of lily Tarrango; C and D: mildly ULN leaf of Lily Tarrango; E and F: severely Lily ULN leaf of Lily Tarrango.2.3 转录组测序组装及unigene注释分析
使用软件Cutadapt对测序原始数据（Pass filter data）去除接头以及低质量序列，获得后续信息分析用的过滤数据（Clean data）。4个处理12个文库产生了541 393 662条序列，约80.68 Gb转录组数据，Unigene平均长度在148.63 ～149.00 bp，GC含量变化范围为49.17%～52.32%（表1）。由表2可以看出，转录组数据经组装产生了19 585 575个重叠群，349537条unigenes，平均长度为513.25 bp。在所有unigenes序列中，序列长度小于500 bp的占75.92%（表3）。Unigene注释分析显示有124 405 unigenes获得注释，占unigenes总数的35.59%，其中获得注释较多的数据库分别是Nr注释121 501（34.76%）、COG注释49 594条（14.19%）、Swissport注释72 421条（20.72%）和KEGG注释17 051条（4.88%）。
表 1 12个cDNA文库的过滤数据Table 1. Clean data of 12 cDNA library样品
Sample平均长度
Average length/bp总序列数
The total number of sequences总碱基数
Total base number /bpQ20含量
Q20 content /%Q30含量
Q30 content /%GC含量
GC /%TarCK1 148.70 46691744 6943031355 98.11 94.32 50.55 TarCK2 148.77 49717384 7396527911 98.26 94.67 49.80 TarCK3 148.63 40433794 6009572727 98.15 94.35 52.27 TarCa1 148.74 47758784 7103493445 98.15 94.44 51.41 TarCa2 148.87 40229314 5988834979 98.07 94.30 52.04 TarCa3 149.00 46500744 6928790175 98.07 94.28 51.59 TNCK1 148.98 43959714 6549244364 98.15 94.42 50.78 TNCK2 148.79 40479744 6022881697 98.23 94.59 49.17 TNCK3 148.87 45992514 6846847661 98.08 94.21 49.91 TNCa1 149.00 44762836 6669452846 98.10 94.41 52.32 TNCa2 148.96 46342564 6903233715 98.04 94.15 51.03 TNCa3 148.89 48524526 7224858395 98.09 94.31 52.03 合计 Total 541393662 80686769270 表 2 转录组序列组装分析Table 2. Summary of transcriptome assembly序列类型
Sequence type重叠群
Contig序列
Unigene最短序列长度 Minimum sequence Length/bp 25 201 最长序列长度 Maximum sequence length/bp 15 734 11 377 序列平均长度 Mean sequence length/bp 53.76 513.25 N50长度 N50 length/bp 48 686 (A+T)/% 51.94 55.72 (C+G)/% 48.06 44.28 序列总数 The total number of sequences 19 585 575 349 537 总碱基数量 Total base number/bp 1 052 959 813 179 400 360 表 3 Unigene 的长度及数量统计Table 3. Unigene length and quantity statistics长度
Length/bp数量
Number比例
Percentage/%<200 0 0.00 200～500 265 367 75.92 500～1 000 44113 12.62 1 000～1 500 17 752 5.08 1 500～2 000 10 495 3.00 ≥2 000 11 809 3.38 总数 Total 349 537 100 2.4 差异表达基因筛选及分析
对4组处理转录组数据进行层次聚类（Hierarchical clustering）分析，喷钙2个处理TarCa与TNCa基因表达模式相近，并且与喷清水2个处理TarCK与TNCK基因表达模式存在显著区别，通过基因表达量可以将差异表达基因划分成A、B两个簇（图5-A），同时通过对转录组数据分组对比分析发现，TarCK/TarCa比较中差异表达基因最多，总数为4 816个，其中上调表达基因2 419个，下调表达基因2 397个；其次TNCK/TNCa比较中差异表达基因2 782个，其中上调表达基因1 090个，下调表达基因1 692个；TarCK/TNCK比较中差异表达基因822个，其中上调表达基因399个，下调表达基因423个；TarCa/TNCa比较中差异表达基因最少，总数为113个，其中上调表达基因56个，下调表达基因57个（图5-B）。
![]()
