极小种群濒危植物盐桦叶绿体基因组特征分析
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摘要
【目的】为了解析极小种群植物盐桦叶绿体基因组结构,筛选SSRs分子标记和桦属叶绿体基因组高变区,采用高通量测序技术完成盐桦叶绿体基因组测序并与其近缘种进行比对分析。【方法】采集盐桦幼叶,使用TIANGEN试剂盒提取DNA并利用HiSeq Xten测序平台进行测序。以欧洲矮桦叶绿体基因组作为参考,使用MITObim v1.8和NOVOplasty软件拼接组装盐桦叶绿体基因组,并利用生物信息学手段进行特征分析。【结果】利用质量控制后的30 000 000条clean data,成功拼接完成序列全长为160 648 bp的盐桦叶绿体基因组,NCBI登录号为MG674393。其中,大单拷贝(LSC)区段长度89 553 bp,GC含量33.7%,小单拷贝(SSC)区段长19 027 bp,GC含量为29.7%,2个反向重复区(IRs)区段均为26 034 bp,GC含量42.5%。成功注释了盐桦叶绿体基因组共114个基因,其中包括79个蛋白编码基因,31个tRNA基因和4个rRNA基因。盐桦叶绿体基因组含有91个SSRs位点,其中33个SSRs均由A或T组成。SSRs主要分布于LSC区,56个SSRs位于LSC区,13个SSRs位于SSC区,而IRs区有22个SSRs。对盐桦和欧洲矮桦叶绿体全基因组进行比对分析,结果显示ndhC-trnV、petA-psbJ、rpl22-rps19区域的核酸变异度(π>0.2)显著高于其他区域,高变区的位置都位于LSC区,而IRs区和SSC区内2种植物变异度较小。正选择分析显示ycf1,rpoC1,rpl2与ndhA 4个基因出现正选择信号。盐桦、欧洲矮桦与其他双子叶植物共14条叶绿体全基因组序列通过MP、NJ方法进行聚类分析,MP和NJ树中的11个节点中有9个支持度为100%。支持盐桦和欧洲矮桦亲缘关系最近,桦木属物种与天目铁木在同一分支。【结论】通过高通量测序和生物信息学分析,得到盐桦叶绿体基因组。比对分析表明结构、基因组成和SSRs分布都与欧洲矮桦叶绿体基因组相似,二者在3个片段区域存在较高的核酸多态性,可作为桦属植物叶绿体基因组高分辨率的分子条形码。正选择分析确定有4个基因出现正选择信号(ycf1,rpoC1,rpl2,ndhA)。本研究结果可为极小濒危植物盐桦的保护工作提供重要的遗传背景信息。
【Objective】 Betula halophila was recorded as an endangered species in the list of plant species with extremely small populations(PSESPs). In this study, we used high-throughput sequencing technology to sequence its chloroplast(cp) genome to understand the structure of B. halophila cp genome as well as the potential simple sequence repeats(SSRs) markers. We also compared the cp genome of B. halophila with those of the phylogenetically related species to understand the hotspot region in Betulaceae.【Method】 Total genomic DNA was extracted from fresh leaf samples using the Plant Genomic DNA Kit(Tiangen Biotech). The HiSeq Xten platform was used for library construction and sequencing. MITObim v1.8 and NOVOplasty were used to assemble trimmed reads with the cp genome of B. nana as the reference.【Result】 Totally, 30 000 000 reads were obtained after trimming. The B. halophila cp genome(Accession Number MG674393 in NCBI) was a typical circular double-stranded DNA molecule with 160 648 bp in length, and its quadripartite structure was similar to most angiosperms, which comprised of LSC region(89 553 bp, GC content 33.7%), SSC regions(18 159 bp, GC content 29.7%) and IRs regions(IRa and IRb, 26 689 bp each, GC content 42.5%). Four unique rRNAs, 31 tRNAs, and 79 protein-coding genes were annotated. We further detected 91 SSRs, of which 33 SSRs were composed of A or T, and 56, 13, and 22 SSRs were at the LSC, SSC and IRs, respectively. The sliding window analysis showed three highly variable regions(hotspots) under a criterion of nucleotide variability >0.2: ndhC-trnV, petA-psbJ, and rpl22-rps19. All of these hotspots locate at the LSC region. In contrast, the SSC and IRs regions are relatively conservative. In addition, four genes of ycf1, rpoC1, rpl2, and ndhA were signaled with positive selection. Phylogenomic reconstruction with 13 dicotyledonous plants revealed that nine of the 11 nodes had 100% supports for grouping in both MP and NJ trees. The phylogenomic analysis indicated that B. halophila was close to B. nana and sister with Ostrya rehderiana.【Conclusion】 Via the high-throughput sequencing and the bioinformatic analyses of the cp genome of B. halophila, we found the similar organization, gene contents and SSRs distribution with B. nana cp genomes. Three identified highly variable regions in LSC are applicable for DNA barcodes for species identification, plant breeding, and phylogenetic inference. Four positively selected genes deserve further investigation. It provides important genetic information for the future protection of B. halophila.
