浙江农业学报 ›› 2024, Vol. 36 ›› Issue (3): 544-558.DOI: 10.3969/j.issn.1004-1524.20230443
基于牡丹涝害胁迫的转录组分析及SSR引物开发
刘慧春(
), 许雯婷, 周江华, 张加强, 史小华, 朱开元()
- 浙江省园林植物与花卉研究所,浙江 杭州 311251
-
收稿日期:2023-04-10出版日期:2024-03-25发布日期:2024-04-09 -
作者简介:刘慧春(1979—),女,湖北武汉人,博士,副研究员,主要从事江南牡丹资源收集、遗传育种及抗逆性机理研究。E-mail:lhuichun@163.com -
通讯作者:*朱开元,E-mail:kyzhu1999@163.com -
基金资助:浙江省公益技术应用研究项目(LGN22C150015)
Transcriptomic analysis and simple sequence repeat markers development of Paeonia suffruticosa L. in responses to waterlogging stress
LIU Huichun(), XU Wenting, ZHOU Jianghua, ZHANG Jiaqiang, SHI Xiaohua, ZHU Kaiyuan()
- Zhejiang Institute of Landscaping Plants and Flowers, Hangzhou 311251, China
-
Received:2023-04-10Online:2024-03-25Published:2024-04-09
摘要：
涝害胁迫是限制牡丹种植、生长和高产的最主要的非生物胁迫之一。为了阐明牡丹在涝害胁迫下的作用机制,研究进行了转录组学分析,并开发了SSR标记。利用高通量测序对6个由涝害处理的幼苗和对照的mRNA构建的cDNA文库进行测序。通过组装总共获得73 925个基因,创建了牡丹的初始参考转录组数据库。其中780个被鉴定为涝害早期反应基因,其中155个基因上调,625个基因下调。功能分析表明,参与转录因子信号调节、DNA复制、核糖体和嘧啶代谢的基因表达的改变可能在牡丹对涝害胁迫的反应中发挥重要作用。基于这些高通量转录组测序数据,挖掘出5 204个SSR标记。在这些标记中,二核苷酸重复占58.13%,三核苷酸重复占27.11%,四核苷酸重复占7.24%。在牡丹耐涝性状靶基因相关的功能注释基因中,发现了110对SSR标记引物,筛选出45对引物,其中12对能扩增出清晰的条带,多态性SSR标记引物占引物总数的26.67%。研究结果不仅有助于阐明牡丹的涝害反应机制,同时也为耐涝牡丹品种的分子标记辅助选择育种提供了宝贵的基因组资源和科学依据。
中图分类号:
引用本文
刘慧春, 许雯婷, 周江华, 张加强, 史小华, 朱开元. 基于牡丹涝害胁迫的转录组分析及SSR引物开发[J]. 浙江农业学报, 2024, 36(3): 544-558.
LIU Huichun, XU Wenting, ZHOU Jianghua, ZHANG Jiaqiang, SHI Xiaohua, ZHU Kaiyuan. Transcriptomic analysis and simple sequence repeat markers development of Paeonia suffruticosa L. in responses to waterlogging stress[J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 544-558.
图1 牡丹转录组unigene的长度分布和丰度
Fig.1 Length distribution and abundance of unigenes from the P. suffruticosa transcriptome
图1 牡丹转录组unigene的长度分布和丰度
Fig.1 Length distribution and abundance of unigenes from the P. suffruticosa transcriptome
图2 unigene BLASTX比对结果的物种分布图 A,葡萄;B,核桃;C,可可;D,土瓶草; E,莲藕;F,酸枣;G,梅花;H,麻疯树;I,柑橘; J,木豆。
Fig.2 Species distribution of unigene BLASTX results A, Vitis vinifera; B, Juglans regia; C, Theobroma cacao; D, Cephalotus follicularis; E, Nelumbo nucifera; F, Ziziphus jujuba; G, Prunus mume; H, Jatropha curcas; I, Citrus sinensis; J, Cajanus cajan.
图2 unigene BLASTX比对结果的物种分布图 A,葡萄;B,核桃;C,可可;D,土瓶草; E,莲藕;F,酸枣;G,梅花;H,麻疯树;I,柑橘; J,木豆。
Fig.2 Species distribution of unigene BLASTX results A, Vitis vinifera; B, Juglans regia; C, Theobroma cacao; D, Cephalotus follicularis; E, Nelumbo nucifera; F, Ziziphus jujuba; G, Prunus mume; H, Jatropha curcas; I, Citrus sinensis; J, Cajanus cajan.
图3 涝害胁迫下牡丹差异表达基因的GO分类图 a-q,生物过程(a,再生产;b,免疫系统过程;c,代谢过程;d,细胞过程;e,再生过程;f,生物黏附;g,信号传导;h,多细胞生物过程;i,发展过程;j,增长;k,单体过程;l,韵律过程;m,对刺激的反应;n,本地化;o,多生物过程;p,生物调控;q,细胞成分组织或生物发生); r-z,A-F,细胞成分 (r,细胞外区域;s,细胞;t,膜;u,病毒粒子;v,细胞连接;w,细胞外基质;x,膜封闭管腔;y,大分子复合物;z,细胞器;A,细胞外基质成分;B,细胞器部分;C,病毒粒子部分;D,膜部分;E,细胞部分;F,超分子纤维); G-Q,分子功能 (G,转录因子活性、蛋白质结合;H,核酸结合转录因子活性;I,催化活性;J,信号转换器活动;K,结构分子活性;L,转运蛋白活性;M,绑定;N,电子载体活性;O,抗氧化活性;P,分子转导活性;Q,分子功能调节剂)。
Fig.3 GO classification map of differentially expressed genes during waterlogging stress in P. suffruticosa a-q, Biological process (a, reproduction; b, immune system process; c, metabolic process; d, cellular process; e, reproductive process; f, biological adhesion; g, signaling; h, multicellular organismal process; I, developmental process; j, growth; k, single-organism process; l, rhythmic process; m, response to stimulus; n, localization; o, multi-organism process; p, biological regulation; q, cellular component organization or biogenesis) ; r-z, A-F, Cellular component (r, extracellular region; s, cell; t, membrane; u, virion; v, cell junction; w, extracellular matrix; x, membrane-enclosed lumen; y, macromolecular complex; z, organelle; A, extracellular matrix component; B, organelle part; C, virion part; D, membrane part; E, cell part; F, supramolecular fiber) ; G-Q, Molecular function (G, transcription factor activity, protein binding; H, nucleic acid binding transcription factor activity; I, catalytic activity; J, signal transducer activity; K, structural molecule activity; L, transporter activity; M, binding; N, electron carrier activity; O, antioxidant activity; P, molecular transducer activity; Q, molecular function regulator).