图 5 差异表达基因的表达谱分析注：A，差异表达基因的聚类分析；B，差异表达基因的数量；C，差异表达基因的主成分分析；D，差异基因维恩图分析。Figure 5. Expression profiling of differentially expressed genesNote: A: Cluster analysis on differentially expressed genes; B: Number of differentially expressed genes; C: Principal component analysis on differentially expressed genes; D: Venn diagram of differential genes.通过PCA（Principal Component Analysis）分析发现，TarCa与TNCa两个处理的重复组间的变异较小，并且两个处理间的差异性也较小，而TarCK与TNCK两个处理间不但处理间的差异性较大，重复组间的变异也比较大（图5-C）。同时，在4组对照分析中，发现了特异的和共同的差异表达基因，共发现7 185个差异表达基因，包括5 860个特异差异表达基因和1 325个共同差异表达基因（图5-D）。其中TarCK/TarCa与TNCK/TNCa共差异表达基因最多，为991个；其次为TarCK/TNCK与TarCK/TarCa共差异表达基因为230个和TNCK/TNCa与TarCK/TNCK共差异表达基因为96个，TarCa/TNCa与TarCK/TNCK共差异表达基因最少，只有1个，通过上述数据表明，喷钙处理能显著降低叶烧叶片与正常叶片的差异表达基因数量。
2.5 差异基因GO富集分析
通过GO富集分析对差异表达基因进行生物学功能研究，在错误发现率FDR＜0.01时，25个GO条目富集在TarCa/TNCa，25个GO条目富集在TarCK/TNCK，31个GO条目富集在TarCK/TarCa，31个GO条目富集在TNCK/TNCa。有18个GO条目在4组对比分析中均有富集，其中，“催化活性” “结合”和“代谢过程”是富集基因最多的GO条目。在4组对比分析中有5个特异性GO条目，其中“多细胞生物过程”和“繁殖”GO条目仅在TarCK/TarCa中富集，“突触部分”GO条目仅在TNCK/TNCa中富集，“细胞外基质”和“细胞外基质成分”GO条目仅在TarCa/TNCa中富集，TarCK/TNCK无特异性GO条目（图6）。
![]()
图 6 差异表达基因的GO富集分析注：1，催化活性；2，结合；3，转运活性；4，结构分子活性；5，电子载体；6，核酸结合转录因子活性；7，酶调节活性；8，抗氧化活性；9，分子传感器活性；10，细胞组分；11，细胞器；12，膜部分；13，细胞器部分；14，细胞膜；15，高分子复合物；16，胞外区；17，类核；18，细胞连接；19，代谢过程；20，细胞过程；21，单一生物过程；22，生物调节；23，应激反应；24，定位；25，发育过程；26，多生物过程；27，免疫系统过程；28，组织细胞组成或生物起源；29，生殖过程；30，多细胞生物过程；31，繁殖；32，细胞外区域部分；33，细胞外基质；34，细胞外基质成分；35，运动；36，生长；37，突触部分。Figure 6. GO enrichment analysis on differentially expressed genesNote: 1: catalytic activity; 2: binding; 3: transporter activity; 4: structural molecule activity; 5: electron carrier activity; 6: nucleic acid binding transcription factor activity; 7: enzyme regulator activity; 8: antioxidant activity; 9: molecular transducer activity; 10: cell part; 11: organelle; 12: membrane part;13: organelle part; 14: membrane; 15: macromolecular complex; 16: extracellular region; 17: nucleoid; 18: cell junction; 19: metabolic process; 20: cellular process; 21: single-organism process; 22: biological regulation; 23: response to stimulus; 24: localization; 25: developmental process; 26: multi-organism process; 27: immune system process; 28: cellular component organization or biogenesis; 29: reproductive process; 30: multicellular organismal process; 31: reproduction; 32: extracellular region part; 33: extracellular matrix; 34: extracellular matrix component; 35: locomotion; 36: growth; 37: synapse part.2.6 差异基因KEGG富集分析
在植物体内，Ca2+参与的代谢调控十分复杂，通过KEGG代谢通路分析，在本次转录组测序中“剪接体” “代谢途径” “丙酮酸代谢” “次生代谢产物的生物合成”和“光合作用生物的碳固定作用”代谢途径在4组试验处理对比分析中都有基因富集，经过差异基因COG分析，4组试验处理对比分析共有25个COG分组，其中4组试验处理共有COG功能分类22个，包括“细胞外结构” “信号转导机制” “未知功能基因” “细胞内运输、分泌和囊泡运输” “翻译、核糖体结构和生物发生” “细胞壁/膜/信封生源论” “一般功能预测基因” “复制、重组和修复” “碳水化合物运输和代谢” “无机离子的转运和代谢” “细胞骨架” “氨基酸的运输和代谢” “脂质运输与代谢” “细胞周期控制，细胞分裂，染色体分裂” “能源生产与转换” “染色质结构和动力学” “翻译后修饰，蛋白质转换，伴侣” “辅酶运输和代谢” “RNA加工和修饰” “次级代谢产物生物合成、运输和分解代谢”。
