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作者简介:

贾守宁(1966-),主任药师,研究方向为中藏药药效物质的研究、利用与开发,(E-mail)jiashouning@163.com。

通讯作者:

李国栋,博士,副教授,研究方向为分子生药学,(E-mail)liguodong@ynutcm.edu.cn。

中图分类号:Q943

文献标识码:A

文章编号:1000-3142(2023)10-1907-14

DOI:10.11931/guihaia.gxzw202204010

参考文献
BAI L, JIAO ML, ZANG HY, et al. , 2019. Chemical composition of essential oils from four Rhododendron species and their repellent activity against three stored-product insects [J]. Environ Sci Pollut Res, 26(22): 23198-23205.
参考文献
BAI XX, SHEN FJ, LI ZB, et al. , 2019. DNA barcode identification of genus Rhododendron L. : reference library, molecular markers and cultivar wild relatives tracing [J]. Tianjin Agric Sci, 25(10): 1-7. [白霄霞, 沈风娇, 李志斌, 等, 2019. 杜鹃花属 (Rhododendron L. ) 植物 DNA 条形码鉴定——参考序列库, 分子标记与栽培品种野生亲本溯源 [J]. 天津农业科学, 25(10): 1-7. ]
参考文献
BEIER S, THIEL T, MÜNCH T, et al. , 2017. MISA-web: a web server for microsatellite prediction [J]. Bioinformatics, 33(16): 2583-2585.
参考文献
BRUDNO M, MALDE S, POLIAKOV A, et al. , 2003. Glocal alignment: Finding rearrangements during alignment [J]. Bioinformatics, 19: i54-i62.
参考文献
CAO WX, 2001. Ecological adaptive strategy of genus Rhododendron L. in eastern Qilian Mountains alpine ecosystem [D]. Gansu: Northwest Normal University: 35-37. [曹文侠, 2001. 东祁连山高寒生态系统杜鹃花属植物适应对策的研究 [D]. 甘肃: 西北师范大学: 35-37. ]
参考文献
CAO XJ, LIU JJ, YANG M, 2009. Photosynthetic characteristics and anatomical structure of five species of Rhododendron in the Taibai Mountain [J]. Acta Bot Boreal-Occident Sin, 29(12): 2483-2491. [曹晓娟, 刘建军, 杨梅, 2009. 太白山 5 种杜鹃属植物叶片光合特性及解剖结构的生态适应性研究 [J]. 西北植物学报, 29(12): 2483-2491. ]
参考文献
CHENG SY, 2017. Study on resources and classification of azalea cultivars in China [D]. Ganzhou: Gannan Normal University: 11-12. [程淑媛, 2017. 中国杜鹃花栽培品种资源与分类研究 [D]. 赣州: 赣南师范大学: 11-12]
参考文献
CHUMLEY TW, PALMER JD, MOWER JP, et al. , 2006. The complete chloroplast genome sequence of Pelargonium × hortorum: organization and evolution of the largest and most highly rearranged chloroplast genome of land plants [J]. Mol Biol Evol, 23(11): 2175-2190.
参考文献
DANIELL H, LIN CS, YU M, et al. , 2016. Chloroplast genomes: diversity, evolution, and applications in genetic engineering [J]. Genome Biol, 17(1): 134.
参考文献
DRUMMOND A, 2012. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data [J]. Bioinformatics, 28(12): 1647-1649.
参考文献
FAN WB, WU Y, YANG J, et al. , 2018. Comparative chloroplast genomics of Dipsacales species: insights into sequence variation, adaptive evolution, and phylogenetic relationships [J]. Front Plant Sci, 9: 689.
参考文献
FU CN, MO ZQ, YANG JB, et al. , 2022. Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron [J]. Mol Ecol Resour, 22(1): 404-414.
参考文献
GENG XM, HUAN ZQ, SU JL, et al. , 2021. Researches advances in germplasm innovation of Rhododendrons [J]. Mol Plant Breed, 19(2): 604-613. [耿兴敏, 宦智群, 苏家乐, 等, 2021. 杜鹃花属植物种质创新研究进展 [J]. 分子植物育种, 19(2): 604-613. ]
参考文献
GREINER S, LEHWARK P, BOCK R, 2019. Organellar-GenomeDRAW (OGDRAW) version 1. 3. 1: expanded toolkit for the graphical visualization of organellar genomes [J]. Nucl Acid Res, 47(W1): W59-W64.
参考文献
GU CH, MA L, WU ZQ, et al. , 2019. Comparative analyses of chloroplast genomes from 22 Lythraceae species: inferences for phylogenetic relationships and genome evolution within Myrtales [J]. BMC Plant Biol, 19(1): 1-19.
参考文献
HE JL, ZHOU TY, SONG YK, et al. , 2021. Variations in floral traits of Rhododendron przewalskii with slope aspect in the southeastern Tibetan Plateau [J]. Chin J Appl Environ Biol, 27(4): 860-868. [何家莉, 周天阳, 宋怡珂, 等, 2021. 青藏高原东南缘不同坡向对陇蜀杜鹃花性状的影响 [J]. 应用与环境生物学报, 27(4): 860-868. ]
参考文献
HIRAO T, WATANABE A, KURITA M, et al. , 2008. Complete nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species [J]. BMC Plant Biol, 8(1): 1-20.
参考文献
HUANG CL, YAO G, TIAN XL, et al. , 2021. Phylogenomic analysis of Rhododendron species in Guizhou Baili Rhododendron reserve based on RAD sequencing [M]. Sci Silv Sin, 57(2): 72-81. [黄承玲, 姚刚, 田晓玲, 等, 2021. 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, 57(2): 72-81. ]
参考文献
HUELSENBECK JP, RONQUIST F, 2001. MRBAYES: Bayesian inference of phylogenetic trees [J]. Bioinformatics, 17: 754-755.
参考文献
JANSEN RK, RAUBESON LA, BOORE JL, et al. , 2005. Methods for obtaining and analyzing whole chloroplast genome sequences [J]. Meth Enzym, 395: 348-384.
参考文献
JIN JJ, YU WB, YANG JB, et al. , 2020. GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes [J]. Genome Biol, 21(1): 1-31.
参考文献
KANE N, SVEINSSON S, DEMPEWOLF H, et al. , 2012. Ultra-barcoding in cacao (Theobroma spp. ; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA [J]. Am J Bot, 99(2): 320-329.
参考文献
KATOH K, STANDLEY DM, 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability [J]. Mol Biol Evol, 30(4): 772-780.
参考文献
KUMAR S, STECHER G, LI M, et al. , 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms [J]. Mol Biol Evol, 35(6): 1547.
参考文献
LEE SB, KAITTANIS C, JANSEN RK, et al. 2006. The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms [J]. BMC Genomic, 7(1): 1-12.
参考文献
LI Y, LÜ GH, ZHANG XN, et al. , 2017. Chloroplast genome structure and variation analysis of Brasicaceae species [J]. Acta Bot Boreal-Occident Sin, 37(6): 1090-1101. [李岩, 吕光辉, 张雪妮, 等, 2017. 十字花科植物叶绿体基因组结构及变异分析 [J]. 西北植物学报, 37(6): 1090-1101. ]
参考文献
LI Y, SYLVESTER SP, LI M, et al. , 2019. The complete plastid genome of Magnolia zenii and genetic comparison to Magnoliaceae species [J]. Molecules, 24(2): 261.
参考文献
LI YF, LI SM, JIN X, et al. , 2019. Classification of 85 species of Rhododendron in China based on RAD high throughput sequencing [J]. For Res, 32(3): 1-8. [李云飞, 李世明, 金鑫, 等, 2019. 基于 RAD 高通量测序探讨中国 85 种杜鹃花属植物的分类 [J]. 林业科学研究, 32(3): 1-8. ]
参考文献
LI YJ, 2011. Current situation and garden use of wild Rhododendron resources in Qinghai Province [D]. Yangling: Northwest A & F University: 25-26. [李永金, 2011. 青海杜鹃属植物种质资源现状及园林应用 [D]. 杨凌: 西北农林科技大学: 25-26. ]
参考文献
LIAO HB, HUANG GH, YU MH, et al. 2017. Five pairs of meroterpenoid enantiomers from Rhododendron capitatum [J]. J Org Chem, 82(3): 1632-1637.
参考文献
LUO DS, 2018. The original interpretation of Jingzhu Materia Medica [M]. Chengdu: Sichuan Science and Technology Press: 485. [罗达尚, 2018. 晶珠本草正本诠释 [M]. 成都: 四川科学技术出版社: 485. ]
参考文献
MILNE RI, DAVIES C, PRICKETT R, et al. , 2010. Phylogeny of Rhododendron subgenus Hymenanthes based on chloroplast DNA markers: between-lineage hybridisation during adaptive radiation? [J] Plant Syst Evol, 285: 233-244.
参考文献
MA YP, XIE WJ, SUN WB, et al. , 2016. Strong reproductive isolation despite occasional hybridization between a widely distributed and a narrow endemic Rhododendron species [J]. Sci Rep, 6(1): 1-11.
参考文献
MAYOR C, BRUDNO M, SCHWARTZ JR, et al. , 2000. VISTA: visualizing global DNA sequence alignments of arbitrary length [J]. Bioinformatics, 16(11): 1046-1047.
参考文献
MENG J, LI XP, LI HT, et al. , 2018. Comparative analysis of the complete chloroplast genomes of four Aconitum medicinal species [J]. Molecules, 23(5): 1015.
参考文献
PATEL RK, JAIN M, 2012. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data [J]. PLoS ONE, 7(2): e30619.
参考文献
PENG LW, YAMAMOTO H, SHIKANAI T, 2011. Structure and biogenesis of the chloroplast NAD (P) H dehydrogenase complex [J]. Biochim et Biophys Acta, 1807(8): 945-953.
参考文献
PING JY, FENG PP, LI JY, et al. , 2021. Molecular evolution and SSRs analysis based on the chloroplast genome of Callitropsis funebris [J]. Ecol Evol, 11(9): 4786-4802.
参考文献
Qinghai Provincial Drug Inspection Institute, Qinghai Tibetan Medicinal Institute, 1996. Chinese Tibetan Medicine [M]. Shanghai: Shanghai Science & Technology Press: 210-211. [青海省药品检验所, 青海省藏医药研究所, 1996. 中国藏药 [M]. 上海: 上海科学技术出版社: 210-211. ]
参考文献
ROZAS J, FERRER-MATA A, SÁNCHEZ-DELBARRIO JC, et al. , 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets [J]. Mol Biol Evol, 34(12): 3299-3302.
参考文献
SHARP PM, COWE E, 1991. Synonymous codon usage in Saccharomyces cerevisiae [J]. Yeast, 7(7): 657-678.
参考文献
SHI LC, CHEN HM, JIANG M, et al. , 2019. CPGAVAS2, an integrated plastome sequence annotator and analyzer [J]. Nucl Acids Res, 47(W1): W65-W73.
参考文献
SILVA SR, DIAZ YC, PENHA HA, et al. , 2016. The chloroplast genome of Utricularia reniformis sheds light on the evolution of the ndh gene complex of terrestrial carnivorous plants from the Lentibulariaceae family [J]. PLoS ONE, 11: e0165176.
参考文献
STAMATAKIS A, 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies [J]. Bioinformatics, 30: 1312-1313.
参考文献
SUGIURA C, KOBAYASHI Y, AOKI S, et al. , 2003. Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus [J]. Nucl Acids Res, 31(18): 5324-5331.
参考文献
TANG HQ, TANG L, SHAO SC, et al. , 2021. Chloroplast genomic diversity in Bulbophyllum section Macrocaulia (Bl. ) Aver. (Orchidaceae, Epidendroideae, Malaxideae): insights into species divergence and adaptive evolution [J]. Plant Divers, 43(5): 350-361.
参考文献
WANG L, DONG WP, ZHOU SL, 2012. Structural mutations and reorganizations in chloroplast genomes of flowering plants [J]. Acta Bot Boreal-Occident Sin, 32(6): 1282-1288. [王玲, 董文攀, 周世良, 2012. 被子植物叶绿体基因组的结构变异研究进展 [J]. 西北植物学报, 32(6): 1282-1288. ]
参考文献
WANG XQ, SONG WW, XIAO JJ, et al. , 2021. Phylogeny of Myrtales and related groups based on chloroplast genome [J]. Guihaia, 41(1): 68-80. [王雪芹, 宋卫武, 肖建加, 等, 2021. 基于叶绿体基因组探讨桃金娘目及其近缘类群的系统发育关系 [J]. 广西植物, 41(1): 68-80. ]
参考文献
WICKE S, MÜLLER KF, DEPAMPHILIS CW, et al. , 2016. Mechanistic model of evolutionary rate variation en route to anonphotosynthetic lifestyle in plants [J]. Proc Natl Acad Sci, 113(32): 9045-9050.
参考文献
XIE DF, YU HX, PRICE M, et al. , 2019. Phylogeny of Chinese Allium species in section Daghestanica and adaptive evolution of Allium (Amaryllidaceae, Allioideae) species revealed by the chloroplast complete genome [J]. Front Plant Sci, 10: 460.
参考文献
YAN LJ, LIU J, MICHAE M, et al. , 2015. DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya-Hengduan Mountain [M]. Mol Ecol Resour, 15(4): 932-944.
参考文献
YANG HB, FANG RZ, JIN CL, et al. , 1999. Flora Reipublicae Popularis Sinicae (Vol. 57, Part 1) [M]. Beijing: Science Press: 13-213. [杨汉碧, 方瑞征, 金存礼, 1999. 中国植物志(第57 卷第1分册) [M]. 北京: 科学出版社: 13-213].
参考文献
YANG KT, CHEN GP, XIAN JR, et al. , 2021. Scaling relationship between leaf mass and leaf area: A case study using six alpine Rhododendron species in the Eastern Tibetan Plateau [J]. Global Ecol Conserv, 30: e01754.
参考文献
YI X, GAO L, WANG B, et al. , 2013. The complete chloroplast genome sequence of Cephalotaxus oliveri (Cephalotaxaceae): evolutionary comparison of Cephalotaxus chloroplast DNAs and insights into the loss of inverted repeat copies in gymnosperms [J]. Genome Biol Evol, 5(4): 688-698.
参考文献
ZHANG DG, CAO WX, PU XP, et al. , 2003. Botanical morpha and ecological adaptation of Rhododendron in Eastern Qilian Mountains [J]. Grassl Turf, (1): 27-30. [张德罡, 曹文侠, 蒲小鹏, 等, 2003. 东祁连山杜鹃属植物形态特征及其生态适应性 [J]. 草原与草坪, (1): 27-30. ]
参考文献
ZHANG Q, LIU Y, SODMERGEN, 2003. Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species [J]. Plant Cell Physiol, 44(9): 941-951.
参考文献
ZHANG YM, HAN LJ, YANG CW, et al. , 2022. Comparative chloroplast genome analysis of medicinally important Veratrum (Melanthiaceae) in China: insights into genomic characterization and phylogenetic relationships [J]. Plant Divers, 44: 70-82.
参考文献
ZHUANG P, 2019. Progress on the fertility of Rhododendron [J]. Biodivers Sci, 27(3): 327-338. [庄平, 2019. 杜鹃花属植物的可育性研究进展 [J]. 生物多样性, 27(3): 327-338. ]
目录contents