图3 涝害胁迫下牡丹差异表达基因的GO分类图 a-q,生物过程(a,再生产;b,免疫系统过程;c,代谢过程;d,细胞过程;e,再生过程;f,生物黏附;g,信号传导;h,多细胞生物过程;i,发展过程;j,增长;k,单体过程;l,韵律过程;m,对刺激的反应;n,本地化;o,多生物过程;p,生物调控;q,细胞成分组织或生物发生); r-z,A-F,细胞成分 (r,细胞外区域;s,细胞;t,膜;u,病毒粒子;v,细胞连接;w,细胞外基质;x,膜封闭管腔;y,大分子复合物;z,细胞器;A,细胞外基质成分;B,细胞器部分;C,病毒粒子部分;D,膜部分;E,细胞部分;F,超分子纤维); G-Q,分子功能 (G,转录因子活性、蛋白质结合;H,核酸结合转录因子活性;I,催化活性;J,信号转换器活动;K,结构分子活性;L,转运蛋白活性;M,绑定;N,电子载体活性;O,抗氧化活性;P,分子转导活性;Q,分子功能调节剂)。
Fig.3 GO classification map of differentially expressed genes during waterlogging stress in P. suffruticosa a-q, Biological process (a, reproduction; b, immune system process; c, metabolic process; d, cellular process; e, reproductive process; f, biological adhesion; g, signaling; h, multicellular organismal process; I, developmental process; j, growth; k, single-organism process; l, rhythmic process; m, response to stimulus; n, localization; o, multi-organism process; p, biological regulation; q, cellular component organization or biogenesis) ; r-z, A-F, Cellular component (r, extracellular region; s, cell; t, membrane; u, virion; v, cell junction; w, extracellular matrix; x, membrane-enclosed lumen; y, macromolecular complex; z, organelle; A, extracellular matrix component; B, organelle part; C, virion part; D, membrane part; E, cell part; F, supramolecular fiber) ; G-Q, Molecular function (G, transcription factor activity, protein binding; H, nucleic acid binding transcription factor activity; I, catalytic activity; J, signal transducer activity; K, structural molecule activity; L, transporter activity; M, binding; N, electron carrier activity; O, antioxidant activity; P, molecular transducer activity; Q, molecular function regulator).
图4 涝害胁迫下牡丹差异表达基因的KOG分类和分析 A,RNA加工和修饰;B,染色质结构和动力学;C,能源产生与转换;D,细胞周期控制、细胞分裂、染色体分割;E,氨基酸运输和代谢;F,核苷酸运输和代谢;G,碳水化合物运输和代谢;H,辅酶运输和代谢;I,脂质运输和代谢;J,翻译、核糖体结构与生物发生;K,转录;L,复制、重组和修复;M,细胞壁/膜/包膜生物发生;N,细胞运动性;O,翻译后修饰、蛋白质转运蛋白、伴侣蛋白;P,无机离子运输和代谢;Q,次生代谢产物的生物合成、运输和分解代谢;R,仅通用功能预测;S,功能未知;T,信号转导机制;U,细胞内运输、分泌和囊泡运输;V,防御机制;W,细胞外结构;Y,核结构;Z,细胞骨架。
Fig.4 KOG classification and analysis of differentially expressed genes during waterlogging stress in P. suffruticosa A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeletion.
图4 涝害胁迫下牡丹差异表达基因的KOG分类和分析 A,RNA加工和修饰;B,染色质结构和动力学;C,能源产生与转换;D,细胞周期控制、细胞分裂、染色体分割;E,氨基酸运输和代谢;F,核苷酸运输和代谢;G,碳水化合物运输和代谢;H,辅酶运输和代谢;I,脂质运输和代谢;J,翻译、核糖体结构与生物发生;K,转录;L,复制、重组和修复;M,细胞壁/膜/包膜生物发生;N,细胞运动性;O,翻译后修饰、蛋白质转运蛋白、伴侣蛋白;P,无机离子运输和代谢;Q,次生代谢产物的生物合成、运输和分解代谢;R,仅通用功能预测;S,功能未知;T,信号转导机制;U,细胞内运输、分泌和囊泡运输;V,防御机制;W,细胞外结构;Y,核结构;Z,细胞骨架。
Fig.4 KOG classification and analysis of differentially expressed genes during waterlogging stress in P. suffruticosa A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeletion.