2.7 显著差异基因分析
结合扫描电镜和透射电镜分析，本研究重点关注叶烧病发病过程和喷施钙处理中，细胞膜通透性相关基因的表达变化。如表4所示，在百合叶烧病发病过程中即TarCK/TNCK对比分析中，显著下调基因有参与脱落酸信号的负调控的FOLK基因（Farnesol kinase 法呢醇激酶）[8]、影响脂类物质生物合成和细胞膜稳定性的PLD1_2基因（phospholipase D1/2 磷脂酶D1/2）[9]、ATPeF1B基因（F-type H+-transporting ATPase subunit bet膜上ATP合酶）和KCS基因（3-ketoacyl-CoA synthase 3-酮脂酰辅酶A合酶）[10]。同时CALM基因（Calmodulin 钙调蛋白）[11]、ENO基因（enolase 烯醇酶）[12]和pel基因（Pectate lyase 果胶酸裂解酶）[13]等响应钙离子信号和影响木质素和果胶生物合成的基因下调。在百合叶烧病发病过程中表达量上调的基因有促进生成脱落酸的AAO基因（abscisic-aldehyde oxidase 脱落醛氧化酶）[14]。
表 4 显著差异表达基因Table 4. Significantly differentially expressed genes对照组
Group基因编号
Gene ID差异倍数
Log2 fold
change基因名称
KO_name基因定义
KO_definitionTarCK/TNCK DN74111_c0_g1_i2 −6.76 FOLK 法呢醇醇激酶 Farnesol kinase TarCK/TNCK DN42826_c0_g2_i2 −6.56 PLD1_2 磷脂酶D1/2 Phospholipase D1/2 TarCK/TNCK DN73032_c0_g2_i1 −3.14 ATPeF1B 膜上ATP合酶 F-type H+-transporting ATPase subunit beta TarCK/TNCK DN59908_c0_g1_i2 −1.88 KCS 3-酮脂酰辅酶A合成酶基因 3-ketoacyl-CoA synthase TarCK/TNCK DN88233_c0_g1_i2 −1.08 ENO 烯醇酶 Enolase TarCK/TNCK DN45172_c0_g1_i1 −1.00 CALM 钙调蛋白 Calmodulin TarCK/TNCK DN75575_c0_g1_i1 6.00 AAO3 脱落醛氧化酶 Abscisic-aldehyde oxidase TNCK/TNCa DN75425_c0_g1_i6 −4.83 ABF ABA响应元件结合因子 ABA responsive element binding factor TNCK/TNCa DN89025_c0_g1_i3 −2.34 MFP2 烯酰辅酶A水合酶/3-羟酰辅酶A脱氢酶 Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase TNCK/TNCa DN10149_c0_g1_i1 1.09 AUX1 生长素流入载体蛋白 Auxin influx carrier TNCK/TNCa DN36010_c0_g1_i1 1.20 CALM 钙调蛋白 Calmodulin TNCK/TNCa DN81084_c0_g4_i2 1.29 CPK 钙依赖性蛋白激酶 Calcium-dependent protein kinase TNCK/TNCa DN132576_c0_g1_i1 1.64 CML 钙结合蛋白CML Calcium-binding protein CML TNCK/TNCa DN55671_c1_g1_i8 2.01 PLD1_2 磷脂酶D1/2 Phospholipase D1/2 TNCK/TNCa DN1778_c0_g3_i1 2.45 SORD L-艾杜糖醇-2-脱氢酶 L-iditol 2-dehydrogenase TNCK/TNCa DN48191_c0_g1_i1 2.61 EIN2 乙烯不敏感蛋白2 Ethylene-insensitive protein 2 而在叶烧叶片喷钙后即TNCK/TNCa对比分析中，表达量上调的基因有CALM基因、CPK基因（Calcium-dependent protein kinase 钙依赖性蛋白激酶）、EIN2基因 （ethylene-insensitive protein 2 乙烯不敏感蛋白2）[15]、AUX1基因（Auxin influx carrier 生长素流入载体）[16]、PLD1_2基因和果糖和甘露糖的代谢途径的SORD基因（L-iditol 2-dehydrogenase L-艾杜糖醇-2-脱氢酶）[17]。与此同时下调表达的基因有ABF基因（ABA responsive element binding factor ABA响应元件结合因子）[18]和参与脂肪酸降解途径的MFP2基因 （Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase烯酰辅酶A水合酶/3-羟酰辅酶A脱氢酶）[19]。
3. 讨论与结论
通过百合叶片扫描电镜分析，叶片近轴面表皮细胞大小随叶烧程度的加深而减小，重度叶烧时，表皮细胞发生皱缩，表明此时表皮细胞失水。叶片远轴面表皮细胞在轻度叶烧时发生皱缩，而近轴面表皮细胞在重度叶烧时才发生皱缩，说明叶烧症状先出现在远轴面一侧。通过透射电镜分析，百合叶片在发生叶烧的过程中，液泡失水是导致表皮细胞体积缩小的原因。因此，推测细胞膜和液泡膜的通透性发生改变是导致百合叶片叶烧病症状的原因。