    摘要

    头花杜鹃(Rhododendron capitatum)和陇蜀杜鹃(R. przewalskii)是极具观赏价值的野生花卉和药用植物。为探讨头花杜鹃和陇蜀杜鹃叶绿体基因组的遗传结构及进化特征,该研究利用 Illumina HiSeq 4000 平台对头花杜鹃和陇蜀杜鹃的叶绿体全基因组进行测序,经组装和注释后,结合 7 个已发表的杜鹃属植物叶绿体全基因组进行比较基因组学分析和系统发育分析。结果表明:(1)头花杜鹃和陇蜀杜鹃叶绿体全基因组呈典型的环状四分体结构,均由一个大单拷贝区(105990、109191 bp)、一个小单拷贝区(2617、2606 bp)和一对反向重复区(45825、47516 bp)构成,全长分别为200257、206829 bp。(2)头花杜鹃和陇蜀杜鹃叶绿体基因组中共鉴定出 263 个SSR位点,大部分 SSR 偏好使用 A/T 碱基,密码子偏好使用 A/U 结尾。(3)杜鹃属植物叶绿体全基因组中普遍存在基因丢失以及基因组重排等结构变异现象。该研究丰富了杜鹃属植物的基因组资源,为头花杜鹃、陇蜀杜鹃的资源开发、遗传进化、育种及系统发育相关研究提供了理论参考。

    Abstract

    Rhododendron capitatum and R. przewalskii are ornamental flowers and medicinal plants. The chloroplast genomes of R. capitatum and R. przewalskii have been sequenced using Illumina HiSeq 4000 platform to explore the genetic structure and evolution features. After assembly and annotation, comparative genomic analysis and phylogenetic analysis of the chloroplast genomes of R. capitatum and R. przewalskii were analyzed and compared with those of other Rhododendron species. The results were as follows: (1) The chloroplast genomes of R. capitatum and R. przewalskii exhibited a typical quadripartite structure, including a large single copy region (105990, 109191 bp), a small single copy region (2617, 2606 bp), and a pair of inverted repeat regions (45825, 47516 bp). And the two Rhododendron species chloroplast genomes were 200257 bp and 206829 bp in length, respectively. (2) In addition, the 263 SSRs were detected in the chloroplast genomes of R. capitatum and R. przewalskii, most of which had A/T base preference; the codons preferred ending in A/U. (3) The chloroplast genomes of Rhododendron species exhibit structural variation such as gene loss and genomic rearrangement. The study enriches the genomic resources of Rhododendron species and provides the theoretical reference for resource exploitation, genetic evolution, breeding, and phylogeny of R. capitatum and R. przewalskii.