【Objective】 Betula halophila was recorded as an endangered species in the list of plant species with extremely small populations(PSESPs). In this study, we used high-throughput sequencing technology to sequence its chloroplast(cp) genome to understand the structure of B. halophila cp genome as well as the potential simple sequence repeats(SSRs) markers. We also compared the cp genome of B. halophila with those of the phylogenetically related species to understand the hotspot region in Betulaceae.【Method】 Total genomic DNA was extracted from fresh leaf samples using the Plant Genomic DNA Kit(Tiangen Biotech). The HiSeq Xten platform was used for library construction and sequencing. MITObim v1.8 and NOVOplasty were used to assemble trimmed reads with the cp genome of B. nana as the reference.【Result】 Totally, 30 000 000 reads were obtained after trimming. The B. halophila cp genome(Accession Number MG674393 in NCBI) was a typical circular double-stranded DNA molecule with 160 648 bp in length, and its quadripartite structure was similar to most angiosperms, which comprised of LSC region(89 553 bp, GC content 33.7%), SSC regions(18 159 bp, GC content 29.7%) and IRs regions(IRa and IRb, 26 689 bp each, GC content 42.5%). Four unique rRNAs, 31 tRNAs, and 79 protein-coding genes were annotated. We further detected 91 SSRs, of which 33 SSRs were composed of A or T, and 56, 13, and 22 SSRs were at the LSC, SSC and IRs, respectively. The sliding window analysis showed three highly variable regions(hotspots) under a criterion of nucleotide variability >0.2: ndhC-trnV, petA-psbJ, and rpl22-rps19. All of these hotspots locate at the LSC region. In contrast, the SSC and IRs regions are relatively conservative. In addition, four genes of ycf1, rpoC1, rpl2, and ndhA were signaled with positive selection. Phylogenomic reconstruction with 13 dicotyledonous plants revealed that nine of the 11 nodes had 100% supports for grouping in both MP and NJ trees. The phylogenomic analysis indicated that B. halophila was close to B. nana and sister with Ostrya rehderiana.【Conclusion】 Via the high-throughput sequencing and the bioinformatic analyses of the cp genome of B. halophila, we found the similar organization, gene contents and SSRs distribution with B. nana cp genomes. Three identified highly variable regions in LSC are applicable for DNA barcodes for species identification, plant breeding, and phylogenetic inference. Four positively selected genes deserve further investigation. It provides important genetic information for the future protection of B. halophila.