差异表达基因的PCA分析发现，正常叶片3个重复处理和叶烧叶片3个重复处理之间的差异性都很大，而正常叶片喷钙后3各处理和叶烧叶片喷钙后3个处理之间差异性较小。同时在对比分析中也发现，TarCa/TNCa比较组差异表达基因显著少于TarCK与TNCK，都说明喷钙处理能降低正常叶片和叶烧叶片之间的差异表达基因数量，甚至喷钙处理能够降低正常叶片和叶烧叶片之间差异表达基因数量，说明喷钙处理能缓解叶烧病的发病过程，这与白菜干烧心[20-21]、莴苣[22]等缺钙生理性病害的一致。
在分析单个基因表达中发现，在叶烧病发病过程中，响应钙离子信号CALM基因显著下调，而喷钙处理后CALM和CPK基因显著上调，说明CALM基因是叶烧病发病过程中的主要信号传导基因。同时，叶烧病发病过程中促进生成脱落酸的AAO基因显著上调，叶烧叶片喷钙后乙烯信号调控基因EIN2和生长素输入载体基因AUX1表达量上调，表明叶烧病发病可能受脱落酸、乙烯和生长素的调控。同时，叶烧病发病过程中，在细胞膜和液泡膜稳定相关基因phospholipase D1/2、ATPeF1B 、KCS等表达均显著下调。而喷钙后phospholipase D1/2 显著上调，这与拟南芥中PLD及其产物磷脂酸（PA）调控ABA响应的负调控因子[23-24]结果一致。
钙在植物生理活动中，既起着结构成分的作用，也具有酶的辅助因素功能，它能维持细胞壁、细胞膜及膜结合蛋白的稳定性，参与细胞内各种生长发育的调控作用。因此目前虽然基本证实百合叶烧病是由缺钙导致，但其具体的致病分子机理仍不能十分明晰，今后仍需开展相关基因分析验证工作，为百合分子育种提供理论支撑。
-
![]()
图 1 人参属物种叶绿体基因组长重复序列类型及分布
A：正向、回文、反向、互补重复数量；B：长重复类型及数量。
Figure 1. Types and distribution of long repeats in chloroplast genomes of Panax species
A: Counts of forward, palindromic, reverse and complementary repeats; B: types and counts of long repeats.
![]()
图 2 人参属物种叶绿体基因组SSR位点类型及分布
A：单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸、六核苷酸重复数量；B：SSR类型及数量。
Figure 2. Types and distribution of SSRs in chloroplast genomes of Panax species
A: Counts of mono-, di-, tri-, tetra-, penta- and hexanucleotides; B: types and counts of SSR.
![]()
图 3 人参属物种叶绿体基因组LSC、SSC和IRs边界比较
Figure 3. Borders of LSC, SSC, and IRs in chloroplast genomes of Panax species
![]()
图 5 人参属物种叶绿体基因组序列多态性分析
Figure 5. Nucleotide diversity of chloroplast genomes of Panax species
![]()
图 6 人参属叶绿体蛋白编码基因dN/dS分析
Figure 6. dN/dS ratios on protein coding genes of chloroplasts of Panax species
![]()
图 7 基于IQ-TREE软件构建的人参属物种叶绿体基因组系统发育树
Figure 7. Phylogenetic tree of chloroplast genes of Panax species based on IQ-TREE
表 1 人参属物种叶绿体基因组特征
Table 1 Chloroplast genomes of Panax species
物种 Species 登录号 Accession 长度 Length/bp GC含量 GC content/% 基因组 Genome LSC SSC IR 基因组 Genome LSC SSC IR 人参
P. ginsengMK408938 156 241 86 128 18 077 26 018 38.07 36.27 32.18 43.10 竹节参
P. japonicusKP036469 156 188 86 199 18 013 25 988 38.07 36.29 32.21 43.05 疙瘩七
P. japonicus var. bipinnatifidusMK408962 156 244 86 186 18 006 26 026 38.06 36.28 32.21 43.05 珠子参
P. majorMN496312 156 402 86 189 18 007 26 103 38.07 36.28 32.22 43.05 三七
P. notoginsengMK408945 156 319 86 157 18 004 26 079 38.07 36.27 32.23 43.07 假人参
P. pseudoginsengMW145449 156 074 86 124 18 008 25 971 38.06 36.27 32.22 43.06 西洋参
P. quinquefoliusMK408953 156 070 86 077 17 993 26 000 38.07 36.27 32.22 43.08 屏边三七
P. stipuleanatusMK408965 156 007 86 083 18 150 25 887 38.04 36.26 32.07 43.08 三叶参
P. trifoliusMF100782 156 157 86 322 18 047 25 894 38.08 36.27 32.26 43.10 越南参
P. vietnamensisKP036470 155 993 86 178 17 935 25 940 38.05 36.27 32.22 43.02 野三七
P. vietnamensis var. fuscidiscusMT798585 156 284 86 171 17 971 26 071 38.06 36.28 32.27 42.98 越南参变种
