Volume 41 Issue 5
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LIU Xuan, ZOU Longhai, ZHOU Mingbing. Chloroplast genome of Phyllostachys edulis f. luteosulcata and comparison of chloroplast genome sequence of subspecies of Ph. edulis[J]. Journal of Zhejiang A&F University, 2024, 41(5): 1037-1046. doi: 10.11833/j.issn.2095-0756.20240110
Citation: LIU Xuan, ZOU Longhai, ZHOU Mingbing. Chloroplast genome of Phyllostachys edulis f. luteosulcata and comparison of chloroplast genome sequence of subspecies of Ph. edulis[J]. Journal of Zhejiang A&F University, 2024, 41(5): 1037-1046. doi: 10.11833/j.issn.2095-0756.20240110
shu

Chloroplast genome of Phyllostachys edulis f. luteosulcata and comparison of chloroplast genome sequence of subspecies of Ph. edulis

doi: 10.11833/j.issn.2095-0756.20240110

  • State Key Laboratory of Subtropical Silviculture/Bamboo Industry Institute, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

  • Received Date: 2024-01-06
  • Accepted Date: 2024-05-29
  • Rev Recd Date: 2024-05-08
    • Available Online: 2024-09-25
  • Publish Date: 2024-09-25
  •   Objective  This study aims to sequence, assemble, annotate, and analyze the chloroplast genome of Phyllostachys edulis f. luteosulcata. The research also involves comparing its chloroplast genetic information and phylogenetic relationships with those of other subspecies of Ph. edulis (moso bamboo).   Method  High-throughput sequencing data were used to assemble and annotate the complete chloroplast genome of Ph. edulis f. luteosulcata. Subsequently, we analyze the composition, codon preference, and repetitive sequences of the genome. Furthermore, sequence comparison and phylogenetic analysis were conducted to compare the phylogenetic relationships and genome sequence differences among different subspecies of moso bamboo.   Result  The chloroplast genome of Ph. edulis f. luteosulcata is a double-loop DNA of 139 678 bp in length containing 132 genes, including 85 protein-coding genes, eight ribosomal RNAs (rRNAs), and 39 transfer RNAs (tRNAs). The codon preference for this genome has an A/U base at the end. There are 49 repetitive sequences with the most common type being A/T and 55 Simple Sequence Repeat (SSR) sites. Phylogenetic analyses constructed using the chloroplast genome sequences showed that Ph. edulis f. luteulosulcata is in a monophyletic branch together with other subspecies of Ph. edulis and is closely related to the original variety of Ph. edulis var. pubescens. The analysis of chloroplast genome sequence and coding gene characteristics showed that there were differences in the number and structure of coding genes and low degree of sequence variation among the subspecies of Ph. edulis.   Conclusion  This study is the first to comparatively analyze the chloroplast genomes of subspecies of Ph. edulis and reveals a degree of sequence variation in these subspecies of Ph. edulis. This variation information would be available for the identification and comparison of subspecies of Ph. edulis. [Ch, 5 fig. 2 tab. 27 ref.]
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Chloroplast genome of Phyllostachys edulis f. luteosulcata and comparison of chloroplast genome sequence of subspecies of Ph. edulis

doi: 10.11833/j.issn.2095-0756.20240110
  • State Key Laboratory of Subtropical Silviculture/Bamboo Industry Institute, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • Received Date: 2024-01-06
  • Accepted Date: 2024-05-29
  • Rev Recd Date: 2024-05-08
  • Available Online: 2024-09-25
  • Publish Date: 2024-09-25

