Phylogenetic Position of <i>Chosenia arbutifolia</i> in the Salicaceae Inferred from Whole Chloroplast Genome

FENG Chu-hang; HE Cai-yun; WANG Ying; ZENG Yan-fei; ZHANG Jian-guo

doi:10.13275/j.cnki.lykxyj.2019.02.011

Volume 32 Issue 2

Jul. 2019

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Phylogenetic Position of Chosenia arbutifolia in the Salicaceae Inferred from Whole Chloroplast Genome

1.
Key Laboratory of Tree Breeding and Cultivation, National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
2.
BGI-Shenzhen, Shenzhen 518083, Guangdong, China

Corresponding author: ZENG Yan-fei, zengyf@caf.acf.ac.cn ;
Received Date: 2018-03-21
Accepted Date: 2019-01-15

Abstract

Objective To resolve the controversy over the phylogenetic position of Chosenia arbutifolia in Salicaceae. Method The whole chloroplast genome sequences of C. arbutifolia was determined by next-generation sequencing, and the phylogenetic position of C. arbutifolia was investigated by comparing its sequences with all available complete chloroplast genome sequences from the genera Populus and Salix. Result The total genome was 155, 661 bp, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 27, 455 bp. The large single-copy (LSC) and small single-copy (SSC) regions spanned 84, 536 bp and 16, 215 bp, respectively. The total GC content of the chloroplast genome was 36.68% and 113 unique genes were annotated, including 79 protein coding genes, 30 tRNA genes, and four rRNA genes. Twenty genes were duplicated in the inverted repeat regions, 14 genes contained one intron, and three genes (rps12, clpP, and ycf3) contained two introns. Conclusion A phylogenetic tree constructed from all available complete chloroplast genome sequences from the genera Populus and Salix based on maximum likelihood, maximum parsimony and Bayesian inference strongly supports the merging of C. arbutifolia into the genus Salix. This study would supply an important basis for the genetic study as well as conservation of C. arbutifolia.
- Chosenia arbutifolia,
- chloroplast genome,
- next-generation sequencing,
- Salicaceae,
- Salix

References

[1]	Skvortsov A K. Willows of Russia and Adjacent Countries:Taxonomical and Geographical Revision[M]. Joensuu:University of Joensuu, 1999:1-307.
[2]	Sohma K. Pollen diversity in Salix (Salicaceae)[J]. Sci Rep Tohoku Univ, Ⅳ, 1993, 40(2):77-178.
[3]	Azuma T, Kajita T, Yokoyama J, et al. Phylogenetic relationships of Salix (Salicaceae) based on rbcL sequence data[J]. American Journal of Botany, 2000, 87(1):67-75. doi: 10.2307/2656686
[4]	Chen J H, Sun H, Wen J, et al. Molecular phylogeny of Salix L.(Salicaceae) inferred from three chloroplast datasets and its systematic implications[J]. Taxon, 2010, 59(1):29-37. doi: 10.1002/tax.2010.59.issue-1
[5]	Hoshikawa T, Kikuchi S, Nagamitsu T, et al. Eighteen microsatellite loci in Salix arbutifolia (Salicaceae) and cross-species amplification in Salix and Populus species[J]. Molecular Ecology Resources, 2009, 9(4):1202-1205. doi: 10.1111/men.2009.9.issue-4
[6]	McFadden G I, van Dooren G G. Evolution:red algal genome affirms a common origin of all plastids[J]. Current Biology, 2004, 14(13):R514-R516. doi: 10.1016/j.cub.2004.06.041
[7]	Odintsova M S, Yurina N P. Chloroplast genomics of land plants and algae[J]. Biotechnological applications of photosynthetic proteins:biochips, biosensors and biodevices, 2006:57-72.
[8]	Jansen R K, Raubeson L A, Boore J L, et al. Methods for obtaining and analyzing whole chloroplast genome sequences[M]//Methods in enzymology. Academic Press, 2005, 395:348-384.
[9]	Wolfe K H, Li W H, Sharp P M. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs[J]. Proceedings of the National Academy of Sciences, 1987, 84(24):9054-9058. doi: 10.1073/pnas.84.24.9054
[10]	Ravi V, Khurana J P, Tyagi A K, et al. An update on chloroplast genomes[J]. Plant Systematics and Evolution, 2008, 271(1-2):101-122. doi: 10.1007/s00606-007-0608-0
[11]	Chen D, Zhang X, Kang H, et al. Phylogeography of Quercus variabilis based on chloroplast DNA sequence in East Asia:multiple glacial refugia and mainland-migrated island populations[J]. PLoS One, 2012, 7(10):e47268. doi: 10.1371/journal.pone.0047268
[12]	Li R, Li Y, Kristiansen K, et al. SOAP:short oligonucleotide alignment program[J]. Bioinformatics, 2008, 24(5):713-714. doi: 10.1093/bioinformatics/btn025
[13]	Lu G, Moriyama E N. Vector NTI, a balanced all-in-one sequence analysis suite[J]. Briefings in Bioinformatics, 2004, 5(4):378-388. doi: 10.1093/bib/5.4.378
[14]	Wyman S K, Jansen R K, Boore J L. Automatic annotation of organellar genomes with DOGMA[J]. Bioinformatics, 2004, 20(17):3252-3255. doi: 10.1093/bioinformatics/bth352
[15]	Lohse M, Drechsel O, Kahlau S, et al. OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets[J]. Nucleic Acids Research, 2013, 41(W1):W575-W581. doi: 10.1093/nar/gkt289
[16]	Frazer K A, Pachter L, Poliakov A, et al. VISTA:computational tools for comparative genomics[J]. Nucleic Acids Research, 2004, 32(suppl_2):W273-W279.
[17]	Kumar S, Stecher G, Tamura K. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7):1870-1874. doi: 10.1093/molbev/msw054
[18]	Ronquist F, Huelsenbeck J, Teslenko M. Draft MrBayes version 3.2 manual: tutorials and model summaries[M/OL]. Distributed with the software from http://brahms.biology.rochester.edu/software.html, 2011.
[19]	Kersten B, Rampant P F, Mader M, et al. Genome sequences of Populus tremula chloroplast and mitochondrion:implications for holistic poplar breeding[J]. PloS One, 2016, 11(1):e0147209. doi: 10.1371/journal.pone.0147209