P. vietnamensis var. langbianensisMT798583 155 984 86 174 17 934 25 938 38.05 36.27 32.21 43.03 峨眉三七
P. wangianusMK408964 156 189 86 190 17 969 26 015 38.07 36.29 32.21 43.03 姜状三七
P. zingiberensisMK408969 156 192 86 116 17 966 26 055 38.07 36.31 32.27 43.00 
下载: 导出CSV
表 2 基于位点模型的人参属叶绿体蛋白编码基因正选择分析
Table 2 Potential positive selection test on chloroplast genomes of Panax species based on site model
基因
GeneM1 vs M2 M7 vs M8 正选择位点
Positively selected sites2ΔlnL P 值 2ΔlnL P 值 accD 2.39 3.0×10-1 2.47 2.9×10-1 139 Q, 0.505; 141 S, 0.517; 144 R, 0.528; 209 A, 0.856; 264 E, 0.504; 364 I, 0.507; 372 L, 0.526; 494 Q, 0.855 atpF 3.37 1.9×10-1 3.42 1.8×10-1 14 W, 0.541; 77 Q, 0.537; 158 G, 0.854 cemA 7.48 2.4×10-2 7.48 2.4×10-2 14 A, 0.547; 22 W, 0.538; 54 K, 0.536; 102 D, 0.533; 103 R, 0.544; 118 V, 0.973*; 205 F, 0.531; 208 W, 0.540 clpP 13.40 1.2×10-3 13.40 1.2×10-3 38 P, 0.990*; 39 V, 0.622; 40 A, 0.935; 41 S, 0.612; 138 G, 0.629; 160 Q, 0.626 matK 19.22 6.7×10-5 19.25 6.6×10-5 83 R, 0.994** ndhD 0.77 6.8×10-1 0.99 6.1×10-1 22 F, 0.830; 41 I, 0.829 ndhF 19.81 5.0×10-5 20.29 3.9×10-5 17 P, 0.597; 123 R, 0.592; 133 T, 0.583; 223 F, 0.959*; 421 I, 0.964*; 463 Q, 0.591; 483 R, 0.569; 499 H, 0.552; 502 A, 0.965*; 511 Q, 0.591; 512 M, 0.526; 532 K, 0.559; 534 I, 0.962*; 572 N, 0.543; 598 D, 0.573; 606 F, 0.548; 621 L, 0.630; 641 I, 0.549; 642 G, 0.597; 657 G, 0.594; 670 A, 0.963*; 677 I, 0.532; 703 S, 0.548; 737 Y, 0.964*; 743 F, 0.537; 745 D, 0.958*; 746 L, 1.000** rbcL 15.94 3.5×10-4 16.10 3.2×10-4 225 L, 0.632; 226 Y, 0.969*; 230 A, 0.635; 270 I, 0.866; 281 A, 0.644; 328 S, 0.999**; 353 F, 0.584; 439 R, 0.968*; 472 V, 0.609; 474 I, 0.621; 478 V, 0.714 rpl2 0 1 0 1 99 K, 0.651; 265 D, 0.650; 267 L, 0.652 rpoA 14.53 7.0×10-4 14.56 6.9×10-4 34 L, 0.506; 256 E, 0.991**; 264 N, 0.999**; 272 S, 0.533; 320 A, 0.849; 337 L, 0.537 rpoB 0.90 6.4×10-1 0.90 6.4×10-1 37 Y, 0.505; 165 R, 0.502; 193 R, 0.502; 295 I, 0.502; 365 T, 0.504; 459 S, 0.502; 695 K, 0.506; 735 T, 0.504; 935 D, 0.832 rpoC2 0 1 0 1 85 Q, 0.736 rps12 0 1 0.02 9.9×10-1 25 R, 0.502 ycf1 75.81 0 75.78 0 205 K, 1.000**; 206 Y, 0.999**; 288 I, 0.876; 532 T, 0.803; 533 K, 0.956*; 604 R, 0.801; 624 R, 0.825; 668 I, 0.779; 685 W, 0.837; 699 V, 0.824; 701 Q, 0.802; 706 V, 0.814; 720 V, 0.856; 722 T, 0.814; 723 D, 0.808; 731 R, 0.982*; 733 K, 0.809; 734 I, 0.996**; 736 L, 0.855; 737 I, 0.803; 751 N, 0.760; 809 A, 0.828; 906 N, 0.768; 914 E, 0.795; 915 L, 0.788; 929 L, 0.835; 957 K, 0.979*; 1030 E, 0.979*; 1116 K, 0.804; 1172 M, 0.731; 1186 Q, 0.798; 1206 Q, 0.798; 1502 Q, 0.754; 1601 I, 1.000**; 1616 D, 0.773;