图5 六个RNA-Seq样本的主成分分析(PCA)图
Fig.5 Principal component analysis (PCA) plot of 6 RNA-Seq samples
图5 六个RNA-Seq样本的主成分分析(PCA)图
Fig.5 Principal component analysis (PCA) plot of 6 RNA-Seq samples
图6 涝渍胁迫下牡丹差异表达基因统计图
Fig.6 Changes in gene expression profiles between the waterlogging stress and control libraries of P. suffruticosa
图6 涝渍胁迫下牡丹差异表达基因统计图
Fig.6 Changes in gene expression profiles between the waterlogging stress and control libraries of P. suffruticosa
表1 GO类别下最富集的30个功能组
Table 1 Top 30 most enriched functional groups under GO categories
| 功能组 Functional groups | 富集P值 Enriched P value | 基因数量 Gene number |
|---|---|---|
| 生物进程Biological processes | ||
| 核小体组织Nucleosome organization | 5.73×10-57 | 49 |
| 蛋白质-DNA复合物亚单位组织Protein-DNA complex subunit organization | 1.48×10-56 | 49 |
| 染色体组织Chromosome organization | 3.60×10-50 | 55 |
| 蛋白质复合物亚单位组织Protein complex subunit organization | 2.69×10-49 | 54 |
| 染色质组织Chromatin organization | 4.54×10-48 | 51 |
| 大分子复合物亚单位组织Macromolecular complex subunit organization | 1.94×10-44 | 56 |
| 细胞器组织Organelle organization | 5.84×10-44 | 60 |
| 单元元件组织Cellular component organization | 1.77×10-41 | 68 |
| 细胞成分组织或生物发生Cellular component organization or biogenesis | 2.53×10-39 | 68 |
| 二价金属离子输送Divalent metal ion transport | 1.20×10-9 | 13 |
| 分子功能Molecular functions | ||
| 蛋白质二聚化活性Protein dimerization activity | 8.09×10-51 | 49 |
| 蛋白质结合Protein binding | 2.16×10-36 | 63 |
| 核酸结合Nucleic acid binding | 1.09×10-32 | 58 |
| 杂环化合物结合Heterocyclic compound binding | 7.96×10-14 | 83 |
| 结合Binding | 3.10×10-13 | 112 |
| 有机环状化合物结合Organic cyclic compound binding | 4.95×10-13 | 83 |
| 微管蛋白结合Tubulin binding | 2.54×10-4 | 6 |
| DNA聚合酶活性DNA polymerase activity | 3.59×10-4 | 4 |
| 运动活性Motor activity | 8.52×10-4 | 5 |
| 细胞骨架蛋白结合Cytoskeletal protein binding | 1.45×10-3 | 6 |
| 细胞成分Cellular components | ||
| 无膜细胞器Non-membrane-bounded organelle | 3.68×10-35 | 69 |
| 细胞内无膜细胞器Intracellular non-membrane-bounded organelle | 3.68×10-35 | 69 |
| 膜封闭官腔Membrane-enclosed lumen | 8.35×10-20 | 35 |
| 核腔Nuclear lumen | 8.35×10-20 | 35 |
| 细胞器腔Organelle lumen | 8.35×10-20 | 35 |
| 细胞器内腔Intracellular organelle lumen | 8.35×10-20 | 35 |
| 核部件Nuclear part | 9.44×10-19 | 35 |
| 核Nucleus | 2.22×10-18 | 35 |
| 细胞内细胞器Intracellular organelle | 5.98×10-14 | 95 |
| 细胞器Organelle | 6.90×10-14 | 95 |
表1 GO类别下最富集的30个功能组
Table 1 Top 30 most enriched functional groups under GO categories
| 功能组 Functional groups | 富集P值 Enriched P value | 基因数量 Gene number |
|---|---|---|
| 生物进程Biological processes | ||
| 核小体组织Nucleosome organization | 5.73×10-57 | 49 |
| 蛋白质-DNA复合物亚单位组织Protein-DNA complex subunit organization | 1.48×10-56 | 49 |
| 染色体组织Chromosome organization | 3.60×10-50 | 55 |
| 蛋白质复合物亚单位组织Protein complex subunit organization | 2.69×10-49 | 54 |
| 染色质组织Chromatin organization | 4.54×10-48 | 51 |
| 大分子复合物亚单位组织Macromolecular complex subunit organization | 1.94×10-44 | 56 |
| 细胞器组织Organelle organization | 5.84×10-44 | 60 |
| 单元元件组织Cellular component organization | 1.77×10-41 | 68 |
| 细胞成分组织或生物发生Cellular component organization or biogenesis | 2.53×10-39 | 68 |
| 二价金属离子输送Divalent metal ion transport | 1.20×10-9 | 13 |
| 分子功能Molecular functions | ||
| 蛋白质二聚化活性Protein dimerization activity | 8.09×10-51 | 49 |
| 蛋白质结合Protein binding | 2.16×10-36 | 63 |
| 核酸结合Nucleic acid binding | 1.09×10-32 | 58 |