  • 杜鹃属(Rhododendron L.)是杜鹃花科(Ericaceae)中最大的属,也是多样性最为丰富的一个属,全球共有 1 000 余种,我国约 571 种,集中产于西南、华南地区(杨汉碧等,1999)。杜鹃属植物种类繁多,花型优美且色泽艳丽,是国际知名花卉,也是我国传统十大名花之一,素有“木本花卉之王”、“花中西施”的美誉(李云飞等,2019)。中国是世界上拥有野生杜鹃花资源最丰富的国家,得天独厚的资源优势为我国发展杜鹃花卉产业和开展杜鹃花品种研究提供了有利条件,但对于杜鹃属植物的引种驯化、育种和开发利用的研究工作却远远落后于其他园林发达的国家和地区(程淑媛,2017;庄平,2019)。一方面,杜鹃属植物属于快速的辐射分化(Milne et al.,2010)以及频繁的种间杂交(Ma et al.,2016)繁衍方式,因此形成多种变异和种间杂交类型,给野生杜鹃属植物的种质资源分类鉴定带来困难;另一方面,我国杜鹃花育种起步较晚,基础薄弱(耿兴敏等,2021),在很大程度上制约了杜鹃花新品种的选育及资源挖掘利用的进程。

  • 头花杜鹃(Rhododendron capitatum)和陇蜀杜鹃(R. przewalskii)均为杜鹃属中兼有较高药用价值和观赏价值的常绿灌木。头花杜鹃和陇蜀杜鹃常以叶、花入药,用于治疗培根病、龙病等,是藏族地区特有的民族特色习用药材(青海省药品检验所和青海省藏医药研究所,1996;罗达尚,2018);并且这两种植物树形优美、花色艳丽、花期长,加上其耐寒、耐旱、适应性强等方面的优点,可作为优良的灌木类观花植物育种的亲本材料(曹文侠,2001;李永金,2011),因而极具园林观赏价值和良好的产业化前景。目前,对这两种杜鹃的研究主要集中在功能性状(何家莉等,2021;Yang et al.,2021)、化学成分(Liao et al.,2017; Bai et al.,2019)、生态适应性(张德罡等,2003;曹晓娟等,2009)等方面的研究,而关于其遗传背景的研究尚少。明确其遗传背景有利于掌握和有效利用优异性状基因资源,通过分子遗传学育种方法对这两种杜鹃属植物进行种质创新和品种改良具有重要意义。

  • 叶绿体起源于细胞内共生的蓝藻,是植物细胞进行光合作用的细胞器,参与植物的生长发育,在植物漫长的演化过程中发挥着至关重要的作用(Daniell et al.,2016)。高等植物叶绿体基因组一般为一个长度120~160 kb 的双链环状分子,由一个大单拷贝区(large single copy,LSC)(81~90 kb)、一个小单拷贝区(small single copy,SSC)(18~20 kb)及一对完全相同的反向重复区(inverted repeats,IRs)(20~30 kb)组成(Jansen et al.,2005)。大部分高等植物叶绿体基因组为母系遗传,结构保守,包含大量的遗传信息,是植物遗传改良转化、遗传多样性分析、适应性进化、分子育种研究的惯用信息来源,为经济作物改良、园艺品种选育、濒危物种保育提供了分子基础(Gu et al.,2019; 王雪芹等,2021;Zhang et al.,2022)。此外,前人采用了DNA条形码技术(Yan et al.,2015; 白霄霞等,2019)、简化基因组测序技术(李云飞等,2019;黄承玲等,2020)、基因组浅层测序技术(Fu et al.,2022)等基因组片段对杜鹃属植物的系统发育关系进行了探索,虽然基本能辨清其亲缘关系,但现有对杜鹃属的分类研究大多仅限于属、亚属或组等分类阶元,对于物种、亚组、组间的鉴别能力仍较弱。叶绿体基因组序列分子数小,进化速率适中,长度是普通条形码序列的几百倍,包含了更多的遗传信息,具有更强的分辨率,可以作为DNA超级条形码对植物物种进行准确的鉴别(Kane et al.,2012; Xie et al.,2019)。

  • 本研究以头花杜鹃和陇蜀杜鹃为研究材料,在对其进行测序、组装和注释的基础上,全面解析其序列与结构特征,并结合已发表的 7 个杜鹃属植物叶绿体基因组序列进行比较分析。拟探讨以下科学问题:(1)头花杜鹃、陇蜀杜鹃叶绿体基因组序列有什么特征;(2)杜鹃属植物叶绿体基因组有什么进化特征;(3)杜鹃属植物叶绿体基因组的变异模式及产生的原因是什么。本研究将进一步为头花杜鹃、陇蜀杜鹃及杜鹃属植物的遗传育种、物种鉴别及资源开发利用等研究提供遗传资源。

  • 1 材料与方法

  • 1.1 植物材料

  • 头花杜鹃于 2021 年 9 月 6 日采自青海省海东市互助县(102°15′90.11″ E、37°00′16.11″ N),陇蜀杜鹃于 2021 年 8 月 11 日采自青海省海北藏族自治州门源县(101°80′12.39″ E、37°25′19.25″ N),采集时选取健康植株的新鲜嫩叶放入硅胶中干燥保存,用于 DNA 提取,同时采集带花果的植株用于后期的形态鉴定。所有凭证标本由青海省中医院赵国福副主任药师鉴定,并存放于青海省中医院标本馆(头花杜鹃:632126LY0192,陇蜀杜鹃:632126LY0128)。

  • 1.2 方法

  • 1.2.1 总基因组DNA 提取与测序

  • 总 DNA 的提取采用百泰克植物基因组快速提取试剂盒(Plant Genomic DNA Kit,Beijing)进行,采用NanoDrop 2000(Thermo Fisher Scientific,USA)检测 DNA 纯度,采用Quantus Fluorometer(Picogreen)检测 DNA 浓度,采用琼脂糖凝胶电泳检测 DNA 完整性。接着进行 DNA 文库构建,并利用 Illumina HiSeq 4000 平台进行测序。原始数据(raw reads)在 NGS QC ToolKit(Patel &Jain,2012)中进行过滤,去除其中的低质量区后得到待组装序列(clean reads)。

  • 1.2.2 叶绿体基因组的组装、注释以及物理图谱的构建

  • 以阔柄杜鹃的叶绿体全基因组序列(NC_053746)为参考序列,将过滤后的clean reads在 GetOrganelle 软件包(Jin et al.,2020)中进行组装。组装完成的叶绿体全基因组序列利用 CPGAVAS2 软件(Shi et al.,2019)进行注释,并在 Geneious Prime 软件(Drummond,2012)中以阔柄杜鹃(R. platypodum,NC_053746)、秀雅杜鹃(R. concinnum,MT239366)叶绿体全基因组为参考校正注释结果。将叶绿体基因组序列导入OrganellarGenomeDRAW 软件(Greiner et al.,2019)中,进行物理图谱的绘制。通过 Geneious Prime 软件将检查无误的头花杜鹃、陇蜀杜鹃叶绿体全基因组序列上传至 GenBank 数据库。

  • 1.2.3 基本特征分析

  • 使用 MISA 在线工具(Beier et al.,2017)对头花杜鹃、陇蜀杜鹃叶绿体基因组中的简单重复序列(simple sequence repeat,SSR)进行检测,单核苷酸(mono-nucleotide)SSR、二核苷酸(di-nucleotide)SSR、三核苷酸(tri-nucleotide)SSR、四核苷酸(tetra-nucleotide)SSR、五核苷酸(penta-nucleotide)SSR和六核苷酸(hexa-nucleotide)SSR的最小重复值分别设置为 10、5、4、3、3 和 3。

  • 对9个杜鹃属植物叶绿体基因组中的蛋白编码基因(protein coding gene,PCG)进行筛选,剔除重复基因以及长度小于 300 bp的基因,获得符合条件的编码蛋白质的基因序列,接着使用 MEGA X 软件(Kumar et al.,2018)对这些符合条件的基因序列进行密码子使用偏好性分析,以反映编码相应氨基酸的密码子使用偏好。

  • 以阔柄杜鹃(NC_053746)的叶绿体基因组作为参考序列,使用 mVISTA 在线工具(Mayor et al.,2000)的 shufffle-LAGAN 模式(Brudno et al.,2003)对9个杜鹃属植物叶绿体基因组序列进行分析。