1631 R, 0.814; 1652 R, 0.795; 1665 V, 0.738; 1685 E, 0.978*; 1710 I, 0.979*; 1739 R, 0.793ycf2 14.95 5.7×10-4 14.95 5.7×10-4 30 R, 0.767; 56 R, 0.770; 103 L, 0.765; 126 S, 0.765; 248 S, 0.769; 285 L, 0.765; 320 L, 0.766; 332 R, 0.764; 432 V, 0.765; 466 Y, 0.765; 573 I, 0.764; 598 L, 0.766; 606 M, 0.759; 759 F, 0.763; 850 S, 0.965*; 943 E, 0.764; 1037 P, 0.766; 1050 F, 0.765; 1064 S, 0.763; 1074 I, 0.965*; 1093 L, 0.767; 1101 N, 0.964*; 1171 W, 0.767; 1173 S, 0.765; 1245 P, 0.766; 1265 D, 0.762; 1309 D, 0.762; 1344 R, 0.764; 1422 L, 0.965*; 1431 F, 0.763; 1522 D, 0.767; 1550 Y, 0.766; 1656 N, 0.763; 1717 S, 0.768; 1799 R, 0.769; 2045 E, 0.764 *和**分别表示正选择位点概率＞ 95%和＞ 99%。
* and ** indicate that the probabilities of positively selected sites are ＞ 95% and ＞ 99%, respectively.
下载: 导出CSV
-
[1] WEN J, ZIMMER E A. Phylogeny and biogeography of Panax L. (The Ginseng Genus, Araliaceae): Inferences from ITS sequences of nuclear ribosomal DNA [J]. Molecular Phylogenetics and Evolution, 1996, 6(2): 167−177. DOI: 10.1006/mpev.1996.0069
[2] NGUYEN V B, LINH GIANG V N, WAMINAL N E, et al. Comprehensive comparative analysis of chloroplast genomes from seven Panax species and development of an authentication system based on species-unique single nucleotide polymorphism markers [J]. Journal of Ginseng Research, 2020, 41(1): 135−144. DOI: 10.1016/j.jgr.2018.06.003
[3] NGUYEN V B, PARK H S, LEE S C, et al. Authentication markers for five major Panax species developed via comparative analysis of complete chloroplast genome sequences [J]. Journal of Agricultural and Food Chemistry, 2017, 65(30): 6298−6306. DOI: 10.1021/acs.jafc.7b00925
[4] LI S, WANG P, YANG W Z, et al. Characterization of the components and pharmacological effects of mountain-cultivated ginseng and garden ginseng based on the integrative pharmacology strategy [J]. Frontiers in Pharmacology, 2021, 12: 659954. DOI: 10.3389/fphar.2021.659954
[5] SHIN B K, KWON S W, PARK J H. Chemical diversity of ginseng saponins from Panax ginseng [J]. Journal of Ginseng Research, 2015, 39(4): 287−298. DOI: 10.1016/j.jgr.2014.12.005
[6] CHOI H I, KIM N H, LEE J, et al. Evolutionary relationship of Panax ginseng and P. quinquefolius inferred from sequencing and comparative analysis of expressed sequence tags [J]. Genetic Resources and Crop Evolution, 2013, 60(4): 1377−1387. DOI: 10.1007/s10722-012-9926-3
[7] YANG Z J, LIU G Z, ZHANG G H, et al. The chromosome-scale high-quality genome assembly of Panax notoginseng provides insight into dencichine biosynthesis [J]. Plant Biotechnology Journal, 2021, 19(5): 869−871. DOI: 10.1111/pbi.13558