| 杂环化合物结合Heterocyclic compound binding | 7.96×10-14 | 83 |
| 结合Binding | 3.10×10-13 | 112 |
| 有机环状化合物结合Organic cyclic compound binding | 4.95×10-13 | 83 |
| 微管蛋白结合Tubulin binding | 2.54×10-4 | 6 |
| DNA聚合酶活性DNA polymerase activity | 3.59×10-4 | 4 |
| 运动活性Motor activity | 8.52×10-4 | 5 |
| 细胞骨架蛋白结合Cytoskeletal protein binding | 1.45×10-3 | 6 |
| 细胞成分Cellular components | ||
| 无膜细胞器Non-membrane-bounded organelle | 3.68×10-35 | 69 |
| 细胞内无膜细胞器Intracellular non-membrane-bounded organelle | 3.68×10-35 | 69 |
| 膜封闭官腔Membrane-enclosed lumen | 8.35×10-20 | 35 |
| 核腔Nuclear lumen | 8.35×10-20 | 35 |
| 细胞器腔Organelle lumen | 8.35×10-20 | 35 |
| 细胞器内腔Intracellular organelle lumen | 8.35×10-20 | 35 |
| 核部件Nuclear part | 9.44×10-19 | 35 |
| 核Nucleus | 2.22×10-18 | 35 |
| 细胞内细胞器Intracellular organelle | 5.98×10-14 | 95 |
| 细胞器Organelle | 6.90×10-14 | 95 |
表2 最富集的11个KEGG途径
Table 2 Top 11 most enriched KEGG pathways
| KEGG途径 KEGG pathways | 富集P值 Enriched P value | 基因数量 Gene number |
|---|---|---|
| DNA复制DNA replication | 1.04×10-15 | 84 |
| 核糖体Ribosome | 3.74×10-6 | 604 |
| 嘧啶代谢Pyrimidine metabolism | 5.20×10-4 | 230 |
| 吞噬体Phagosome | 1.48×10-3 | 112 |
| 基底切除修复Base excision repair | 3.31×10-3 | 75 |
| 内质网中的蛋白质加工Protein processing in endoplasmic reticulum | 4.59×10-3 | 363 |
| 嘌呤代谢Purine metabolism | 5.29×10-3 | 264 |
| 不匹配修复Mismatch repair | 6.52×10-3 | 61 |
| 半胱氨酸和蛋氨酸代谢Cysteine and methionine metabolism | 1.68×10-2 | 136 |
| 同源重组Homologous recombination | 1.69×10-2 | 77 |
| 核苷酸切除修复Nucleotide excision repair | 3.09×10-2 | 90 |
表2 最富集的11个KEGG途径
Table 2 Top 11 most enriched KEGG pathways
| KEGG途径 KEGG pathways | 富集P值 Enriched P value | 基因数量 Gene number |
|---|---|---|
| DNA复制DNA replication | 1.04×10-15 | 84 |
| 核糖体Ribosome | 3.74×10-6 | 604 |
| 嘧啶代谢Pyrimidine metabolism | 5.20×10-4 | 230 |
| 吞噬体Phagosome | 1.48×10-3 | 112 |
| 基底切除修复Base excision repair | 3.31×10-3 | 75 |
| 内质网中的蛋白质加工Protein processing in endoplasmic reticulum | 4.59×10-3 | 363 |
| 嘌呤代谢Purine metabolism | 5.29×10-3 | 264 |
| 不匹配修复Mismatch repair | 6.52×10-3 | 61 |
| 半胱氨酸和蛋氨酸代谢Cysteine and methionine metabolism | 1.68×10-2 | 136 |
| 同源重组Homologous recombination | 1.69×10-2 | 77 |
| 核苷酸切除修复Nucleotide excision repair | 3.09×10-2 | 90 |
图7 前10个转录因子家族的基因数量
Fig.7 Gene number of top 10 transcription factor families
图7 前10个转录因子家族的基因数量
Fig.7 Gene number of top 10 transcription factor families
图8 qRT-PCR对基因相对表达水平的验证
Fig.8 Verification of the relative expression levels of genes by qRT-PCR
图8 qRT-PCR对基因相对表达水平的验证
Fig.8 Verification of the relative expression levels of genes by qRT-PCR
表3 牡丹耐涝相关unigene中SSR的类型、数量和分布频率
Table 3 Type, number and distribution frequency of SSRs in Paeonia suffruticosa waterlogging related unigenes
| 重复类型 Repeat type | 重复数量Repeat number | 总数 Total | 占比 Percentage/% | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | ≥15 | |||
| 二核苷酸Dinucleotide | 1 084 | 656 | 513 | 350 | 241 | 110 | 17 | 1 | 9 | 44 | 3 025 | 58.13 | ||
| 三核苷酸Trinucleotide | 901 | 292 | 149 | 38 | 12 | 9 | 1 | 1 | 2 | 3 | 3 | 1 411 | 27.11 | |
| 四核苷酸Tetranucleotide | 296 | 61 | 11 | 6 | 1 | 1 | 1 | 377 | 7.24 | |||||
| 五核苷酸Pentanucleotide | 104 | 21 | 3 | 128 | 2.46 | |||||||||
| 六核苷酸Hexanucleotide | 176 | 61 | 8 | 10 | 4 | 1 | 2 | 1 | 263 | 5.05 | ||||
| 合计Total | 5 204 | 100 | ||||||||||||
表3 牡丹耐涝相关unigene中SSR的类型、数量和分布频率