  • 1.2.4 系统发育分析

  • 为了明确头花杜鹃和陇蜀杜鹃在杜鹃属中的系统发育位置,本研究选取17条叶绿体基因组序列进行系统进化树的构建。除了本研究新测定的头花杜鹃和陇蜀杜鹃叶绿体基因组序列外,其余15条均下载自NCBI,包括7条杜鹃属植物序列,3条白珠树属(Gaultheria Kalm ex Linn.)植物序列,3条越桔属(Vaccinium Linn.)植物序列,并以 1 条鹿蹄草属(Pyrola Linn.)植物序列和 1 条喜冬草属(Chimaphila Pursh)植物序列为外类群。将以上17条叶绿体全基因组导入 MAFFT v.7软件(Katoh &Standley,2013)中进行比对,比对的结果在 ModelFinder 软件(Rozas et al.,2017)中计算最佳拟合的核苷酸替代模型和相应参数。分别以最大似然法(maximum likelihood,ML)和贝叶斯法(Bayesian inference,BI)进行系统发育分析。ML 树在 RAxML v8.2.4 软件(Stamatakis,2014)中构建,参数设置为 Models = GTR + GAMMA,bootstrap = 1 000;BI 树在 MrBayes v3.2.6 软件(Huelsenbeck &Ronquist,2001)中构建,参数设置为 Models = GTR + GAMMA,Generations = 2 000 000,Sampling Freq = 1 000,Burnin Fraction = 0.25。

  • 2 结果与分析

  • 2.1 叶绿体基因组基本特征

  • 头花杜鹃与陇蜀杜鹃叶绿体基因组序列均为环式双链分子,呈经典的四段式结构,包括一个大单拷贝区(LSC)、一个小单拷贝区(SSC)以及两个反向重复区(IRs)(图1)。头花杜鹃叶绿体基因组(GenBank登录号:OL804295)全长(Full)为200 257 bp,LSC 区、SSC 区以及两个IRs区长度分别为105 990、2 617、45 825 bp;陇蜀杜鹃叶绿体基因组(GenBank登录号:OL871190)全长(Full)为206 829 bp,LSC 区、SSC 区以及两个IRs区长度分别为 109 191、2 606、47 516 bp。头花杜鹃和陇蜀杜鹃的叶绿体基因组总 GC 含量分别为 35.8%和 35.7%,IRs区的 GC 含量(36.6%、36.5%)均高于 LSC 区(为 35.4%、35.1%)及 SSC 区(29.8%、30.0%)。9 个杜鹃花属植物的叶绿体基因组长度范围为 193 798~208 015 bp,其中最大的是灵宝杜鹃,最小的是马缨杜鹃(原变种);LSC 区长度范围为105 990~110 593 bp,SSC 区长度范围为 26~2 621 bp,IRs区长度范围为 40 583~47 516 bp;总GC含量为 35.7%~36.0%,差异不大(表1)。

  • 头花杜鹃叶绿体基因组编码 138 个基因,包括86个蛋白编码基因(PCG)、44 个 tRNA 基因及 8 个 rRNA基因;陇蜀杜鹃叶绿体基因组则编码 143个基因,包括89个PCG、46 个 tRNA 基因及 8 个 rRNA基因。目前已发表的 9 种杜鹃属植物叶绿体基因组编码的基因从 123 个 [马缨杜鹃(原变种)]至 150 个(朱红大杜鹃)不等,蛋白编码基因 80 个 [马缨杜鹃(原变种)]~95个(朱红大杜鹃),tRNA 基因 35 个 [马缨杜鹃(原变种)]~47 个(朱红大杜鹃),rRNA 基因均为 8 个(表1),引起差异的主要原因是 ycf1、ycf15、trnR-UCU、trnM-CAU、trnH-GUG、accDinfA 等基因在部分物种中缺失(表2)。

  • 2.2 简单重复序列分析

  • 通过对头花杜鹃和陇蜀杜鹃叶绿体基因组序列进行 MISA 分析,共检测出 263 个简单重复序列,6 种类型(单核苷酸 SSR、二核苷酸 SSR、三核苷酸 SSR、四核苷酸 SSR、五核苷酸 SSR 和六核苷酸 SSR)。在头花杜鹃中检测到 130 个SSR,其中,74 个 SSR 分布在LSC,2 个 SSR 分布在SSC区,两个IRs共分布54个 SSR,最丰富的类型是单核苷酸 SSR(86 个),其次是二核苷酸 SSR(19 个)、三核苷酸 SSR(12 个)、四核苷酸 SSR(12 个)、六核苷酸 SSR(1 个),未检测到五核苷酸 SSR。在陇蜀杜鹃杜鹃中共检测到 133 个 SSR,有 87 个 SSR 分布在LSC区, 3 个 SSR 分布在SSC区,两个IRs区共分布43个SSR,其中包括 71 个单核苷酸 SSR、19 个二核苷酸 SSR、25 个三核苷酸 SSR、16 个四核苷酸 SSR,五核苷酸 SSR 和六核苷酸 SSR 各一个(表3)。

  • 2.3 密码子使用偏好性分析

  • 为了进行 9 种杜鹃属植物叶绿体基因组的密码子使用偏好性分析,共筛选出 436 条符合条件的蛋白编码基因序列,其中头花杜鹃 47 条、陇蜀杜鹃 49 条、秀雅杜鹃 49 条、马缨杜鹃(原变种) 44 条、朱红大杜鹃 54 条、灵宝杜鹃 49 条、照山白 50 条、羊踯躅 49 条和阔柄杜鹃 45 条。通过对杜鹃属植物叶绿体基因组密码子使用偏好性分析,由图2可知,在大部分杜鹃属植物叶绿体基因组中,RSCU>1 的高频密码子共有 30 个(AGA、UAA、GCU 等),其中 29 个以 A/U 碱基结尾,而以 G/C 结尾的仅 1 个,表明杜鹃属植物叶绿体密码子偏爱于以 A/U 结尾;RSCU<1 的密码子共有 32 个(AGC、CGC、UGA等),以 G/C 结尾的有 29 个,以 A/U 结尾的有 3 个。而头花杜鹃、照山白和羊踯躅叶绿体基因组中,除 UGA 被偏好使用外(RSCU>1),其余密码子的偏好性均一致。以上结果分析表明,9个杜鹃属物种之间密码子的偏好性保持高度的一致。

  • 图1 9种杜鹃属植物叶绿体全基因组物理图谱

  • Fig.1 Physical maps of nine Rhododendron species complete chloroplast genomes

  • 表1 9种杜鹃属植物叶绿体基因组基本特征

  • Table1 Comparison of general features of nine Rhododendron species chloroplast genomes

  • 2.4 杜鹃属叶绿体基因组比较

  • 利用 mVISTA 软件对9种杜鹃属植物叶绿体基因组进行了全序列比对。由图3可知,杜鹃属植物叶绿体基因组种间整体上呈现较高的保守性,编码区比非编码区保守,反向重复区比两个单拷贝区保守。在9种杜鹃属植物中,分化程度较高的是matK-ycf3、trnM-CAU-rpoBtrnT-GGU-accDrpoA-psbJtrnM-CAU-rrn16、trnI-CAU-rps16 和 rps16-psaI7个基因间隔区(图3)。

  • 2.5 系统发育分析

  • 基于 17 个物种的叶绿体全基因组序列的系统发育分析结果(图4)显示,ML和BI系统发育树拓扑结构完全一致,所有的分支都具有较高的支持率(BS≥63%,PP=1)。杜鹃属的单系性得到强烈支持(BS=100%,PP=1),白珠树属与越桔属形成姊妹群(BS=100%,PP=1),共同构成杜鹃属的姊妹群,支持率较高(BS=100%,PP=1)。在杜鹃内部,9 个杜鹃属物种聚为一支,常绿杜鹃亚属 5 个种 [陇蜀杜鹃、阔柄杜鹃、朱红大杜鹃、马缨杜鹃(原变种)、灵宝杜鹃]聚为一支(BS=100%,PP=1),杜鹃亚属 3 个种(秀雅杜鹃、照山白、头花杜鹃)也聚在一起,但未形成单系。本研究测得的陇蜀杜鹃与阔柄杜鹃互为姐妹关系,支持率较高(BS=67%,PP=1);陇蜀杜鹃与照山白亲缘关系相对较近(BS=100%,PP=1)。