[8] LIU C, CHEN H H, TANG L Z, et al. Plastid genome evolution of a monophyletic group in the subtribe Lauriineae (Laureae, Lauraceae)[J]. Plant Diversity, 2022 , 44 (4): 377 − 388. DOI: 10.1016/j.pld.2021.11.009
[9] 刘潮, 韩利红, 彭悦, 等. 黄丹木姜子叶绿体基因组特征分析 [J]. 南方农业学报, 2022, 53(1):12−20. DOI: 10.3969/j.issn.2095-1191.2022.01.002 LIU C, HAN L H, PENG Y, et al. Characteristics of chloroplast genome of Litsea elongata(wall. ex nees)Benth. et hook. F [J]. Journal of Southern Agriculture, 2022, 53(1): 12−20.(in Chinese) DOI: 10.3969/j.issn.2095-1191.2022.01.002
[10] 刘潮, 韩利红, 代小波, 等. 辣椒属叶绿体基因组特征及进化 [J]. 热带作物学报, 2022, 43(3):447−454. DOI: 10.3969/j.issn.1000-2561.2022.03.002 LIU C, HAN L H, DAI X B, et al. Characteristics and phylogenetics of the complete chloroplast genomes of Capsicum species [J]. Chinese Journal of Tropical Crops, 2022, 43(3): 447−454.(in Chinese) DOI: 10.3969/j.issn.1000-2561.2022.03.002
[11] YU J J, FU J, FANG Y P, et al. Complete chloroplast genomes of Rubus species (Rosaceae) and comparative analysis within the genus [J]. BMC Genomics, 2022, 23(1): 32. DOI: 10.1186/s12864-021-08225-6
[12] 刘潮, 唐利洲, 韩利红. 四川山胡椒叶绿体基因组特征及山胡椒属系统发育 [J]. 林业科学, 2021, 57(12):167−174. LIU C, TANG L Z, HAN L H. Characterization of the chloroplast genome of Lindera setchuenensis and phylogenetics of the genus Lindera [J]. Scientia Silvae Sinicae, 2021, 57(12): 167−174.(in Chinese)
[13] LIU C K, YANG Z Y, YANG L F, et al. The complete plastome of Panax stipuleanatus: Comparative and phylogenetic analyses of the genus Panax (Araliaceae) [J]. Plant Diversity, 2018, 40(6): 265−276. DOI: 10.1016/j.pld.2018.11.001
[14] 岳杰. 四种人参属植物叶绿体全基因组结构及其系统发育分析[D]. 汉中: 陕西理工大学, 2021 YUE J. Structural and phylogenetic analyses of the complete chloroplast genomes of four species within the genus Panax linn. [D]. Hanzhong: Shaanxi University of Technology, 2021. (in Chinese)
[15] KURTZ S, CHOUDHURI J V, OHLEBUSCH E, et al. REPuter: The manifold applications of repeat analysis on a genomic scale [J]. Nucleic Acids Research, 2001, 29(22): 4633−4642. DOI: 10.1093/nar/29.22.4633
[16] BEIER S, THIEL T, MÜNCH T, et al. MISA-web: A web server for microsatellite prediction [J]. Bioinformatics, 2017, 33(16): 2583−2585. DOI: 10.1093/bioinformatics/btx198
[17] AMIRYOUSEFI A, HYVÖNEN J, POCZAI P. IRscope: An online program to visualize the junction sites of chloroplast genomes [J]. Bioinformatics (Oxford, England), 2018, 34(17): 3030−3031. DOI: 10.1093/bioinformatics/bty220
[18] FRAZER K A, PACHTER L, POLIAKOV A, et al. VISTA: Computational tools for comparative genomics[J]. Nucleic Acids Research, 2004, 32(Web Server issue): W273-W279.
