基于RAD高通量测序的贵州百里杜鹃保护区杜鹃花属分类

doi:10.11707/j.1001-7488.20210208

摘要/Abstract

摘要：

目的: 准确界定物种是野生植物资源开发与利用的前提。鉴于叶绿体片段无法区分贵州百里杜鹃区域的杜鹃花属植物，本研究采用基于基因组水平的RAD-seq技术探讨该区域杜鹃花属的系统进化关系，以期为复杂植物类群的物种鉴定提供参考。方法: 对百里杜鹃保护区的34种杜鹃花进行RAD-seq技术测序，统计测序深度、覆盖度及多态性位点等基本指标；用de novo方法获得高质量SNP位点，以Tajima’s D为指标筛选中性的SNPs，最后用中性SNPs通过fastStructure、PCA、GCTA及IQ-Tree软件对其进行基因分型、聚类与系统树构建。结果: 共获得42 083个高质量SNP位点，其中28 983个为中性SNP位点。基于PCA、fastStructure及系统发生树的分析结果，支持目前34种杜鹃花在亚属水平的形态学分类处理，6个亚属中的常绿杜鹃亚属、马银花亚属、羊踯躅亚属和映山红亚属能明显分开，而糙叶杜鹃亚属与杜鹃亚属不能区分开，表明二者亲缘关系较近。物种水平能区分绝大部分的种，相比前期研究有了大幅度提升，但一些形态特征极相似的种如繁花杜鹃与皱叶杜鹃无法区分。结论: RAD-seq数据产生大量的中性SNP位点，可在亚属水平和物种水平区分百里杜鹃保护区杜鹃花属的大部分植物，同时证实RAD-seq技术在复杂植物类群的物种分类方面相比传统分子标记，具有明显优势。

关键词: RAD-seq, 杜鹃花属, 百里杜鹃保护区, 单核苷酸多态性, 系统分类

Abstract:

Objective: Accurate species definition is the premise for development and utilization of wild plant resources. Due to the fact that chloroplast DNA fragments cannot distinguish Rhododendron species of the Baili Rhododendron Reserve in Guizhou reported before, the present study attempted to explore the phylogenetic relationships of Rhododendron in this region based on genome-level RAD(restriction-site associated DNA) sequencing technology, aiming to detect the power of RAD for species definition of complicated plant groups. Method: RAD-seq sequencing was performed on 34 species of Rhododendron from the reserve, and the basic characteristics of the data such as depth, coverage and polymorphic sites were evaluated. De novo method was employed to call SNPs which were subsequently evaluated to be neutral or selective by Tajima's D. Based on these neutral SNPs, fastStructure, PCA, GCTA and IQ-Tree software were finally employed to perform genetic separation, clustering and phylogenetic tree construction. Result: A total of 42 083 SNPs were obtained, among which 28 983 SNPs were detected to be neutral. The analyses of PCA, fastStructure and phylogenetic tree supports the morphological classification of 34 species of Rhododendron at the subgenus level. Subgen. Hymenanthes, Subgen. Azaleastrum, Subgen. Pentanthera and Subgen. Tsutsusi of the 6 subgenera can be distinguished, whereas Subgen. Pseudorhodorastrum and Subgen. Rhododendron cannot, suggesting that the 2 subgenera are closely related. In addition, at species-level most species can be distinguished, indicating a great improvement compared to previous studies, but the species with very similar morphological characteristics are indistinguishable, such as R. floribundum and R. denudatum. Conclusion: RAD-seq data produced a large number of SNPs that can distinguish most Rhododendron species in the reserve at the subgenus and species levels, proving that RAD technology had significant advantages over traditional molecular markers in the classification of complicated plant groups.

Key words: RAD-seq, Rhododendron, Baili Rhododendron Reserve, SNPs, phylogenetic classification

中图分类号:

S718.49

黄承玲,姚刚,田晓玲,任永权,黄家湧,马永鹏. 基于RAD高通量测序的贵州百里杜鹃保护区杜鹃花属分类[J]. 林业科学, 2021, 57(2): 72-81.