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沈阳化工大学材料科学与工程学院沈阳 110142

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钻天柳(Chosenia arbutifolia (Pall.) A. Skv.)是一种主要分布在亚洲东北部的雌雄异株树种，原属杨柳科(Salicaceae)的一个单种属，钻天柳属(Chosenia) ^[1]。由于其形态特征介于杨属(Populus)植物和柳属(Salix)植物之间，钻天柳分类地位的确定对于研究杨柳科家族的演化有着十分重要的价值。然而，近期的研究对钻天柳在杨柳科中的系统发育位置提出了质疑。例如，Sohma^[2]研究了72种柳及杂交种的孢粉形态，其结果认为钻天柳属可合并到柳属中；Azuma等^[3]、Chen等^[4]基于叶绿体片段分析探讨了一些主要的柳属分类系统，同样支持将钻天柳属归入柳属。然而，这些系统发育研究只是基于个别片段的分析，也并未得到国内分类学家的重视。另外，森林的过度砍伐，使钻天柳生长的林地环境条件发生了变化，供其生长的河流受到污染，极大地影响了钻天柳种子的萌发与幼苗的生长，导致钻天柳的分布区正在日益缩减。钻天柳，因其分布高度受限而濒临灭绝^[5]，因此，被列为国家Ⅱ级重点保护野生植物。钻天柳系统发育地位的确定，将为钻天柳的保护提供重要的理论基础。

叶绿体起源于与之内共生的蓝藻^[6]，并且有其自身的遗传物质。叶绿体基因组由120~160 kb的环状双链分子组成，这是一种高度保守的结构^[7]。植物叶绿体基因组一般都有其独特的DNA区域，称为长单拷贝区(LSC)和短单拷贝区(SSC)，它们由两个反向重复序列(IRS)分隔开^[8]。与植物核基因组相比，植物叶绿体基因组的替换率低得多^[9]，并且由于叶绿体基因组是单亲遗传的，这意味着叶绿体基因组是系统发育分析遗传标记的重要来源^[10]。所以，叶绿体基因组可以用来研究植物种群之间的相互关系和生物多样性^[11]。

本研究从头测序、组装并注释了钻天柳叶绿体基因组完整的DNA序列并将其与从美国国家生物技术信息中心(NCBI)上得到的所有其他可用的杨属和柳属物种完整的叶绿体基因组DNA序列进行分析比对。这些分析揭示了钻天柳叶绿体基因组的结构和功能信息，并明确了钻天柳在杨柳科的系统发育地位。