[19] KATOH K, ROZEWICKI J, YAMADA K D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization [J]. Briefings in Bioinformatics, 2019, 20(4): 1160−1166. DOI: 10.1093/bib/bbx108
[20] ROZAS J, FERRER-MATA A, SÁNCHEZ-DELBARRIO J C, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets [J]. Molecular Biology and Evolution, 2017, 34(12): 3299−3302. DOI: 10.1093/molbev/msx248
[21] XU B, YANG Z H. PAMLX: A graphical user interface for PAML [J]. Molecular Biology and Evolution, 2013, 30(12): 2723−2724. DOI: 10.1093/molbev/mst179
[22] MINH B Q, SCHMIDT H A, CHERNOMOR O, et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era [J]. Molecular Biology and Evolution, 2020, 37(5): 1530−1534. DOI: 10.1093/molbev/msaa015
[23] GANLEY A R D, KOBAYASHI T. Monitoring the rate and dynamics of concerted evolution in the ribosomal DNA repeats of Saccharomyces cerevisiae using experimental evolution [J]. Molecular Biology and Evolution, 2011, 28(10): 2883−2891. DOI: 10.1093/molbev/msr117
[24] ZEB U, DONG W L, ZHANG T T, et al. Comparative plastid genomics of Pinus species: Insights into sequence variations and phylogenetic relationships [J]. Journal of Systematics and Evolution, 2020, 58(2): 118−132. DOI: 10.1111/jse.12492
[25] ZHU B, QIAN F, HOU Y F, et al. Complete chloroplast genome features and phylogenetic analysis of Eruca sativa (Brassicaceae) [J]. PLoS One, 2021, 16(3): e0248556. DOI: 10.1371/journal.pone.0248556
[26] LI D M, YE Y J, XU Y C, et al. Complete chloroplast genomes of Zingiber montanum and Zingiber zerumbet: Genome structure, comparative and phylogenetic analyses [J]. PLoS One, 2020, 15(7): e0236590. DOI: 10.1371/journal.pone.0236590
[27] XU K W, LIN C X, LEE S Y, et al. Comparative analysis of complete Ilex (Aquifoliaceae) chloroplast genomes: Insights into evolutionary dynamics and phylogenetic relationships [J]. BMC Genomics, 2022, 23(1): 203. DOI: 10.1186/s12864-022-08397-9
[28] KASHI Y, KING D G. Simple sequence repeats as advantageous mutators in evolution [J]. Trends in Genetics, 2006, 22(5): 253−259. DOI: 10.1016/j.tig.2006.03.005
[29] HAN C Y, DING R, ZONG X Y, et al. Structural characterization of Platanthera ussuriensis chloroplast genome and comparative analyses with other species of Orchidaceae [J]. BMC Genomics, 2022, 23(1): 84. DOI: 10.1186/s12864-022-08319-9
[30] YAN J W, LI J H, YU L, et al. Comparative chloroplast genomes of Prunus subgenus Cerasus (Rosaceae): Insights into sequence variations and phylogenetic relationships [J]. Tree Genetics & Genomes, 2021, 17(6): 50.
[31] 马孟莉, 张薇, 孟衡玲, 等. 草果叶绿体基因组特征及系统发育分析 [J]. 中草药, 2021, 52(19):6023−6031. DOI: 10.7501/j.issn.0253-2670.2021.19.025 MA M L, ZHANG W, MENG H L, et al. Characterization and phylogenetic analysis of the complete chloroplast genome of Amomum tsao-ko [J]. Chinese Traditional and Herbal Drugs, 2021, 52(19): 6023−6031.(in Chinese) DOI: 10.7501/j.issn.0253-2670.2021.19.025
[32] KIM K J. Complete chloroplast genome sequences from Korean ginseng (Panax schinseng nees) and comparative analysis of sequence evolution among 17 vascular plants [J]. DNA Research, 2004, 11(4): 247−261. DOI: 10.1093/dnares/11.4.247
[33] LI R, MA P F, WEN J, et al. Complete sequencing of five Araliaceae chloroplast genomes and the phylogenetic implications [J]. PLoS One, 2013, 8(10): e78568. DOI: 10.1371/journal.pone.0078568
[34] TROFIMOV D, CADAR D, SCHMIDT-CHANASIT J, et al. A comparative analysis of complete chloroplast genomes of seven Ocotea species (Lauraceae) confirms low sequence divergence within the Ocotea complex [J]. Scientific Reports, 2022, 12: 1120. DOI: 10.1038/s41598-021-04635-4
[35] YANG J, CHIANG Y C, HSU T W, et al. Characterization and comparative analysis among plastome sequences of eight endemic Rubus (Rosaceae) species in Taiwan [J]. Scientific Reports, 2021, 11: 1152. DOI: 10.1038/s41598-020-80143-1
[36] NIU Y F, GAO C W, LIU J. Comparative analysis of the complete plastid genomes of Mangifera species and gene transfer between plastid and mitochondrial genomes [J]. PeerJ, 2021, 9: e10774. DOI: 10.7717/peerj.10774
[37] GARUD N R, POLLARD K S. Population genetics in the human microbiome [J]. Trends in Genetics:TIG, 2020, 36(1): 53−67. DOI: 10.1016/j.tig.2019.10.010
[38] JI Y H, LIU C K, YANG Z Y, et al. Testing and using complete plastomes and ribosomal DNA sequences as the next generation DNA barcodes in Panax (Araliaceae) [J]. Molecular Ecology Resources, 2019, 19(5): 1333−1345. DOI: 10.1111/1755-0998.13050
[39] KIM N H, CHOI H I, KIM K H, et al. Evidence of genome duplication revealed by sequence analysis of multi-loci expressed sequence tag-simple sequence repeat bands in Panax ginseng Meyer [J]. Journal of Ginseng Research, 2014, 38(2): 130−135. DOI: 10.1016/j.jgr.2013.12.005
计量
- 文章访问数: 587
- HTML全文浏览量: 170
- PDF下载量: 25