  • 3 讨论与结论

  • 本研究通过对头花杜鹃、陇蜀杜鹃叶绿体全基因组的特征分析,以及与 7 个已报道的杜鹃属植物叶绿体基因组比较分析结果表明,杜鹃属植物叶绿体基因组结构和基因顺序发生了显著变化。通过与大部分被子植物叶绿体基因组对比发现,杜鹃属植物叶绿体基因组中除 SSC 区急剧缩小外,其余各区均高度延展,特别是 IR 区几乎延展约 2 倍,由此推测 IR 区的扩张是导致杜鹃属植物叶绿体基因组增大的主要原因。这种整个基因组发生的显著结构变异的重排现象也在其他物种中发现,如天竺葵(Pelargonium × hortorum)叶绿体基因组的 IR 区延展至 75 kb,基因组全长约为 200 kb(Chumley et al.,2006),日本柳杉(Cryptomeria japonica) 叶绿体基因组完全缺失了 IR 区,其叶绿体基因组全长为 130 kb 左右(Hirao et al.,2008)。此外,杜鹃属植物叶绿体基因组 GC 含量(35.7%~36.0%)比许多被子植物如乌头属(Aconitum L.)植物(约 38.0%)(Meng et al.,2018)、五福花科(Adoxaceae)植物(约 38.3%)(Fan et al.,2018)低,这种低 GC 含量与叶绿体基因组结构重排数量的增加有关(Wicke et al.,2016)。目前叶绿体基因组重排的机制还不清楚,有研究推断,叶绿体基因组重排可能与叶绿体基因组的遗传方式有关(Zhang et al.,2003;王玲等,2011)。杜鹃属植物具有典型的自然杂交现象,当两个遗传背景差异较大的个体产生可育杂交后代时,基因组发生了重组与整合,或许是该属植物叶绿体基因组结构发生变异的原因。

  • 表2 9种杜鹃属植物叶绿体基因组的基因

  • Table2 Genes of nine Rhododendron species chloroplast genomes

  • 续表2

  • 续表2

  • 注: · 表示包含该基因; -表示缺失该基因。Rca. 头花杜鹃; Rpr. 陇蜀杜鹃; Rco. 秀雅杜鹃; Rde. 马缨杜鹃(原变种); Rgr. 朱红大杜鹃; Rhe. 灵宝杜鹃; Rmi. 照山白; Rmo. 羊踯躅; Rpl. 阔柄杜鹃。

  • Note: · indicates the gene presence; -indicates the gene loss. Rca. Rhododendron capitatum; Rpr. R. przewalskii; Rco. R. concinnum; Rde. R. delavayi var. delavayi; Rgr. R. griersonianum; Rhe. R. henanense subsp. lingbaoense; Rmi. R. micranthum; Rmo. R. mole; Rpl. R. platypodum.

  • 表3 头花杜鹃、陇蜀杜鹃叶绿体基因组中的简单重复序列

  • Table3 Simple sequence repeats of the chloroplast genomes in Rhododendron capitatum and R. przewalskii

  • 杜鹃属植物叶绿体基因组的结构变异导致了基因复制和缺失事件。在大部分被子植物中 ycf1 基因具有 2 个拷贝,位于 IRa-SSC 边界的为完整基因,位于 IRb-SSC 边界的 ycf1 基因则被截断,成为一个假基因,除阔柄杜鹃外的 8 种杜鹃属植物叶绿体基因组均完全缺失了 ycf1 基因,结合该属植物叶绿体基因组特征推断,不同物种出现不同类型的基因缺失或复制与 IR 区的扩张或收缩的程度差异有关。此外,对于 tRNA 来说,某一基因拷贝的缺失,有其他拷贝可以作为补充,不影响功能的执行,对蛋白编码基因来说,则需要核基因的编码产物转运至叶绿体补充相应的功能(李岩等,2017),如秀雅杜鹃、马缨杜鹃(原变种)等叶绿体基因组缺失了 infA 基因,或许是该基因从叶绿体基因组转移到了细胞核中并进行转录,随后再将编码的蛋白转运回叶绿体参与生命活动(Sugiura et al.,2003; Lee et al.,2006)。与基因缺失相反,ndhF 等基因由 SSC 区转移至 IR 区时出现基因复制的情况。叶绿体 NADH 脱氢酶复合体参与光合循环电子传递和叶绿体呼吸等过程,突变率高,对环境条件和胁迫敏感(Peng et al.,2011; Silva et al.,2016),杜鹃属植物大多地处严寒低氧的高原地带,这意味着 ndhF等基因的复制可能与物种的环境适应性有关。总之,叶绿体基因组结构重排通常也伴随着部分基因的缺失或复制,这种现象是比较常见的,而杜鹃属不同物种间基因缺失的情况差异较大,说明了杜鹃属植物叶绿体基因组具有较高的突变性。

  • 密码子偏好性是指在 DNA 编码过程中的各同义密码子非均衡使用的现象(Sharp &Cowe,1991)。这是物种或基因长期进化和对自然环境适应性的结果,这种偏好性出现在不同物种之间、不同基因组以及不同基因中(Li et al.,2019)。密码子使用偏好性是基因组中一个重要的演化特征,对基因表达和生物体进化具有重要意义。本研究利用生物信息学统计了已发表的杜鹃属植物叶绿体的密码子使用频率,筛选出了其偏好使用的密码子,从而为提高外源基因的表达效率、转基因研究以及改良育种提供参考。

  • 叶绿体基因组中的 SSR 具有丰富的多态性位点,因其易复制、遗传信息含量高等优点而被广泛应用于物种遗传多样性、分子辅助育种和亲缘关系鉴定等领域(Ping et al.,2021; Tang et al.,2021)。本研究在头花杜鹃和陇蜀杜鹃 2 个叶绿体基因组中发现,SSR 类型以单核苷酸 SSR 为主,其次为二核苷酸 SSR 和三核苷酸 SSR,且基序长度越长,对应的 SSR 越少。几乎所有SSR 的重复单元主要由 A和T 碱基组合构成,说明叶绿体基因组序列中 SSR 的基序类型以多聚的 A/T 为主,在其他被子植物中也有这种现象存在(Yi et al.,2013)。这些 SSR 可为杜鹃属植物品种选育、遗传多样性等研究提供候选分子标记。

  • 图2 9种杜鹃属植物相对同义密码子使用度(RSCU)热图分析

  • Fig.2 Heatmap analysis of relative synonymous codon usage (RSCU) values of nine Rhododendron species

  • 图3 9种杜鹃属植物叶绿体基因组比对图

  • Fig.3 Comparison of nine Rhododendron species chloroplast genomes

  • 图4 基于17个叶绿体基因组以最大似然法和贝叶斯法构建的系统发育树

  • Fig.4 Phylogenetic tree of 17 species using maximum likelihood (ML) and Bayesian inference (BI) methods based on the chloroplast genomes

  • 杜鹃属是分类学研究中较为特殊和困难的一个类群。本研究基于叶绿体基因组构建的杜鹃属系统进化树分辨率较好,支持杜鹃属的单系性,且常绿杜鹃亚属与其他亚属可以完全分开,表明叶绿体基因组用于解决杜鹃属物种的鉴定和系统发育关系的问题非常有效,但杜鹃属植物种类繁多,还需要更多的杜鹃属物种叶绿体基因组序列的公布,才能得到更符合实际的进化谱系。

  • 参考文献

    • BAI L, JIAO ML, ZANG HY, et al. , 2019. Chemical composition of essential oils from four Rhododendron species and their repellent activity against three stored-product insects [J]. Environ Sci Pollut Res, 26(22): 23198-23205.

    • BAI XX, SHEN FJ, LI ZB, et al. , 2019. DNA barcode identification of genus Rhododendron L. : reference library, molecular markers and cultivar wild relatives tracing [J]. Tianjin Agric Sci, 25(10): 1-7. [白霄霞, 沈风娇, 李志斌, 等, 2019. 杜鹃花属 (Rhododendron L. ) 植物 DNA 条形码鉴定——参考序列库, 分子标记与栽培品种野生亲本溯源 [J]. 天津农业科学, 25(10): 1-7. ]

    • BEIER S, THIEL T, MÜNCH T, et al. , 2017. MISA-web: a web server for microsatellite prediction [J]. Bioinformatics, 33(16): 2583-2585.

    • BRUDNO M, MALDE S, POLIAKOV A, et al. , 2003. Glocal alignment: Finding rearrangements during alignment [J]. Bioinformatics, 19: i54-i62.

    • CAO WX, 2001. Ecological adaptive strategy of genus Rhododendron L. in eastern Qilian Mountains alpine ecosystem [D]. Gansu: Northwest Normal University: 35-37. [曹文侠, 2001. 东祁连山高寒生态系统杜鹃花属植物适应对策的研究 [D]. 甘肃: 西北师范大学: 35-37. ]

    • CAO XJ, LIU JJ, YANG M, 2009. Photosynthetic characteristics and anatomical structure of five species of Rhododendron in the Taibai Mountain [J]. Acta Bot Boreal-Occident Sin, 29(12): 2483-2491. [曹晓娟, 刘建军, 杨梅, 2009. 太白山 5 种杜鹃属植物叶片光合特性及解剖结构的生态适应性研究 [J]. 西北植物学报, 29(12): 2483-2491. ]

    • CHENG SY, 2017. Study on resources and classification of azalea cultivars in China [D]. Ganzhou: Gannan Normal University: 11-12. [程淑媛, 2017. 中国杜鹃花栽培品种资源与分类研究 [D]. 赣州: 赣南师范大学: 11-12]

    • CHUMLEY TW, PALMER JD, MOWER JP, et al. , 2006. The complete chloroplast genome sequence of Pelargonium × hortorum: organization and evolution of the largest and most highly rearranged chloroplast genome of land plants [J]. Mol Biol Evol, 23(11): 2175-2190.