Table 3 Type, number and distribution frequency of SSRs in Paeonia suffruticosa waterlogging related unigenes
| 重复类型 Repeat type | 重复数量Repeat number | 总数 Total | 占比 Percentage/% | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | ≥15 | |||
| 二核苷酸Dinucleotide | 1 084 | 656 | 513 | 350 | 241 | 110 | 17 | 1 | 9 | 44 | 3 025 | 58.13 | ||
| 三核苷酸Trinucleotide | 901 | 292 | 149 | 38 | 12 | 9 | 1 | 1 | 2 | 3 | 3 | 1 411 | 27.11 | |
| 四核苷酸Tetranucleotide | 296 | 61 | 11 | 6 | 1 | 1 | 1 | 377 | 7.24 | |||||
| 五核苷酸Pentanucleotide | 104 | 21 | 3 | 128 | 2.46 | |||||||||
| 六核苷酸Hexanucleotide | 176 | 61 | 8 | 10 | 4 | 1 | 2 | 1 | 263 | 5.05 | ||||
| 合计Total | 5 204 | 100 | ||||||||||||
表4 牡丹涝害转录组SSR重复基序的类型和频率分布
Table 4 Types and frequency distribution of SSR repeat motifs in the waterlogging transcriptome of Paeonia suffruticosa
| 重复基序类型 Repeat motif type | 主要重复基序 Main repeat motif | 重复次数 Repeat times | SSR重复的百分比 Percentage of SSR repetition/% | SSR重复总体占比 Percentage of total SSR repetition/% |
|---|---|---|---|---|
| 二核苷酸Dinucleotide | AG/CT | 2153 | 71.2 | 41.4 |
| AT/AT | 513 | 17.0 | 9.9 | |
| AC/CT | 352 | 11.6 | 6.8 | |
| 三核苷酸Trinucleotide | AAG/CCT | 362 | 25.7 | 7.0 |
| ACC/GGT | 277 | 19.6 | 5.3 | |
| ATC/ATG | 196 | 13.9 | 3.8 | |
| AAT/ATT | 154 | 10.9 | 3.0 | |
| AGC/CTG | 133 | 9.4 | 2.6 | |
| AAC/GTT | 109 | 7.7 | 2.1 | |
| AGG/CCT | 101 | 7.2 | 1.9 | |
| 四核苷酸Tetranucleotide | AAAT/ATTT | 171 | 45.4 | 3.3 |
| 38 | 10.1 | 0.7 | ||
| 35 | 9.3 | 0.7 |
表4 牡丹涝害转录组SSR重复基序的类型和频率分布
Table 4 Types and frequency distribution of SSR repeat motifs in the waterlogging transcriptome of Paeonia suffruticosa
| 重复基序类型 Repeat motif type | 主要重复基序 Main repeat motif | 重复次数 Repeat times | SSR重复的百分比 Percentage of SSR repetition/% | SSR重复总体占比 Percentage of total SSR repetition/% |
|---|---|---|---|---|
| 二核苷酸Dinucleotide | AG/CT | 2153 | 71.2 | 41.4 |
| AT/AT | 513 | 17.0 | 9.9 | |
| AC/CT | 352 | 11.6 | 6.8 | |
| 三核苷酸Trinucleotide | AAG/CCT | 362 | 25.7 | 7.0 |
| ACC/GGT | 277 | 19.6 | 5.3 | |
| ATC/ATG | 196 | 13.9 | 3.8 | |
| AAT/ATT | 154 | 10.9 | 3.0 | |
| AGC/CTG | 133 | 9.4 | 2.6 | |
| AAC/GTT | 109 | 7.7 | 2.1 | |
| AGG/CCT | 101 | 7.2 | 1.9 | |
| 四核苷酸Tetranucleotide | AAAT/ATTT | 171 | 45.4 | 3.3 |
| 38 | 10.1 | 0.7 | ||
| 35 | 9.3 | 0.7 |
图9 二核苷酸、三核苷酸、四核苷酸和其他类型重复基序的频率分布
Fig.9 Frequency distribution of di-, tri-, tetra-nucleotide and other type repeat motifs
图9 二核苷酸、三核苷酸、四核苷酸和其他类型重复基序的频率分布
Fig.9 Frequency distribution of di-, tri-, tetra-nucleotide and other type repeat motifs
表5 牡丹涝害胁迫相关的SSR引物开发
Table 5 SSR primers developed from genes related to waterlogging stress in Paeonia suffruticosa
| 引物名称 Primers name | 引物序列 Primer sequence (5'-3') | 重复基序 Repeat motif | Tm/℃ | 片段长度 Fragment length/bp |
|---|---|---|---|---|
| Ps01017 | F:CTTTCGAGAACCGCATTTTC R:ATCGAAACACGACCCACTTC | (TGA)5 | 59.9 | 223 |
| Ps01382 | F:TGGGAGAGCATTATCAAGGG R:GAAAACCTAATCCCCAATGC | (GA)8 | 60.0 | 122 |
| Ps02163 | F:GAGCCGCGGTGATATATGTT R:CCGCGAAATTTCTTCGATAA | (GA)9 | 60.1 | 228 |
| Ps04601 | F:TCCACGAACCAAACACTGAA R:TCTTTGTGATCTGGTGCTGC | (TTTA)4 | 60.1 | 251 |
| Ps06615 | F:AAGAAGGAAGGCGTACACGA R:CACTCCCAACCAAATAACCAA | (AG)10 | 59.8 | 174 |
| Ps08889 | F:GAGAGAAGCTCCGTTGTTGG R:GAATTACAACACCCCATGCC | (AGA)6 | 60.0 | 260 |
| Ps11111 | F:AGAATCCTCCCCTTCTTCCA R:CACCAGAGGTGAAGGTGGTT | (TC)6 | 60.0 | 186 |