1. 研究方法

1.1. 钻天柳全基因组序列

本研究从中国辽宁省丹东市宽甸白石砬子自然保护区附近的天然林中(40°50′00″ ~40°57′12″ N，124°44′07″ ~124°57′30″ E)采集了1棵雄性钻天柳的叶片样本，并利用CTAB法提取总DNA；然后采用Illumina的hiseq2000平台，通过全基因组鸟枪法测序获得60 G读长为100 bp的DNA数据。通过与毛果杨叶绿体基因组全序列(GenBank登录号为EF489041)比对，从这些短片段中筛选出100 Mb高质量的叶绿体基因组片段；并用SOAP de novo执行程序^[12]将这些短片段从头组装钻天柳叶绿体基因组序列。所有组装得到的叶绿体重叠群使用ContigExpress^[13]进行全基因组的组装，采用簸箕柳(S. suchowensis)叶绿体基因组全序列(KM983390)作为参考。重叠群的间隙在手动设计引物后采用Sanger测序来进行人工补洞。得到的完整钻天柳叶绿体基因组序列通过Dual OrganellarGenoMe Annotator (DOGMA)^[14]用默认参数预测其蛋白质编码基因、转运RNA(tRNA)基因和核糖体RNA(rRNA)基因。由于DOGMA的局限性，一些内含子以及外显子的边界不能很好的识别。因此，Blastn(http://blast.ncbi.nlm.nih.gov/)软件被用来比较钻天柳叶绿体基因组序列与已完成注释的毛果杨和簸箕柳叶绿体基因组序列，从而进一步确定边界位置。注释的钻天柳叶绿体基因组全序列发表在GenBank(NCBI)，登录号为KX781246。叶绿体的环状基因组图谱采用OrganellarGenomeDraw工具^[15]绘制得到。

1.2. 系统发育分析

从NCBI上下载得到银白杨(P. alba，AP008956)、大叶钻天杨(P. balsamifera, KJ664927)、青杨(P. cathayana，KP729175)、胡杨(P. euphratica，KJ624919)、弗氏黑杨(P. fremontii，KJ664926)、冬青叶杨(P. ilicifolia，KX421095)、欧洲山杨(P.tremula，KP861984)、毛果杨(P. trichocarpa，EF489041)、滇杨(P. yunnanensis，KP729176)等9种杨树，垂柳(S. babylonica，KT449800)、S. interior(KJ742926)、红皮柳(S. purpurea, KP019639)、簸箕柳(S. suchowensis，KM983390)等4种柳树，和巴西橡胶树(Hevea brasiliensis，HQ285842)的完整叶绿体基因组序列。将这些叶绿体基因组序列与本研究得到的钻天柳的完整叶绿体基因组序列通过mVISTA^[16]进行比对分析。通过进行两两比较获得对齐的序列矩阵，然后利用巴西橡胶树的叶绿体基因组序列作为外类群，采用MEGA7^[17]软件分别用最大似然法(Maximum likelihood，ML)和最大简约法(Maximum parsimony，MP)构建了系统发育树。在ML建树过程中，位点采用相同的进化速率(Uniform rates)，通过GTR模型(General Time Reversible substitution model)确定最优树，并采用SPR(Subtree-Pruning-Regrafting-Fast)作为ML的启发式搜索策略。MP方法采用TBR(Tree-Bisection-Reconnection)作为搜索方法，初始树数量为10，搜索等级为1，最多保留的树为100。这2种方法中，空白部分都采用“部分缺失”处理，分支的置信度均采用1 000次的靴带分析(Bootstrap)。同时利用MrBayes^[18]软件用贝叶斯推断(Bayesian inference，BI)法构建了系统发育树，采用GTR替代模型和伽马分布率，后验概率分布获得200个样本，每1 000代进行1次计算。

3. 讨论

本研究通过二代测序技术首次得到了钻天柳的叶绿体基因组全序列，该序列对于后期利用叶绿体序列研究钻天柳有着重要的参考价值。比较后发现，钻天柳叶绿体基因组的基因组成和结构与网上发表的杨属和柳属物种的叶绿体基因组非常相似，说明杨柳科叶绿体基因组的基因组成和结构具有较高的保守性。

利用本研究组装出来的叶绿体基因组全序列与9个杨属和4个柳属物种构建系统发育树，拓扑结果表明，钻天柳并未如传统分类认为的那样单独形成一支，也没有处于杨柳科的基部，而是被聚到柳属分支内部，与黄花柳亚属(Vetrix)的红皮柳和簸箕柳(二者为杞柳(S. integra)的近缘种)以100%支持率聚为一支。这些系统发育关系与Chen等^[4]的分析是一致的，他基于trnD-T、atpB-rbcL、rbcL序列联合分析的邻接树以及严格一致树中发现，旧世界柳亚属的垂柳与北美柳亚属的S. interior聚于一个分支。柳属分支中的钻天柳与细柱柳(S. gracilistyla)(同为杞柳的近缘种)聚于一个分支，因此表明钻天柳属可并入柳属。当前笔者基于叶绿体基因组全序列数据的分析可以更进一步明确钻天柳应被纳入柳属。另外，笔者构建的系统发育树所确定的杨属各物种之间的亲缘关系与Kersten等^[19]得到的结果略有不同，主要区别在于青杨和胡杨，可能是由于他们是采用UPGMA法来构建的系统发育树。