    • DANIELL H, LIN CS, YU M, et al. , 2016. Chloroplast genomes: diversity, evolution, and applications in genetic engineering [J]. Genome Biol, 17(1): 134.

    • DRUMMOND A, 2012. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data [J]. Bioinformatics, 28(12): 1647-1649.

    • FAN WB, WU Y, YANG J, et al. , 2018. Comparative chloroplast genomics of Dipsacales species: insights into sequence variation, adaptive evolution, and phylogenetic relationships [J]. Front Plant Sci, 9: 689.

    • FU CN, MO ZQ, YANG JB, et al. , 2022. Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron [J]. Mol Ecol Resour, 22(1): 404-414.

    • GENG XM, HUAN ZQ, SU JL, et al. , 2021. Researches advances in germplasm innovation of Rhododendrons [J]. Mol Plant Breed, 19(2): 604-613. [耿兴敏, 宦智群, 苏家乐, 等, 2021. 杜鹃花属植物种质创新研究进展 [J]. 分子植物育种, 19(2): 604-613. ]

    • GREINER S, LEHWARK P, BOCK R, 2019. Organellar-GenomeDRAW (OGDRAW) version 1. 3. 1: expanded toolkit for the graphical visualization of organellar genomes [J]. Nucl Acid Res, 47(W1): W59-W64.

    • GU CH, MA L, WU ZQ, et al. , 2019. Comparative analyses of chloroplast genomes from 22 Lythraceae species: inferences for phylogenetic relationships and genome evolution within Myrtales [J]. BMC Plant Biol, 19(1): 1-19.

    • HE JL, ZHOU TY, SONG YK, et al. , 2021. Variations in floral traits of Rhododendron przewalskii with slope aspect in the southeastern Tibetan Plateau [J]. Chin J Appl Environ Biol, 27(4): 860-868. [何家莉, 周天阳, 宋怡珂, 等, 2021. 青藏高原东南缘不同坡向对陇蜀杜鹃花性状的影响 [J]. 应用与环境生物学报, 27(4): 860-868. ]

    • HIRAO T, WATANABE A, KURITA M, et al. , 2008. Complete nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species [J]. BMC Plant Biol, 8(1): 1-20.

    • HUANG CL, YAO G, TIAN XL, et al. , 2021. Phylogenomic analysis of Rhododendron species in Guizhou Baili Rhododendron reserve based on RAD sequencing [M]. Sci Silv Sin, 57(2): 72-81. [黄承玲, 姚刚, 田晓玲, 等, 2021. 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, 57(2): 72-81. ]

    • HUELSENBECK JP, RONQUIST F, 2001. MRBAYES: Bayesian inference of phylogenetic trees [J]. Bioinformatics, 17: 754-755.

    • JANSEN RK, RAUBESON LA, BOORE JL, et al. , 2005. Methods for obtaining and analyzing whole chloroplast genome sequences [J]. Meth Enzym, 395: 348-384.

    • JIN JJ, YU WB, YANG JB, et al. , 2020. GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes [J]. Genome Biol, 21(1): 1-31.

    • KANE N, SVEINSSON S, DEMPEWOLF H, et al. , 2012. Ultra-barcoding in cacao (Theobroma spp. ; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA [J]. Am J Bot, 99(2): 320-329.

    • KATOH K, STANDLEY DM, 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability [J]. Mol Biol Evol, 30(4): 772-780.

    • KUMAR S, STECHER G, LI M, et al. , 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms [J]. Mol Biol Evol, 35(6): 1547.

    • LEE SB, KAITTANIS C, JANSEN RK, et al. 2006. The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms [J]. BMC Genomic, 7(1): 1-12.

    • LI Y, LÜ GH, ZHANG XN, et al. , 2017. Chloroplast genome structure and variation analysis of Brasicaceae species [J]. Acta Bot Boreal-Occident Sin, 37(6): 1090-1101. [李岩, 吕光辉, 张雪妮, 等, 2017. 十字花科植物叶绿体基因组结构及变异分析 [J]. 西北植物学报, 37(6): 1090-1101. ]

    • LI Y, SYLVESTER SP, LI M, et al. , 2019. The complete plastid genome of Magnolia zenii and genetic comparison to Magnoliaceae species [J]. Molecules, 24(2): 261.

    • LI YF, LI SM, JIN X, et al. , 2019. Classification of 85 species of Rhododendron in China based on RAD high throughput sequencing [J]. For Res, 32(3): 1-8. [李云飞, 李世明, 金鑫, 等, 2019. 基于 RAD 高通量测序探讨中国 85 种杜鹃花属植物的分类 [J]. 林业科学研究, 32(3): 1-8. ]

    • LI YJ, 2011. Current situation and garden use of wild Rhododendron resources in Qinghai Province [D]. Yangling: Northwest A & F University: 25-26. [李永金, 2011. 青海杜鹃属植物种质资源现状及园林应用 [D]. 杨凌: 西北农林科技大学: 25-26. ]

    • LIAO HB, HUANG GH, YU MH, et al. 2017. Five pairs of meroterpenoid enantiomers from Rhododendron capitatum [J]. J Org Chem, 82(3): 1632-1637.

    • LUO DS, 2018. The original interpretation of Jingzhu Materia Medica [M]. Chengdu: Sichuan Science and Technology Press: 485. [罗达尚, 2018. 晶珠本草正本诠释 [M]. 成都: 四川科学技术出版社: 485. ]

    • MILNE RI, DAVIES C, PRICKETT R, et al. , 2010. Phylogeny of Rhododendron subgenus Hymenanthes based on chloroplast DNA markers: between-lineage hybridisation during adaptive radiation? [J] Plant Syst Evol, 285: 233-244.

    • MA YP, XIE WJ, SUN WB, et al. , 2016. Strong reproductive isolation despite occasional hybridization between a widely distributed and a narrow endemic Rhododendron species [J]. Sci Rep, 6(1): 1-11.

    • MAYOR C, BRUDNO M, SCHWARTZ JR, et al. , 2000. VISTA: visualizing global DNA sequence alignments of arbitrary length [J]. Bioinformatics, 16(11): 1046-1047.

    • MENG J, LI XP, LI HT, et al. , 2018. Comparative analysis of the complete chloroplast genomes of four Aconitum medicinal species [J]. Molecules, 23(5): 1015.

    • PATEL RK, JAIN M, 2012. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data [J]. PLoS ONE, 7(2): e30619.

    • PENG LW, YAMAMOTO H, SHIKANAI T, 2011. Structure and biogenesis of the chloroplast NAD (P) H dehydrogenase complex [J]. Biochim et Biophys Acta, 1807(8): 945-953.

    • PING JY, FENG PP, LI JY, et al. , 2021. Molecular evolution and SSRs analysis based on the chloroplast genome of Callitropsis funebris [J]. Ecol Evol, 11(9): 4786-4802.

    • Qinghai Provincial Drug Inspection Institute, Qinghai Tibetan Medicinal Institute, 1996. Chinese Tibetan Medicine [M]. Shanghai: Shanghai Science & Technology Press: 210-211. [青海省药品检验所, 青海省藏医药研究所, 1996. 中国藏药 [M]. 上海: 上海科学技术出版社: 210-211. ]

    • ROZAS J, FERRER-MATA A, SÁNCHEZ-DELBARRIO JC, et al. , 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets [J]. Mol Biol Evol, 34(12): 3299-3302.

    • SHARP PM, COWE E, 1991. Synonymous codon usage in Saccharomyces cerevisiae [J]. Yeast, 7(7): 657-678.

    • SHI LC, CHEN HM, JIANG M, et al. , 2019. CPGAVAS2, an integrated plastome sequence annotator and analyzer [J]. Nucl Acids Res, 47(W1): W65-W73.

    • SILVA SR, DIAZ YC, PENHA HA, et al. , 2016. The chloroplast genome of Utricularia reniformis sheds light on the evolution of the ndh gene complex of terrestrial carnivorous plants from the Lentibulariaceae family [J]. PLoS ONE, 11: e0165176.

    • STAMATAKIS A, 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies [J]. Bioinformatics, 30: 1312-1313.

    • SUGIURA C, KOBAYASHI Y, AOKI S, et al. , 2003. Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus [J]. Nucl Acids Res, 31(18): 5324-5331.

    • TANG HQ, TANG L, SHAO SC, et al. , 2021. Chloroplast genomic diversity in Bulbophyllum section Macrocaulia (Bl. ) Aver. (Orchidaceae, Epidendroideae, Malaxideae): insights into species divergence and adaptive evolution [J]. Plant Divers, 43(5): 350-361.