引文
杜志如, 席江,万佳,等. 2008. 水稻核糖体蛋白(OsRPL14)基因的克隆及表达分析. 中国农学通报, 24(4): 130-134.(Du Z R,Xi J, Wan J, et al. 2008. Cloning and expression analysis of a ribosomal protein from rice(OsRPL14). Chinese Agricultural Science Bulletin, 24(4): 130-134. [in Chinese])
桂腾琴,孙敏. 2009. 叶绿体基因工程的研究进展. 安徽农业科学,37(8): 3429-3431.(Gui T Q,Sun M. 2009. Progress of studies on chloroplast genetic engineering. Journal of Anhui Agricultural Sciences, 37(8): 3429-3431.[in Chinese])
国政,臧润国. 2013. 中国极小种群野生植物濒危程度评价指标体系. 林业科学, 49(6): 10-17.(Guo Z, Zang R G. 2013. Evaluation index system of endangered levels of the wild plants with tiny population in China. Scientia Silvae Sinicae, 49(6): 10-17. [in Chinese])
李宏,邓江宇,张红,等. 2009. NaCl胁迫对盐桦幼苗生理特性的影响. 西北植物学报,29(11): 2281-2287.(Li H, Deng J Y, Zhang H, et al. 2009. Physiological characteristics of Betula halophila seedlings under NaCl stress. Acta Botanica Boreali-Occidentalia Sinica, 29(11): 2281-2287.[in Chinese])
梅新娣,马纪,张富春. 2006. 新疆濒危植物盐桦试管苗生根培养的研究. 新疆农业科学,43(3): 218-223.(Mei X D, Ma J, Zhang F C. 2006. Rooting cultivation of rare endangered plant Betula halophila in vitro. Xinjiang Agricultural Sciences, 43(3): 218-223.[in Chinese])
梅新娣,张富春. 2008. 应用ITS序列分析新疆濒危植物盐桦的系统发育. 生物技术,18(6): 4-6.(Mei X D,Zhang F C. 2008. Phylogeny of endangered plant species-Betula halophila in Xinjiang inferred from ITS sequence data. Xinjiang Agricultural Sciences, 18(6): 4-6. [in Chinese])
陶玲,李新荣,刘新民,等. 2001. 中国珍稀濒危荒漠植物保护等级的定量研究. 林业科学,37(1): 52-57.(Tao L, Li X R, Liu X M, et al. 2001. Quantitative study of conservation grading of rare and endangered desert plants in China. Scientia Silvae Sinicae, 37(1): 52-57.
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. 西北植物学报,32(6): 1282-1288.(Wang L, Dong W P, Zhou S L. 2012. Structural mutations and reorganizations in chloroplast genomes of flowering plants. Acta Botanica Boreali-Occidentalia Sinica, 32(6): 1282-1288. [in Chinese])
汪智军,李行斌,郭仲军,等. 2003. 新疆14种珍稀濒危植物资源现状及保护. 中国野生植物资源, 22(2): 15-16.(Wang Z J, Li X B, Guo Z J, et al. 2003. 14 rare and endangered plant resources in Xinjiang and their protection. Chinese Wild Plant Resources, 22(2): 15-16. [in Chinese])
张立运,潘伯荣. 2000. 新疆植物资源评价及开发利用. 干旱区地理, 23(4): 334-335.(Zhang L Y,Pan B R. 2000. Evaluation on exploitation and utilization of plant resources in Xinjiang. Arid Land Geography, 23(4): 334-335.[in Chinese])
张韵洁,李德铢. 2011. 叶绿体系统发育基因组学的研究进展. 植物分类与资源学报,33(4): 365-375.(Zhang Y J, Li D Z. 2011. Advances in phylogenomics based on complete chloroplast genomes. Plant Diversity and Resources, 33(4): 365-375.[in Chinese])
Berberich T, Uebeler M, Feierabend J. 2000. cDNA cloning of cytoplasmic ribosomal protein S7 of winter rye (Secale cereale) and its expression in low-temperature-treated leaves. Biochimica Et Biophysica Acta, 1492(1): 276-279.
Birky C W. 1995. Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution.Proc Natl Acad Sci USA, 92 (25): 11331-11338.
Clegg M T, Gaut B S, Learn G H, et al. 1994. Rates and patterns of chloroplast DNA evolution. Proc Natl Acad Sci USA, 91 (15): 6795-6801.
Dierckxsens N, Mardulyn P, Smits G. 2016. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Research, doi: 10.1093/nar/gkw955.