Chengling Huang,Gang Yao,Xiaoling Tian,Yongquan Ren,Jiayong Huang,Yongpeng Ma. Phylogenomic Analysis of Rhododendron Species in Guizhou Baili Rhododendron Reserve Based on RAD Sequencing[J]. Scientia Silvae Sinicae, 2021, 57(2): 72-81.

图/表 5

表1

杜鹃花属34个种的采样信息"

样品编号 Sample ID	物种 Species	采样地点 Sampling site	地理坐标 Geographic coordinates	海拔 Altitude/m
23	羊踯躅R. molle	九牛Jiuniu	105°87′E，27°23′N	1 540
26	锦绣杜鹃R. pulchrum	九牛Jiuniu	105°87′E，27°23′N	1 540
32	繁花杜鹃R. floribundum	黄坪Huangping	105°87′E，27°24′N	1 650
34	大白杜鹃R. decorum	五彩亭Wucaiting	105°86′E，27°23′N	1 740
39	美容杜鹃R. calophytum	九龙山Jiulongshan	105°89′E，27°31′N	1 970
42	糙叶杜鹃R. scabrifolium	大水乡大石Dashi, Dashui township	105°99′E，27°26′N	1 580
45	百合花杜鹃R. liliiflorum	方家坪Fangjiaping	105°88′E，27°26′N	1 850
47	基毛杜鹃R. rigidum	方家坪Fangjiaping	105°89′E，27°27′N	1 840
61	锈叶杜鹃R. siderophyllum	方家坪Fangjiaping	105°90′E，27°25′N	1 820
65	大果杜鹃R. glanduliferum	盘县石龙Shilong, Pan county	104°50′E，25°83′N	1 570
69	桃叶杜鹃R. annae	方家坪Fangjiaping	105°89′E，27°27′N	1 850
71	红棕杜鹃R. rubiginosum	方家坪Fangjiaping	105°89′E，27°27′N	1 850
73	马缨杜鹃R. delavayi	马缨岭Mayingling	105°96′E，27°17′N	1 520
94	皱叶杜鹃R. denudatum	九龙山Jiulongshan	105°89′E，27°31′N	1 970
97	长蕊杜鹃R. stamineum	百花坪Baihuaping	105°94′E，27°18′N	1 550
2088	露珠杜鹃R. irroratum	数花峰Shuhuafeng	105°87′E，27°24′N	1 630
DJ-1	普底杜鹃R. pudiense	黄坪Huangping	105°87′E，27°24′N	1 650
DJ-8	淡粉杜鹃R. subroseum	黄坪Huangping	105°87′E，27°24′N	1 650
DJ-9	香叶杜鹃R. sp. nov.	方家坪Fangjiaping	105°89′E，27°27′N	1 850
DJ-11	睡莲叶杜鹃R. nymphaeoides	箐脚Qingjiao	105°87′E，27°23′N	1 540
DJ-13	匙叶杜鹃R. cochlearifolium	黄坪Huangping	105°87′E，27°24′N	1 650
DJ-14	金波杜鹃R. jinboense	百花坪Baihuaping	105°94′E，27°18′N	1 550
DJ-19	淡紫杜鹃R. lilacinum	云台岭Yuntailing	105°87′E，27°23′N	1 540
DJ-21	枇杷叶杜鹃R. eriobotryoides	云台岭Yuntailing	105°87′E，27°23′N	1 540
DJ-23	黄坪杜鹃R. huangpingense	黄坪Huangping	105°87′E，27°24′N	1 650
DJ-27	百纳杜鹃R. bainaense	云台岭Yuntailing	105°87′E，27°23′N	1 540
DJ-34	九龙山杜鹃R. jiulongshanense	方家坪Fangjiaping	105°89′E，27°27′N	1 840
DJ-37	映山红R. simsii	百花坪Baihuaping	105°94′E，27°18′N	1 550
DJ-44	黔中杜鹃R. feddei	百花坪Baihuaping	105°94′E，27°18′N	1 550
DJ-45	银叶杜鹃R. argyrophyllum	九龙山Jiulongshan	105°89′E，27°31′N	1 970
DJ-46	百里杜鹃R. bailiense	红板壁Hongbanbi	105°89′E，27°24′N	1 720
DJ-47	凹叶杜鹃R. davidsonianum	方家坪Fangjiaping	105°89′E，27°27′N	1 840
DJ-48	平房杜鹃R. truncatovarium	百花坪Baihuaping	105°94′E，27°18′N	1 550
DJ-63	树枫杜鹃R. changii	九龙山Jiulongshan	105°89′E，27°31′N	1 970