    • WANG L, DONG WP, ZHOU SL, 2012. Structural mutations and reorganizations in chloroplast genomes of flowering plants [J]. Acta Bot Boreal-Occident Sin, 32(6): 1282-1288. [王玲, 董文攀, 周世良, 2012. 被子植物叶绿体基因组的结构变异研究进展 [J]. 西北植物学报, 32(6): 1282-1288. ]

    • WANG XQ, SONG WW, XIAO JJ, et al. , 2021. Phylogeny of Myrtales and related groups based on chloroplast genome [J]. Guihaia, 41(1): 68-80. [王雪芹, 宋卫武, 肖建加, 等, 2021. 基于叶绿体基因组探讨桃金娘目及其近缘类群的系统发育关系 [J]. 广西植物, 41(1): 68-80. ]

    • WICKE S, MÜLLER KF, DEPAMPHILIS CW, et al. , 2016. Mechanistic model of evolutionary rate variation en route to anonphotosynthetic lifestyle in plants [J]. Proc Natl Acad Sci, 113(32): 9045-9050.

    • XIE DF, YU HX, PRICE M, et al. , 2019. Phylogeny of Chinese Allium species in section Daghestanica and adaptive evolution of Allium (Amaryllidaceae, Allioideae) species revealed by the chloroplast complete genome [J]. Front Plant Sci, 10: 460.

    • YAN LJ, LIU J, MICHAE M, et al. , 2015. DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya-Hengduan Mountain [M]. Mol Ecol Resour, 15(4): 932-944.

    • YANG HB, FANG RZ, JIN CL, et al. , 1999. Flora Reipublicae Popularis Sinicae (Vol. 57, Part 1) [M]. Beijing: Science Press: 13-213. [杨汉碧, 方瑞征, 金存礼, 1999. 中国植物志(第57 卷第1分册) [M]. 北京: 科学出版社: 13-213].

    • YANG KT, CHEN GP, XIAN JR, et al. , 2021. Scaling relationship between leaf mass and leaf area: A case study using six alpine Rhododendron species in the Eastern Tibetan Plateau [J]. Global Ecol Conserv, 30: e01754.

    • YI X, GAO L, WANG B, et al. , 2013. The complete chloroplast genome sequence of Cephalotaxus oliveri (Cephalotaxaceae): evolutionary comparison of Cephalotaxus chloroplast DNAs and insights into the loss of inverted repeat copies in gymnosperms [J]. Genome Biol Evol, 5(4): 688-698.

    • ZHANG DG, CAO WX, PU XP, et al. , 2003. Botanical morpha and ecological adaptation of Rhododendron in Eastern Qilian Mountains [J]. Grassl Turf, (1): 27-30. [张德罡, 曹文侠, 蒲小鹏, 等, 2003. 东祁连山杜鹃属植物形态特征及其生态适应性 [J]. 草原与草坪, (1): 27-30. ]

    • ZHANG Q, LIU Y, SODMERGEN, 2003. Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species [J]. Plant Cell Physiol, 44(9): 941-951.

    • ZHANG YM, HAN LJ, YANG CW, et al. , 2022. Comparative chloroplast genome analysis of medicinally important Veratrum (Melanthiaceae) in China: insights into genomic characterization and phylogenetic relationships [J]. Plant Divers, 44: 70-82.

    • ZHUANG P, 2019. Progress on the fertility of Rhododendron [J]. Biodivers Sci, 27(3): 327-338. [庄平, 2019. 杜鹃花属植物的可育性研究进展 [J]. 生物多样性, 27(3): 327-338. ]

  • 参考文献

    • BAI L, JIAO ML, ZANG HY, et al. , 2019. Chemical composition of essential oils from four Rhododendron species and their repellent activity against three stored-product insects [J]. Environ Sci Pollut Res, 26(22): 23198-23205.

    • BAI XX, SHEN FJ, LI ZB, et al. , 2019. DNA barcode identification of genus Rhododendron L. : reference library, molecular markers and cultivar wild relatives tracing [J]. Tianjin Agric Sci, 25(10): 1-7. [白霄霞, 沈风娇, 李志斌, 等, 2019. 杜鹃花属 (Rhododendron L. ) 植物 DNA 条形码鉴定——参考序列库, 分子标记与栽培品种野生亲本溯源 [J]. 天津农业科学, 25(10): 1-7. ]

    • BEIER S, THIEL T, MÜNCH T, et al. , 2017. MISA-web: a web server for microsatellite prediction [J]. Bioinformatics, 33(16): 2583-2585.

    • BRUDNO M, MALDE S, POLIAKOV A, et al. , 2003. Glocal alignment: Finding rearrangements during alignment [J]. Bioinformatics, 19: i54-i62.

    • CAO WX, 2001. Ecological adaptive strategy of genus Rhododendron L. in eastern Qilian Mountains alpine ecosystem [D]. Gansu: Northwest Normal University: 35-37. [曹文侠, 2001. 东祁连山高寒生态系统杜鹃花属植物适应对策的研究 [D]. 甘肃: 西北师范大学: 35-37. ]

    • CAO XJ, LIU JJ, YANG M, 2009. Photosynthetic characteristics and anatomical structure of five species of Rhododendron in the Taibai Mountain [J]. Acta Bot Boreal-Occident Sin, 29(12): 2483-2491. [曹晓娟, 刘建军, 杨梅, 2009. 太白山 5 种杜鹃属植物叶片光合特性及解剖结构的生态适应性研究 [J]. 西北植物学报, 29(12): 2483-2491. ]

    • CHENG SY, 2017. Study on resources and classification of azalea cultivars in China [D]. Ganzhou: Gannan Normal University: 11-12. [程淑媛, 2017. 中国杜鹃花栽培品种资源与分类研究 [D]. 赣州: 赣南师范大学: 11-12]

    • CHUMLEY TW, PALMER JD, MOWER JP, et al. , 2006. The complete chloroplast genome sequence of Pelargonium × hortorum: organization and evolution of the largest and most highly rearranged chloroplast genome of land plants [J]. Mol Biol Evol, 23(11): 2175-2190.

    • DANIELL H, LIN CS, YU M, et al. , 2016. Chloroplast genomes: diversity, evolution, and applications in genetic engineering [J]. Genome Biol, 17(1): 134.

    • DRUMMOND A, 2012. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data [J]. Bioinformatics, 28(12): 1647-1649.

    • FAN WB, WU Y, YANG J, et al. , 2018. Comparative chloroplast genomics of Dipsacales species: insights into sequence variation, adaptive evolution, and phylogenetic relationships [J]. Front Plant Sci, 9: 689.

    • FU CN, MO ZQ, YANG JB, et al. , 2022. Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron [J]. Mol Ecol Resour, 22(1): 404-414.

    • GENG XM, HUAN ZQ, SU JL, et al. , 2021. Researches advances in germplasm innovation of Rhododendrons [J]. Mol Plant Breed, 19(2): 604-613. [耿兴敏, 宦智群, 苏家乐, 等, 2021. 杜鹃花属植物种质创新研究进展 [J]. 分子植物育种, 19(2): 604-613. ]

    • GREINER S, LEHWARK P, BOCK R, 2019. Organellar-GenomeDRAW (OGDRAW) version 1. 3. 1: expanded toolkit for the graphical visualization of organellar genomes [J]. Nucl Acid Res, 47(W1): W59-W64.

    • GU CH, MA L, WU ZQ, et al. , 2019. Comparative analyses of chloroplast genomes from 22 Lythraceae species: inferences for phylogenetic relationships and genome evolution within Myrtales [J]. BMC Plant Biol, 19(1): 1-19.

    • HE JL, ZHOU TY, SONG YK, et al. , 2021. Variations in floral traits of Rhododendron przewalskii with slope aspect in the southeastern Tibetan Plateau [J]. Chin J Appl Environ Biol, 27(4): 860-868. [何家莉, 周天阳, 宋怡珂, 等, 2021. 青藏高原东南缘不同坡向对陇蜀杜鹃花性状的影响 [J]. 应用与环境生物学报, 27(4): 860-868. ]

    • HIRAO T, WATANABE A, KURITA M, et al. , 2008. Complete nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species [J]. BMC Plant Biol, 8(1): 1-20.

    • HUANG CL, YAO G, TIAN XL, et al. , 2021. Phylogenomic analysis of Rhododendron species in Guizhou Baili Rhododendron reserve based on RAD sequencing [M]. Sci Silv Sin, 57(2): 72-81. [黄承玲, 姚刚, 田晓玲, 等, 2021. 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, 57(2): 72-81. ]

    • HUELSENBECK JP, RONQUIST F, 2001. MRBAYES: Bayesian inference of phylogenetic trees [J]. Bioinformatics, 17: 754-755.

    • JANSEN RK, RAUBESON LA, BOORE JL, et al. , 2005. Methods for obtaining and analyzing whole chloroplast genome sequences [J]. Meth Enzym, 395: 348-384.

    • JIN JJ, YU WB, YANG JB, et al. , 2020. GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes [J]. Genome Biol, 21(1): 1-31.

    • KANE N, SVEINSSON S, DEMPEWOLF H, et al. , 2012. Ultra-barcoding in cacao (Theobroma spp. ; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA [J]. Am J Bot, 99(2): 320-329.