| Ps11416 | F:ATGATTATGCTCCAGCAGCC R:ACCCCCATCTCTAGGAGGAA | (TGA)5 | 60.2 | 103 |
| Ps11993 | F:AGGCCGAGCTCCACATGTAT R:TGCGGAGGATGAGTTCTTCT | (CT)7 | 61.9 | 257 |
| Ps14499 | F:GCAGGGCAGGACTCTCTATG R:AAGCCCAGTCGAGCAGATTA | (TA)9 | 59.9 | 167 |
| Ps17872 | F:TACCTGTCTCGAACTCCCCA R:CTATGAGGGACCAGAGAGCG | (CT)10 | 60.6 | 200 |
| Ps18852 | F:CCAGAGCCACATGCCTTATT R:ATGTCATCTGAGGTTTCGGC | (CCGTGT)4 | 60.0 | 269 |
| Ps25388 | F:CCGTGTAGATCTGGTTCGGT R:TCACCGCTGAATCAGAACAG | (TA)7 | 59.9 | 168 |
| Ps28246 | F:ACCTCCTTCTTTAGCGCTCC R:CCGCCAATTTCAGAGAGAGA | (TC)8 | 59.9 | 245 |
| Ps29032 | F:TCCCACATTCTTGTGCAGAG R:GTTTGGGGAAATTGGGAACT | (TA)6 | 60.0 | 259 |
| Ps29541 | F:GTGAGAGGATGAAACGGGAA R:ACAACAAATCCCTCACTCGC | (TC)8 | 60.1 | 272 |
| Ps29681 | F:CGAAATGGTGAGACACGAAG R:AATCTCAAACGGCTCATTGG | (GA)6 | 60.0 | 198 |
| Ps30272 | F:CCGGGACTATTTTCTAGCCC R:CAAAACTCCATCAGCAGCAG | (GAC)6 | 59.9 | 259 |
| Ps31923 | F:CCATAACAGCGGTGAGGATT R:TATGCGAGCCATCTCCTTTT | (AG)7 | 59.9 | 183 |
| Ps33766 | F:CTCGGGTGTTGGAGTTCATT R:TGTCGCCCTGTCTCTCTTTT | (GA)8 | 59.9 | 132 |
| Ps34118-1 | F:TCTTGCCATTTCCTTGAAGC R:AAGGTGGCACGAGCATTAAC | (CT)8 | 60.3 | 207 |
| Ps34118-2 | F:ATGGCCGTAGCATAGTGTCC R:CACAAGAGGCCAACACGTAA | (AT)7 | 59.9 | 233 |
| Ps34343 | F:GGGTTGGGAGGGATTAAGAA R:CACAATTCGGACACAGCATC | (TGG)5 | 60.1 | 165 |
| Ps34446 | F:TCAGGGTTTTAGCTCGAGGA R:CTCCTTTGCCCATTGTCATC | (AG)6 | 59.9 | 142 |
| Ps35645-1 | F:CACAAAGCAACCACCCTTTT R:GCATCTGCATCTGGCTCATA | (ATGTAG)4 | 60.0 | 179 |
| Ps35645-2 | F:GCAGAGTCGATCCTGGCTAC R:GGGTACGTCTGCAACCTTGT | (TC)6 | 60.0 | 238 |
| Ps44241 | F:GCCATCGAAACTTTTGAACC R:TTCCGGAAAAATGAAAGACG | (CT)9 | 60.0 | 148 |
| Ps45222 | F:TTAGGATGAAAATCGGCTGG R:TCTTTTTAAGACCCAGACTCTTTGA | (GA)10 | 60.0 | 197 |
| Ps45971 | F:AAAACGTGTGGCCTCAAAAC R:GCGACGAATCAGGAGAGAAC | (AGA)5 | 60.0 | 204 |
| Ps46734 | F:CCATACCGTGCATATGACCA R:CAGCGCATCAATACCTCAAA | (TC)6 | 60.2 | 142 |
| Ps47595 | F:TCGCCTTGTATACCTTTCCG R:AGTGCAGTCCTTCTCGAATCT | (TC)7tt(TC)7 | 60.0 | 147 |
| Ps48286 | F:CTCTCCTCTTCCAGACACGC R:GCAGAAAGAACCAAACCCAA | (TG)6 | 60.1 | 177 |
| Ps49879 | F:ATAGGCAACGCCATGTTTTC R:CTCCCCTCTCTTTGGCTCTT | (GA)8 | 59.9 | 110 |
| Ps51526 | F:GCGAGGGGATAAAACTAGGG R:GTGCTTTCCTGAAACCCAAC | (TA)6 | 59.9 | 176 |
| Ps54582 | F:GAGAGAGATAGAGGGCGCAA R:TCACCTCCGATTCGTAAACC | (TCT)5 | 59.9 | 230 |
| Ps54679 | F:TGTACTTGGGCATCACCAGA R:TTAAGGGCAGCTCGGACTAA | (TC)8 | 60.1 | 257 |
| Ps55401 | F:AGCCTTAATCGGTCGAACCT R:TTGTGCCAGACACCAAGAAG | (GAT)6 | 60.0 | 272 |
| Ps55802 | F:CCCAAGTTTTTCCCTTCTCA R:GGTGGACTTTGACACGGACT | (TTA)5 | 60.0 | 144 |
| Ps57846 | F:TCCATACGCATTCTTTTCCC R:GGAGCCCAAGCGTAACAATA | (AGC)6 | 59.9 | 276 |
| Ps57972 | F:ATTGTTTCCGGGACCACTTC R:GTTTGGTAGGGGTTTTGGCT | (ACC)5 | 61.1 | 223 |
| Ps58678 | F:AAGGCCGGTAAGAAGCTACC R:TTCCCTAGAGGTGATGGTGG | (AGA)5 | 59.9 | 249 |
| Ps58726 | F:TCGAGTACCTTGAGGAGCGT R:CAGATGGGCTTCTGCTTGTT | (AGA)5 | 60.3 | 262 |
| Ps60834 | F:TCCCTCTCTTTTCTCACTGCAT R:GGTGCATGACTGGGAGGTAT | (TG)6 | 60.3 | 152 |
| Ps64449 | F:GTTGCCCCTCTCCTTCTCTT R:CTCTCTCCTTGCCGCTCTTA | (AT)6 | 59.8 | 168 |
| Ps67490 | F:GAACAAAAGCCTTGCCTCTG R:TACGATAGGCCACAACCACA | (CT)10 | 59.9 | 262 |
表5 牡丹涝害胁迫相关的SSR引物开发
Table 5 SSR primers developed from genes related to waterlogging stress in Paeonia suffruticosa
| 引物名称 Primers name | 引物序列 Primer sequence (5'-3') | 重复基序 Repeat motif | Tm/℃ | 片段长度 Fragment length/bp |