表1

表2

杜鹃花属34个种RAD测序的数据特性"

样品编号 Sample ID	种 Species	亚属 Subgenus	组或亚组 Section/Subsection	高质量序列 Clean reads /Mb	高质量碱基 Clean base /Mb	测序深度(倍) Depth(times)	覆盖度 Coverage(%)	多态性位点 Polymorphic sites
23	羊踯躅 R. molle	羊踯躅亚属 Subgen. Pentanthera	五花药组 Sect. Pentanthera	1.76	216.21	4.42	5.83	3 388
26	锦绣杜鹃 R. pulchrum	映山红亚属 Subgen. Tsutsusi	映山红组 Sect. Tsutsusi	2.28	283.37	4.91	7.51	7 775
32	繁花杜鹃 R. floribundum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组银叶杜鹃亚组 Sect. Ponticum Subsect. Argyrophylla	1.61	195.33	4.26	7.14	3 636
34	大白杜鹃 R. decorum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组云锦杜鹃亚组 Sect. Ponticum Subsect. Fortunea	2.69	340.81	6.08	9.24	12 447
39	美容杜鹃 R. calophytum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组云锦杜鹃亚组 Sect. Ponticum Subsect. Fortunea	4.23	529.59	7.89	9.97	14 485
42	糙叶杜鹃 R. scabrifolium	糙叶杜鹃亚属 Subgen. Pseudorhodorastrum	糙叶杜鹃组 Sect. Trachyrhodion	5.39	674.57	8.52	9.87	29 092
45	百合花杜鹃 R. liliiflorum	杜鹃亚属 Subgen. Rhododendron	杜鹃组有鳞大花亚组 Sect. Rhododendron Subsect. Maddenia	3.86	470.73	8.66	6.44	10 511
47	基毛杜鹃 R. rigidum	杜鹃亚属 Subgen. Rhododendron	杜鹃组三花杜鹃亚组 Sect. Rhododendron Subsect. Triflora	1.53	188.81	3.84	6.26	3 956
61	锈叶杜鹃 R. siderophyllum	杜鹃亚属 Subgen. Rhododendron	杜鹃组三花杜鹃亚组 Sect. Rhododendron Subsect. Triflora	3.10	390.70	5.42	8.75	14 355
65	大果杜鹃 R. glanduliferum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组云锦杜鹃亚组 Sect. Ponticum Subsect. Fortunea	2.77	342.90	5.98	9.36	11 966
69	桃叶杜鹃 R. annae	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组露珠杜鹃亚组 Sect. Ponticum Subsect. Irrorata	4.60	571.25	9.15	10.13	18 083
71	红棕杜鹃 R. rubiginosum	杜鹃亚属 Subgen. Rhododendron	杜鹃组亮鳞杜鹃亚组 Sect. Rhododendron Subsect. Heliolepida	5.14	650.30	7.81	10.28	28 193
73	马缨杜鹃 R. delavayi	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组树形杜鹃亚组 Sect. Ponticum Subsect. Arborea	2.82	352.83	4.77	12.04	10 990
94	皱叶杜鹃 R. denudatum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组银叶杜鹃亚组 Sect. Ponticum Subsect. Argyrophylla	4.03	498.60	6.94	10.64	16 959
97	长蕊杜鹃 R. stamineum	马银花亚属 Subgen. Azaleastrum	长蕊组 Sect. Choniastrum	1.45	175.34	4.09	5.55	3 842
2088	露珠杜鹃 R. irroratum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组露珠杜鹃亚组 Sect. Ponticum Subsect. Irrorata	8.42	1 084.16	6.18	8.60	29 675
DJ-1	普底杜鹃 R. pudiense	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组大理杜鹃亚组 Sect. Ponticum Subsect. Taliensia	5.03	656.53	6.79	7.99	22 094