Dong W P, Xu C, Li C H, et al. 2015. Ycf1, the most promising plastid DNA barcode of land plants. Scientific Reports, 5: 8348.
Dong W P, Xu C, Li D L, et al. 2016. Comparative analysis of the complete chloroplast genome sequences in psammophytic Haloxylon species (Amaranthaceae). PeerJ, 4(2): e2699.
Hahn C, Bachmann L, Chevreux B. 2013. Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach. Nucleic Acids Res, 41 (13): e129.
Katoh K, Misawa K, Kuma K I, et al. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res, 30 (14): 3059-3066.
Kusumi J, Sato A, Tachida H. 2006. Relaxation of functional constraint on light-independent protochlorophyllide oxidoreductase in Thuja. Molecular Biology & Evolution, 23(5): 941-948.
Li H, Durbin R. 2009a. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25: 1754-1760.
Li H, Handsaker B, Wysoker A, et al. 2009b. The sequence Alignment/Map format and SAMtools. Bioinformatics, 25 (16): 2078-2079.
Librado P, Rozas J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25 (11): 1451-1452.
Liu C, Shi L, Zhu Y, et al. 2012. CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences. Bmc Genomics, 13(1): 715.
Lohse M, Drechsel O, Kahlau S, et al. 2013. Organellar Genome DRAW—a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets. Nucleic Acids Research, 41: W575-581.
Ma P F, Zhang Y X, Zeng C X, et al. 2014. Chloroplast phylogenomic analyses resolve deep-level relationships of an intractable bamboo tribe Arundinarieae(Poaceae). Systematic Biology, 63(6): 933.
McGinnis S, Madden T L. 2004. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res, 32: W20-25.
Nie X J, Lu S Z, Zhang Y X, et al. 2012. Complete chloroplast genome sequence of a major invasive species, Crofton weed (Ageratina adenophora). PLoS One, 7(5): e36869.
Nock C J, Waters D L, Edwards M A, et al. 2011. Chloroplast genome sequences from total DNA for plant identification. Plant Biotechnol J, 9: 328-333.
Parida S K, Yadava D K, Mohapatra T. 2010. Microsatellites in Brassica unigenes: relative abundance, marker design, and use in comparative physical mapping and genome analysis. Genome, 53 (1): 55-67.
Parks M, Cronn R, Liston A. 2009. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biology, 7: 84.
Parveen I, Singh H K, Malik S, et al. 2017. Evaluating five different loci (rbcL, rpoB, rpoC1, matK and ITS) for DNA barcoding of Indian orchids. Genome, 60(8): 665-671.
Patel R K, Jain M. 2012. QC Toolkit: A Toolkit for quality control of next generation sequencing data. PLoS One, 7(2): e30619.
Peng L W, Yamamoto H, Shikanai T. 2011. Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex. Biochimica et Biophysica Acta(BBA)-Bioenergetics, 1807(8): 945-953.
Swofford D L. 2003. PAUP*: Phylogenetic analysis using parsimony (*and other methods), Version 4. Sunderland Massachusetts, USA: Sinauer Associates.
Tamura K, Dudley J, Nei M, et al. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24 (8): 1596-1599.
Wang N, Mcallister H A, Bartlett P R, et al. 2016. Molecular phylogeny and genome size evolution of the genus Betula (Betulaceae). Annals of Botany, 117(6): 1023-1035.
Xu C, Dong W P, Li W Q, et al. 2017. Comparative analysis of six Lagerstroemia complete chloroplast genomes. Frontiers in Plant Science, 8: article 15.
Yang Y C, Zhou T, Duan D, et al. 2016. Comparative analysis of the complete chloroplast genomes of five Quercus species. Frontiers in Plant Science, 7: article 959.
Zhang S D, Jin J J, Chen S Y, et al. 2017. Diversification of Rosaceae since the Late Cretaceous based on plastid phylogenomics. New Phytologist, 214(3): 1355-1367.