4. 结论

本研究通过将钻天柳叶绿体全基因组与从NCBI上下载得到的已知的杨柳科植物进行系统发育分析，明确了钻天柳应并入柳属。分子系统学在分类上问题解决效果良好，以往的分子系统学在分类上的应用主要以单个基因或者几个基因来进行系统发育分析。本研究则是通过完整的叶绿体基因来进行系统发育分析，这在物种的分类上有着较大的应用价值。随着测序技术的不断发展，杨柳科的系统学研究也将有着极大的促进，本文也能为以后的杨柳科系统进化研究提供一些参考。

Reference (19)

[1]	Skvortsov A K. Willows of Russia and Adjacent Countries:Taxonomical and Geographical Revision[M]. Joensuu:University of Joensuu, 1999:1-307.
[2]	Sohma K. Pollen diversity in Salix (Salicaceae)[J]. Sci Rep Tohoku Univ, Ⅳ, 1993, 40(2): 77-178.
[3]	Azuma T, Kajita T, Yokoyama J. Phylogenetic relationships of Salix (Salicaceae) based on rbcL sequence data[J]. American Journal of Botany, 2000, 87(1): 67-75. doi: 10.2307/2656686
[4]	Chen J H, Sun H, Wen J. Molecular phylogeny of Salix L.(Salicaceae) inferred from three chloroplast datasets and its systematic implications[J]. Taxon, 2010, 59(1): 29-37. doi: 10.1002/tax.2010.59.issue-1
[5]	Hoshikawa T, Kikuchi S, Nagamitsu T. Eighteen microsatellite loci in Salix arbutifolia (Salicaceae) and cross-species amplification in Salix and Populus species[J]. Molecular Ecology Resources, 2009, 9(4): 1202-1205. doi: 10.1111/men.2009.9.issue-4
[6]	McFadden G I, van Dooren G G. Evolution:red algal genome affirms a common origin of all plastids[J]. Current Biology, 2004, 14(13): R514-R516. doi: 10.1016/j.cub.2004.06.041
[7]	Odintsova M S, Yurina N P. Chloroplast genomics of land plants and algae[J]. Biotechnological applications of photosynthetic proteins:biochips, biosensors and biodevices, 2006, (): 57-72.
[8]	Jansen R K, Raubeson L A, Boore J L, et al. Methods for obtaining and analyzing whole chloroplast genome sequences[M]//Methods in enzymology. Academic Press, 2005, 395:348-384.
[9]	Wolfe K H, Li W H, Sharp P M. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs[J]. Proceedings of the National Academy of Sciences, 1987, 84(24): 9054-9058. doi: 10.1073/pnas.84.24.9054
[10]	Ravi V, Khurana J P, Tyagi A K. An update on chloroplast genomes[J]. Plant Systematics and Evolution, 2008, 271(1-2): 101-122. doi: 10.1007/s00606-007-0608-0
[11]	Chen D, Zhang X, Kang H. Phylogeography of Quercus variabilis based on chloroplast DNA sequence in East Asia:multiple glacial refugia and mainland-migrated island populations[J]. PLoS One, 2012, 7(10): e47268-. doi: 10.1371/journal.pone.0047268
[12]	Li R, Li Y, Kristiansen K. SOAP:short oligonucleotide alignment program[J]. Bioinformatics, 2008, 24(5): 713-714. doi: 10.1093/bioinformatics/btn025
[13]	Lu G, Moriyama E N. Vector NTI, a balanced all-in-one sequence analysis suite[J]. Briefings in Bioinformatics, 2004, 5(4): 378-388. doi: 10.1093/bib/5.4.378
[14]	Wyman S K, Jansen R K, Boore J L. Automatic annotation of organellar genomes with DOGMA[J]. Bioinformatics, 2004, 20(17): 3252-3255. doi: 10.1093/bioinformatics/bth352
[15]	Lohse M, Drechsel O, Kahlau S. OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets[J]. Nucleic Acids Research, 2013, 41(W1): W575-W581. doi: 10.1093/nar/gkt289
[16]	Frazer K A, Pachter L, Poliakov A. VISTA:computational tools for comparative genomics[J]. Nucleic Acids Research, 2004, 32(suppl_2): W273-W279.
[17]	Kumar S, Stecher G, Tamura K. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7): 1870-1874. doi: 10.1093/molbev/msw054
[18]	Ronquist F, Huelsenbeck J, Teslenko M. Draft MrBayes version 3.2 manual: tutorials and model summaries[M/OL]. Distributed with the software from http://brahms.biology.rochester.edu/software.html, 2011.
[19]	Kersten B, Rampant P F, Mader M. Genome sequences of Populus tremula chloroplast and mitochondrion:implications for holistic poplar breeding[J]. PloS One, 2016, 11(1): e0147209-. doi: 10.1371/journal.pone.0147209

Phylogenetic Position of Chosenia arbutifolia in the Salicaceae Inferred from Whole Chloroplast Genome