|---|---|---|---|---|
| Ps01017 | F:CTTTCGAGAACCGCATTTTC R:ATCGAAACACGACCCACTTC | (TGA)5 | 59.9 | 223 |
| Ps01382 | F:TGGGAGAGCATTATCAAGGG R:GAAAACCTAATCCCCAATGC | (GA)8 | 60.0 | 122 |
| Ps02163 | F:GAGCCGCGGTGATATATGTT R:CCGCGAAATTTCTTCGATAA | (GA)9 | 60.1 | 228 |
| Ps04601 | F:TCCACGAACCAAACACTGAA R:TCTTTGTGATCTGGTGCTGC | (TTTA)4 | 60.1 | 251 |
| Ps06615 | F:AAGAAGGAAGGCGTACACGA R:CACTCCCAACCAAATAACCAA | (AG)10 | 59.8 | 174 |
| Ps08889 | F:GAGAGAAGCTCCGTTGTTGG R:GAATTACAACACCCCATGCC | (AGA)6 | 60.0 | 260 |
| Ps11111 | F:AGAATCCTCCCCTTCTTCCA R:CACCAGAGGTGAAGGTGGTT | (TC)6 | 60.0 | 186 |
| Ps11416 | F:ATGATTATGCTCCAGCAGCC R:ACCCCCATCTCTAGGAGGAA | (TGA)5 | 60.2 | 103 |
| Ps11993 | F:AGGCCGAGCTCCACATGTAT R:TGCGGAGGATGAGTTCTTCT | (CT)7 | 61.9 | 257 |
| Ps14499 | F:GCAGGGCAGGACTCTCTATG R:AAGCCCAGTCGAGCAGATTA | (TA)9 | 59.9 | 167 |
| Ps17872 | F:TACCTGTCTCGAACTCCCCA R:CTATGAGGGACCAGAGAGCG | (CT)10 | 60.6 | 200 |
| Ps18852 | F:CCAGAGCCACATGCCTTATT R:ATGTCATCTGAGGTTTCGGC | (CCGTGT)4 | 60.0 | 269 |
| Ps25388 | F:CCGTGTAGATCTGGTTCGGT R:TCACCGCTGAATCAGAACAG | (TA)7 | 59.9 | 168 |
| Ps28246 | F:ACCTCCTTCTTTAGCGCTCC R:CCGCCAATTTCAGAGAGAGA | (TC)8 | 59.9 | 245 |
| Ps29032 | F:TCCCACATTCTTGTGCAGAG R:GTTTGGGGAAATTGGGAACT | (TA)6 | 60.0 | 259 |
| Ps29541 | F:GTGAGAGGATGAAACGGGAA R:ACAACAAATCCCTCACTCGC | (TC)8 | 60.1 | 272 |
| Ps29681 | F:CGAAATGGTGAGACACGAAG R:AATCTCAAACGGCTCATTGG | (GA)6 | 60.0 | 198 |
| Ps30272 | F:CCGGGACTATTTTCTAGCCC R:CAAAACTCCATCAGCAGCAG | (GAC)6 | 59.9 | 259 |
| Ps31923 | F:CCATAACAGCGGTGAGGATT R:TATGCGAGCCATCTCCTTTT | (AG)7 | 59.9 | 183 |
| Ps33766 | F:CTCGGGTGTTGGAGTTCATT R:TGTCGCCCTGTCTCTCTTTT | (GA)8 | 59.9 | 132 |
| Ps34118-1 | F:TCTTGCCATTTCCTTGAAGC R:AAGGTGGCACGAGCATTAAC | (CT)8 | 60.3 | 207 |
| Ps34118-2 | F:ATGGCCGTAGCATAGTGTCC R:CACAAGAGGCCAACACGTAA | (AT)7 | 59.9 | 233 |
| Ps34343 | F:GGGTTGGGAGGGATTAAGAA R:CACAATTCGGACACAGCATC | (TGG)5 | 60.1 | 165 |
| Ps34446 | F:TCAGGGTTTTAGCTCGAGGA R:CTCCTTTGCCCATTGTCATC | (AG)6 | 59.9 | 142 |
| Ps35645-1 | F:CACAAAGCAACCACCCTTTT R:GCATCTGCATCTGGCTCATA | (ATGTAG)4 | 60.0 | 179 |
| Ps35645-2 | F:GCAGAGTCGATCCTGGCTAC R:GGGTACGTCTGCAACCTTGT | (TC)6 | 60.0 | 238 |
| Ps44241 | F:GCCATCGAAACTTTTGAACC R:TTCCGGAAAAATGAAAGACG | (CT)9 | 60.0 | 148 |
| Ps45222 | F:TTAGGATGAAAATCGGCTGG R:TCTTTTTAAGACCCAGACTCTTTGA | (GA)10 | 60.0 | 197 |
| Ps45971 | F:AAAACGTGTGGCCTCAAAAC R:GCGACGAATCAGGAGAGAAC | (AGA)5 | 60.0 | 204 |
| Ps46734 | F:CCATACCGTGCATATGACCA R:CAGCGCATCAATACCTCAAA | (TC)6 | 60.2 | 142 |
| Ps47595 | F:TCGCCTTGTATACCTTTCCG R:AGTGCAGTCCTTCTCGAATCT | (TC)7tt(TC)7 | 60.0 | 147 |
| Ps48286 | F:CTCTCCTCTTCCAGACACGC R:GCAGAAAGAACCAAACCCAA | (TG)6 | 60.1 | 177 |
| Ps49879 | F:ATAGGCAACGCCATGTTTTC R:CTCCCCTCTCTTTGGCTCTT | (GA)8 | 59.9 | 110 |
| Ps51526 | F:GCGAGGGGATAAAACTAGGG R:GTGCTTTCCTGAAACCCAAC | (TA)6 | 59.9 | 176 |
| Ps54582 | F:GAGAGAGATAGAGGGCGCAA R:TCACCTCCGATTCGTAAACC | (TCT)5 | 59.9 | 230 |
| Ps54679 | F:TGTACTTGGGCATCACCAGA R:TTAAGGGCAGCTCGGACTAA | (TC)8 | 60.1 | 257 |
| Ps55401 | F:AGCCTTAATCGGTCGAACCT R:TTGTGCCAGACACCAAGAAG | (GAT)6 | 60.0 | 272 |
| Ps55802 | F:CCCAAGTTTTTCCCTTCTCA R:GGTGGACTTTGACACGGACT | (TTA)5 | 60.0 | 144 |
| Ps57846 | F:TCCATACGCATTCTTTTCCC R:GGAGCCCAAGCGTAACAATA | (AGC)6 | 59.9 | 276 |
| Ps57972 | F:ATTGTTTCCGGGACCACTTC R:GTTTGGTAGGGGTTTTGGCT | (ACC)5 | 61.1 | 223 |