桂腾琴,孙敏. 2009. 叶绿体基因工程的研究进展. 安徽农业科学,37(8): 3429-3431.(Gui T Q,Sun M. 2009. Progress of studies on chloroplast genetic engineering. Journal of Anhui Agricultural Sciences, 37(8): 3429-3431.[in Chinese])
国政,臧润国. 2013. 中国极小种群野生植物濒危程度评价指标体系. 林业科学, 49(6): 10-17.(Guo Z, Zang R G. 2013. Evaluation index system of endangered levels of the wild plants with tiny population in China. Scientia Silvae Sinicae, 49(6): 10-17. [in Chinese])
李宏,邓江宇,张红,等. 2009. NaCl胁迫对盐桦幼苗生理特性的影响. 西北植物学报,29(11): 2281-2287.(Li H, Deng J Y, Zhang H, et al. 2009. Physiological characteristics of Betula halophila seedlings under NaCl stress. Acta Botanica Boreali-Occidentalia Sinica, 29(11): 2281-2287.[in Chinese])
梅新娣,马纪,张富春. 2006. 新疆濒危植物盐桦试管苗生根培养的研究. 新疆农业科学,43(3): 218-223.(Mei X D, Ma J, Zhang F C. 2006. Rooting cultivation of rare endangered plant Betula halophila in vitro. Xinjiang Agricultural Sciences, 43(3): 218-223.[in Chinese])
梅新娣,张富春. 2008. 应用ITS序列分析新疆濒危植物盐桦的系统发育. 生物技术,18(6): 4-6.(Mei X D,Zhang F C. 2008. Phylogeny of endangered plant species-Betula halophila in Xinjiang inferred from ITS sequence data. Xinjiang Agricultural Sciences, 18(6): 4-6. [in Chinese])
陶玲,李新荣,刘新民,等. 2001. 中国珍稀濒危荒漠植物保护等级的定量研究. 林业科学,37(1): 52-57.(Tao L, Li X R, Liu X M, et al. 2001. Quantitative study of conservation grading of rare and endangered desert plants in China. Scientia Silvae Sinicae, 37(1): 52-57.
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. 西北植物学报,32(6): 1282-1288.(Wang L, Dong W P, Zhou S L. 2012. Structural mutations and reorganizations in chloroplast genomes of flowering plants. Acta Botanica Boreali-Occidentalia Sinica, 32(6): 1282-1288. [in Chinese])
汪智军,李行斌,郭仲军,等. 2003. 新疆14种珍稀濒危植物资源现状及保护. 中国野生植物资源, 22(2): 15-16.(Wang Z J, Li X B, Guo Z J, et al. 2003. 14 rare and endangered plant resources in Xinjiang and their protection. Chinese Wild Plant Resources, 22(2): 15-16. [in Chinese])
张立运,潘伯荣. 2000. 新疆植物资源评价及开发利用. 干旱区地理, 23(4): 334-335.(Zhang L Y,Pan B R. 2000. Evaluation on exploitation and utilization of plant resources in Xinjiang. Arid Land Geography, 23(4): 334-335.[in Chinese])
张韵洁,李德铢. 2011. 叶绿体系统发育基因组学的研究进展. 植物分类与资源学报,33(4): 365-375.(Zhang Y J, Li D Z. 2011. Advances in phylogenomics based on complete chloroplast genomes. Plant Diversity and Resources, 33(4): 365-375.[in Chinese])
Berberich T, Uebeler M, Feierabend J. 2000. cDNA cloning of cytoplasmic ribosomal protein S7 of winter rye (Secale cereale) and its expression in low-temperature-treated leaves. Biochimica Et Biophysica Acta, 1492(1): 276-279.
Birky C W. 1995. Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution.Proc Natl Acad Sci USA, 92 (25): 11331-11338.
Clegg M T, Gaut B S, Learn G H, et al. 1994. Rates and patterns of chloroplast DNA evolution. Proc Natl Acad Sci USA, 91 (15): 6795-6801.