    • KATOH K, STANDLEY DM, 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability [J]. Mol Biol Evol, 30(4): 772-780.

    • KUMAR S, STECHER G, LI M, et al. , 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms [J]. Mol Biol Evol, 35(6): 1547.

    • LEE SB, KAITTANIS C, JANSEN RK, et al. 2006. The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms [J]. BMC Genomic, 7(1): 1-12.

    • LI Y, LÜ GH, ZHANG XN, et al. , 2017. Chloroplast genome structure and variation analysis of Brasicaceae species [J]. Acta Bot Boreal-Occident Sin, 37(6): 1090-1101. [李岩, 吕光辉, 张雪妮, 等, 2017. 十字花科植物叶绿体基因组结构及变异分析 [J]. 西北植物学报, 37(6): 1090-1101. ]

    • LI Y, SYLVESTER SP, LI M, et al. , 2019. The complete plastid genome of Magnolia zenii and genetic comparison to Magnoliaceae species [J]. Molecules, 24(2): 261.

    • LI YF, LI SM, JIN X, et al. , 2019. Classification of 85 species of Rhododendron in China based on RAD high throughput sequencing [J]. For Res, 32(3): 1-8. [李云飞, 李世明, 金鑫, 等, 2019. 基于 RAD 高通量测序探讨中国 85 种杜鹃花属植物的分类 [J]. 林业科学研究, 32(3): 1-8. ]

    • LI YJ, 2011. Current situation and garden use of wild Rhododendron resources in Qinghai Province [D]. Yangling: Northwest A & F University: 25-26. [李永金, 2011. 青海杜鹃属植物种质资源现状及园林应用 [D]. 杨凌: 西北农林科技大学: 25-26. ]

    • LIAO HB, HUANG GH, YU MH, et al. 2017. Five pairs of meroterpenoid enantiomers from Rhododendron capitatum [J]. J Org Chem, 82(3): 1632-1637.

    • LUO DS, 2018. The original interpretation of Jingzhu Materia Medica [M]. Chengdu: Sichuan Science and Technology Press: 485. [罗达尚, 2018. 晶珠本草正本诠释 [M]. 成都: 四川科学技术出版社: 485. ]

    • MILNE RI, DAVIES C, PRICKETT R, et al. , 2010. Phylogeny of Rhododendron subgenus Hymenanthes based on chloroplast DNA markers: between-lineage hybridisation during adaptive radiation? [J] Plant Syst Evol, 285: 233-244.

    • MA YP, XIE WJ, SUN WB, et al. , 2016. Strong reproductive isolation despite occasional hybridization between a widely distributed and a narrow endemic Rhododendron species [J]. Sci Rep, 6(1): 1-11.

    • MAYOR C, BRUDNO M, SCHWARTZ JR, et al. , 2000. VISTA: visualizing global DNA sequence alignments of arbitrary length [J]. Bioinformatics, 16(11): 1046-1047.

    • MENG J, LI XP, LI HT, et al. , 2018. Comparative analysis of the complete chloroplast genomes of four Aconitum medicinal species [J]. Molecules, 23(5): 1015.

    • PATEL RK, JAIN M, 2012. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data [J]. PLoS ONE, 7(2): e30619.

    • PENG LW, YAMAMOTO H, SHIKANAI T, 2011. Structure and biogenesis of the chloroplast NAD (P) H dehydrogenase complex [J]. Biochim et Biophys Acta, 1807(8): 945-953.

    • PING JY, FENG PP, LI JY, et al. , 2021. Molecular evolution and SSRs analysis based on the chloroplast genome of Callitropsis funebris [J]. Ecol Evol, 11(9): 4786-4802.

    • Qinghai Provincial Drug Inspection Institute, Qinghai Tibetan Medicinal Institute, 1996. Chinese Tibetan Medicine [M]. Shanghai: Shanghai Science & Technology Press: 210-211. [青海省药品检验所, 青海省藏医药研究所, 1996. 中国藏药 [M]. 上海: 上海科学技术出版社: 210-211. ]

    • ROZAS J, FERRER-MATA A, SÁNCHEZ-DELBARRIO JC, et al. , 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets [J]. Mol Biol Evol, 34(12): 3299-3302.

    • SHARP PM, COWE E, 1991. Synonymous codon usage in Saccharomyces cerevisiae [J]. Yeast, 7(7): 657-678.

    • SHI LC, CHEN HM, JIANG M, et al. , 2019. CPGAVAS2, an integrated plastome sequence annotator and analyzer [J]. Nucl Acids Res, 47(W1): W65-W73.

    • SILVA SR, DIAZ YC, PENHA HA, et al. , 2016. The chloroplast genome of Utricularia reniformis sheds light on the evolution of the ndh gene complex of terrestrial carnivorous plants from the Lentibulariaceae family [J]. PLoS ONE, 11: e0165176.

    • STAMATAKIS A, 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies [J]. Bioinformatics, 30: 1312-1313.

    • SUGIURA C, KOBAYASHI Y, AOKI S, et al. , 2003. Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus [J]. Nucl Acids Res, 31(18): 5324-5331.

    • TANG HQ, TANG L, SHAO SC, et al. , 2021. Chloroplast genomic diversity in Bulbophyllum section Macrocaulia (Bl. ) Aver. (Orchidaceae, Epidendroideae, Malaxideae): insights into species divergence and adaptive evolution [J]. Plant Divers, 43(5): 350-361.

    • WANG L, DONG WP, ZHOU SL, 2012. Structural mutations and reorganizations in chloroplast genomes of flowering plants [J]. Acta Bot Boreal-Occident Sin, 32(6): 1282-1288. [王玲, 董文攀, 周世良, 2012. 被子植物叶绿体基因组的结构变异研究进展 [J]. 西北植物学报, 32(6): 1282-1288. ]

    • WANG XQ, SONG WW, XIAO JJ, et al. , 2021. Phylogeny of Myrtales and related groups based on chloroplast genome [J]. Guihaia, 41(1): 68-80. [王雪芹, 宋卫武, 肖建加, 等, 2021. 基于叶绿体基因组探讨桃金娘目及其近缘类群的系统发育关系 [J]. 广西植物, 41(1): 68-80. ]

    • WICKE S, MÜLLER KF, DEPAMPHILIS CW, et al. , 2016. Mechanistic model of evolutionary rate variation en route to anonphotosynthetic lifestyle in plants [J]. Proc Natl Acad Sci, 113(32): 9045-9050.

    • XIE DF, YU HX, PRICE M, et al. , 2019. Phylogeny of Chinese Allium species in section Daghestanica and adaptive evolution of Allium (Amaryllidaceae, Allioideae) species revealed by the chloroplast complete genome [J]. Front Plant Sci, 10: 460.

    • YAN LJ, LIU J, MICHAE M, et al. , 2015. DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya-Hengduan Mountain [M]. Mol Ecol Resour, 15(4): 932-944.

    • YANG HB, FANG RZ, JIN CL, et al. , 1999. Flora Reipublicae Popularis Sinicae (Vol. 57, Part 1) [M]. Beijing: Science Press: 13-213. [杨汉碧, 方瑞征, 金存礼, 1999. 中国植物志(第57 卷第1分册) [M]. 北京: 科学出版社: 13-213].

    • YANG KT, CHEN GP, XIAN JR, et al. , 2021. Scaling relationship between leaf mass and leaf area: A case study using six alpine Rhododendron species in the Eastern Tibetan Plateau [J]. Global Ecol Conserv, 30: e01754.

    • YI X, GAO L, WANG B, et al. , 2013. The complete chloroplast genome sequence of Cephalotaxus oliveri (Cephalotaxaceae): evolutionary comparison of Cephalotaxus chloroplast DNAs and insights into the loss of inverted repeat copies in gymnosperms [J]. Genome Biol Evol, 5(4): 688-698.

    • ZHANG DG, CAO WX, PU XP, et al. , 2003. Botanical morpha and ecological adaptation of Rhododendron in Eastern Qilian Mountains [J]. Grassl Turf, (1): 27-30. [张德罡, 曹文侠, 蒲小鹏, 等, 2003. 东祁连山杜鹃属植物形态特征及其生态适应性 [J]. 草原与草坪, (1): 27-30. ]

    • ZHANG Q, LIU Y, SODMERGEN, 2003. Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species [J]. Plant Cell Physiol, 44(9): 941-951.

    • ZHANG YM, HAN LJ, YANG CW, et al. , 2022. Comparative chloroplast genome analysis of medicinally important Veratrum (Melanthiaceae) in China: insights into genomic characterization and phylogenetic relationships [J]. Plant Divers, 44: 70-82.

    • ZHUANG P, 2019. Progress on the fertility of Rhododendron [J]. Biodivers Sci, 27(3): 327-338. [庄平, 2019. 杜鹃花属植物的可育性研究进展 [J]. 生物多样性, 27(3): 327-338. ]