DJ-8	淡粉杜鹃 R. subroseum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组麻花杜鹃亚组 Sect. Ponticum Subsect. Maculifera	2.61	337.64	6.23	8.53	14 403
DJ-9	香叶杜鹃 R. sp. nov.	杜鹃亚属 Subgen. Rhododendron	杜鹃组亮鳞杜鹃亚组 Sect. Rhododendron Subsect. Heliolepida	5.94	754.76	10.08	9.10	34 156
DJ-11	睡莲叶杜鹃 R. nymphaeoides	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组云锦杜鹃亚组 Sect. Ponticum Subsect. Fortunea	13.39	1 753.26	27.23	9.93	25 640
DJ-13	匙叶杜鹃 R. cochlearifolium	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组露珠杜鹃亚组 Sect. Ponticum Subsect. Irrorata	0.02	2.86	7.46	8.68	2 012
DJ-14	金波杜鹃 R. jinboense	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组露珠杜鹃亚组 Sect. Ponticum Subsect. Irrorata	7.37	972.26	13.00	11.68	26 615
DJ-19	淡紫杜鹃 R. lilacinum	映山红亚属 Subgen. Tsutsusi	映山红组 Sect. Tsutsusi	8.70	1 143.73	18.63	7.02	39 585
DJ-21	枇杷叶杜鹃 R. eriobotryoides	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组银叶杜鹃亚组 Sect. Ponticum Subsect. Argyrophylla	10.74	1 352.98	12.48	11.10	38 490
DJ-23	黄坪杜鹃 R. huangpingense	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组云锦杜鹃亚组 Sect. Ponticum Subsect. Fortunea	12.35	1 607.41	11.46	11.44	51 256
DJ-27	百纳杜鹃 R. bainaense	杜鹃亚属 Subgen. Rhododendron	杜鹃组亮鳞杜鹃亚组 Sect. Rhododendron Subsect. Heliolepida	4.70	618.91	7.84	8.56	32 735
DJ-34	九龙山杜鹃 R. jiulongshanense	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组云锦杜鹃亚组 Sect. Ponticum Subsect. Fortunea	12.69	1.65	22.45	9.41	32 636
DJ-37	映山红 R. simsii	映山红亚属 Subgen. Tsutsusi	映山红组 Sect. Tsutsusi	1.83	241.67	6.03	4.63	8 836
DJ-44	黔中杜鹃 R. feddei	马银花亚属 Subgen. Azaleastrum	长蕊组 Sect. Choniastrum	7.86	1 026.56	20.17	6.07	29 184
DJ-45	银叶杜鹃 R. argyrophyllum	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组银叶杜鹃亚组 Sect. Ponticum Subsect. Argyrophylla	7.69	998.88	12.94	9.31	25 595
DJ-46	百里杜鹃 R. bailiense	常绿杜鹃亚属 Subgen. Hymenanthes	常绿杜鹃组耳叶杜鹃亚组 Sect. Ponticum Subsect. Auriculata	0.02	2.57	9.39	8.70	2 620
DJ-47	凹叶杜鹃 R. davidsonianum	杜鹃亚属 Subgen. Rhododendron	杜鹃组三花杜鹃亚组 Sect. Rhododendron Subsect. Triflora	7.82	1 020.79	12.29	10.34	47 926
DJ-48	平房杜鹃 R. truncatovarium	马银花亚属 Subgen. Azaleastrum	长蕊组 Sect. Choniastrum	3.24	422.08	9.71	4.88	17 214
DJ-63	树枫杜鹃 R. changii	杜鹃亚属 Subgen. Rhododendron	杜鹃组有鳞大花亚组 Sect. Rhododendron Subsect. Maddenia	10.39	1 369.27	21.65	7.42	23 702

表2

图1

图2

图3

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