Dierckxsens N, Mardulyn P, Smits G. 2016. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Research, doi: 10.1093/nar/gkw955.
Dong W P, Xu C, Li C H, et al. 2015. Ycf1, the most promising plastid DNA barcode of land plants. Scientific Reports, 5: 8348.
Dong W P, Xu C, Li D L, et al. 2016. Comparative analysis of the complete chloroplast genome sequences in psammophytic Haloxylon species (Amaranthaceae). PeerJ, 4(2): e2699.
Hahn C, Bachmann L, Chevreux B. 2013. Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach. Nucleic Acids Res, 41 (13): e129.
Katoh K, Misawa K, Kuma K I, et al. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res, 30 (14): 3059-3066.
Kusumi J, Sato A, Tachida H. 2006. Relaxation of functional constraint on light-independent protochlorophyllide oxidoreductase in Thuja. Molecular Biology & Evolution, 23(5): 941-948.
Li H, Durbin R. 2009a. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25: 1754-1760.
Li H, Handsaker B, Wysoker A, et al. 2009b. The sequence Alignment/Map format and SAMtools. Bioinformatics, 25 (16): 2078-2079.
Librado P, Rozas J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25 (11): 1451-1452.
Liu C, Shi L, Zhu Y, et al. 2012. CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences. Bmc Genomics, 13(1): 715.
Lohse M, Drechsel O, Kahlau S, et al. 2013. Organellar Genome DRAW—a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets. Nucleic Acids Research, 41: W575-581.
Ma P F, Zhang Y X, Zeng C X, et al. 2014. Chloroplast phylogenomic analyses resolve deep-level relationships of an intractable bamboo tribe Arundinarieae(Poaceae). Systematic Biology, 63(6): 933.
McGinnis S, Madden T L. 2004. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res, 32: W20-25.
Nie X J, Lu S Z, Zhang Y X, et al. 2012. Complete chloroplast genome sequence of a major invasive species, Crofton weed (Ageratina adenophora). PLoS One, 7(5): e36869.
Nock C J, Waters D L, Edwards M A, et al. 2011. Chloroplast genome sequences from total DNA for plant identification. Plant Biotechnol J, 9: 328-333.
Parida S K, Yadava D K, Mohapatra T. 2010. Microsatellites in Brassica unigenes: relative abundance, marker design, and use in comparative physical mapping and genome analysis. Genome, 53 (1): 55-67.
Parks M, Cronn R, Liston A. 2009. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biology, 7: 84.
Parveen I, Singh H K, Malik S, et al. 2017. Evaluating five different loci (rbcL, rpoB, rpoC1, matK and ITS) for DNA barcoding of Indian orchids. Genome, 60(8): 665-671.
Patel R K, Jain M. 2012. QC Toolkit: A Toolkit for quality control of next generation sequencing data. PLoS One, 7(2): e30619.
Peng L W, Yamamoto H, Shikanai T. 2011. Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex. Biochimica et Biophysica Acta(BBA)-Bioenergetics, 1807(8): 945-953.
Swofford D L. 2003. PAUP*: Phylogenetic analysis using parsimony (*and other methods), Version 4. Sunderland Massachusetts, USA: Sinauer Associates.
Tamura K, Dudley J, Nei M, et al. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24 (8): 1596-1599.
Wang N, Mcallister H A, Bartlett P R, et al. 2016. Molecular phylogeny and genome size evolution of the genus Betula (Betulaceae). Annals of Botany, 117(6): 1023-1035.
Xu C, Dong W P, Li W Q, et al. 2017. Comparative analysis of six Lagerstroemia complete chloroplast genomes. Frontiers in Plant Science, 8: article 15.
Yang Y C, Zhou T, Duan D, et al. 2016. Comparative analysis of the complete chloroplast genomes of five Quercus species. Frontiers in Plant Science, 7: article 959.
Zhang S D, Jin J J, Chen S Y, et al. 2017. Diversification of Rosaceae since the Late Cretaceous based on plastid phylogenomics. New Phytologist, 214(3): 1355-1367.