| Ps58678 | F:AAGGCCGGTAAGAAGCTACC R:TTCCCTAGAGGTGATGGTGG | (AGA)5 | 59.9 | 249 |
| Ps58726 | F:TCGAGTACCTTGAGGAGCGT R:CAGATGGGCTTCTGCTTGTT | (AGA)5 | 60.3 | 262 |
| Ps60834 | F:TCCCTCTCTTTTCTCACTGCAT R:GGTGCATGACTGGGAGGTAT | (TG)6 | 60.3 | 152 |
| Ps64449 | F:GTTGCCCCTCTCCTTCTCTT R:CTCTCTCCTTGCCGCTCTTA | (AT)6 | 59.8 | 168 |
| Ps67490 | F:GAACAAAAGCCTTGCCTCTG R:TACGATAGGCCACAACCACA | (CT)10 | 59.9 | 262 |
图10 SSR引物Ps18852, Ps28246, Ps29032, Ps29541和 Ps30272在9个牡丹品种中的扩增结果
Fig.10 Polymorphism of Ps18852, Ps28246, Ps29032, Ps29541, and Ps30272 of SSR primers in 9 samples of P. suffruticosa
图10 SSR引物Ps18852, Ps28246, Ps29032, Ps29541和 Ps30272在9个牡丹品种中的扩增结果
Fig.10 Polymorphism of Ps18852, Ps28246, Ps29032, Ps29541, and Ps30272 of SSR primers in 9 samples of P. suffruticosa
表6 十二个SSR引物在9个牡丹品种中的遗传多样性指数
Table 6 Genetic diversity index for 12 SSR primers among 9 Paeonia suffruticosa cultivars
| 引物 Primers | 观测等位基因数 Observed number of alleles (Na) | 有效等位基因数 Effective number of alleles (Ne) | Nei’s基因多样性 Nei’s gene diversity index (H) | Shannon信息指数 Shannon’s information index (I) | 多态性位点 Polymorphic sites | 多态性百分比 Polymorphic percentage/% |
|---|---|---|---|---|---|---|
| Ps02163 | 2.000 0 | 1.371 1 | 0.244 2 | 0.396 1 | 14 | 100 |
| P04601 | 1.833 3 | 1.487 2 | 0.285 6 | 0.428 2 | 10 | 83.33 |
| Ps17872 | 1.857 1 | 1.405 1 | 0.259 9 | 0.404 3 | 6 | 85.71 |
| Ps18852 | 2.000 0 | 1.413 7 | 0.264 5 | 0.422 7 | 5 | 100 |
| Ps28246 | 1.777 8 | 1.294 4 | 0.192 8 | 0.310 2 | 7 | 77.78 |
| Ps29032 | 1.333 3 | 1.266 7 | 0.148 1 | 0.212 2 | 1 | 33.33 |
| Ps30272 | 1.833 3 | 1.265 1 | 0.177 9 | 0.295 1 | 5 | 83.33 |
| Ps34118-1 | 1.833 3 | 1.351 7 | 0.217 0 | 0.343 4 | 5 | 83.33 |
| Ps34118-2 | 1.500 0 | 1.060 4 | 0.053 9 | 0.109 6 | 1 | 50.00 |
| Ps35645 | 2.000 0 | 1.692 6 | 0.397 2 | 0.581 8 | 5 | 100 |
| Ps54679 | 2.000 0 | 1.479 4 | 0.297 7 | 0.462 5 | 10 | 100 |
| Ps55401 | 2.000 0 | 1.573 6 | 0.332 2 | 0.497 4 | 3 | 100 |
| Mean | 1.830 7 | 1.388 4 | 0.256 1 | 0.372 0 | 6 | 83.00 |
表6 十二个SSR引物在9个牡丹品种中的遗传多样性指数
Table 6 Genetic diversity index for 12 SSR primers among 9 Paeonia suffruticosa cultivars
| 引物 Primers | 观测等位基因数 Observed number of alleles (Na) | 有效等位基因数 Effective number of alleles (Ne) | Nei’s基因多样性 Nei’s gene diversity index (H) | Shannon信息指数 Shannon’s information index (I) | 多态性位点 Polymorphic sites | 多态性百分比 Polymorphic percentage/% |
|---|---|---|---|---|---|---|
| Ps02163 | 2.000 0 | 1.371 1 | 0.244 2 | 0.396 1 | 14 | 100 |
| P04601 | 1.833 3 | 1.487 2 | 0.285 6 | 0.428 2 | 10 | 83.33 |
| Ps17872 | 1.857 1 | 1.405 1 | 0.259 9 | 0.404 3 | 6 | 85.71 |
| Ps18852 | 2.000 0 | 1.413 7 | 0.264 5 | 0.422 7 | 5 | 100 |
| Ps28246 | 1.777 8 | 1.294 4 | 0.192 8 | 0.310 2 | 7 | 77.78 |
| Ps29032 | 1.333 3 | 1.266 7 | 0.148 1 | 0.212 2 | 1 | 33.33 |
| Ps30272 | 1.833 3 | 1.265 1 | 0.177 9 | 0.295 1 | 5 | 83.33 |
| Ps34118-1 | 1.833 3 | 1.351 7 | 0.217 0 | 0.343 4 | 5 | 83.33 |
| Ps34118-2 | 1.500 0 | 1.060 4 | 0.053 9 | 0.109 6 | 1 | 50.00 |
| Ps35645 | 2.000 0 | 1.692 6 | 0.397 2 | 0.581 8 | 5 | 100 |
| Ps54679 | 2.000 0 | 1.479 4 | 0.297 7 | 0.462 5 | 10 | 100 |
| Ps55401 | 2.000 0 | 1.573 6 | 0.332 2 | 0.497 4 | 3 | 100 |
| Mean | 1.830 7 | 1.388 4 | 0.256 1 | 0.372 0 | 6 | 83.00 |
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