Abstract:   Objective  This study aims to sequence, assemble, annotate, and analyze the chloroplast genome of Phyllostachys edulis f. luteosulcata. The research also involves comparing its chloroplast genetic information and phylogenetic relationships with those of other subspecies of Ph. edulis (moso bamboo).   Method  High-throughput sequencing data were used to assemble and annotate the complete chloroplast genome of Ph. edulis f. luteosulcata. Subsequently, we analyze the composition, codon preference, and repetitive sequences of the genome. Furthermore, sequence comparison and phylogenetic analysis were conducted to compare the phylogenetic relationships and genome sequence differences among different subspecies of moso bamboo.   Result  The chloroplast genome of Ph. edulis f. luteosulcata is a double-loop DNA of 139 678 bp in length containing 132 genes, including 85 protein-coding genes, eight ribosomal RNAs (rRNAs), and 39 transfer RNAs (tRNAs). The codon preference for this genome has an A/U base at the end. There are 49 repetitive sequences with the most common type being A/T and 55 Simple Sequence Repeat (SSR) sites. Phylogenetic analyses constructed using the chloroplast genome sequences showed that Ph. edulis f. luteulosulcata is in a monophyletic branch together with other subspecies of Ph. edulis and is closely related to the original variety of Ph. edulis var. pubescens. The analysis of chloroplast genome sequence and coding gene characteristics showed that there were differences in the number and structure of coding genes and low degree of sequence variation among the subspecies of Ph. edulis.   Conclusion  This study is the first to comparatively analyze the chloroplast genomes of subspecies of Ph. edulis and reveals a degree of sequence variation in these subspecies of Ph. edulis. This variation information would be available for the identification and comparison of subspecies of Ph. edulis. [Ch, 5 fig. 2 tab. 27 ref.]

LIU Xuan, ZOU Longhai, ZHOU Mingbing. Chloroplast genome of Phyllostachys edulis f. luteosulcata and comparison of chloroplast genome sequence of subspecies of Ph. edulis[J]. Journal of Zhejiang A&F University, 2024, 41(5): 1037-1046. doi: 10.11833/j.issn.2095-0756.20240110
Citation: LIU Xuan, ZOU Longhai, ZHOU Mingbing. Chloroplast genome of Phyllostachys edulis f. luteosulcata and comparison of chloroplast genome sequence of subspecies of Ph. edulis[J]. Journal of Zhejiang A&F University, 2024, 41(5): 1037-1046. doi: 10.11833/j.issn.2095-0756.20240110
shu

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  • 毛竹Phyllostachys edulis为竹亚科Bambusoideae刚竹属Phyllostachys的散生竹种，广泛分布于东亚和东南亚，其种植面积高达460多万hm2[1]。由于生长速度快并且富含纤维素和半纤维素，毛竹是中国重要的固碳植物和工业原料来源[2−3]。毛竹的种下分类群具有丰富的变型和栽培变型，主要表现在秆型和秆色等方面的变异[4]。毛竹的秆色变异栽培种是优良的园林观赏竹类资源，主要包括花毛竹Ph. edulis f. taokiang、黄皮毛竹Ph. edulis f. holochrysa、绿槽毛竹Ph. edulis f. bicolor、黄槽毛竹Ph. edulis f. luteosulcata和绿槽龟甲竹Ph. edulis f. lücaoguijiazhu等。

    高等植物的叶绿体是半自主的细胞器，具有相对保守的遗传体系[5]。叶绿体基因组为母系遗传物质，结构保守，进化速率均衡，分别可以应用于目级、科级、属级、种级的系统发育关系重建[6]。HUANG等[7]通过叶绿体基因组系统进化分析，将11个榕属Ficus物种根据不同亚属进一步划分为3个支系，同时发现榕属与桑属Morus植物的属间亲缘关系。ZHANG等[8]通过对17个孩儿参Pseudostellaria heterophylla品种的完整叶绿体基因组序列分析，发现中国种群被聚类为单系群，其中不开花的品种形成了独立的亚支系，并且揭示了孩儿参的种内变异，进一步支持了叶绿体基因组可以阐明近缘物种之间亲缘关系的观点。迄今为止，绿槽毛竹[9]、厚壁毛竹Ph. edulis f. pachyloen[10]、青龙竹Ph. edulis f. curviculmis[11]和龟甲竹Ph. edulis f. tubiformis[12]的叶绿体基因组已发布；这些毛竹种下分类群的叶绿体基因组大小均为139 678 bp，包含126~132个基因，其中包括84~85个蛋白质编码基因、8个rRNA和34~39个tRNA。黄槽毛竹是毛竹中秆色变异类型的重要栽培类型，表现为茎秆节间槽部失绿而秆壁绿色，具有较好的观赏价值[13]。黄槽毛竹的叶绿体基因组资料未见报道，并且毛竹种下分类群的叶绿体基因组之间的比较缺乏研究。本研究将对黄槽毛竹的叶绿体基因组进行组装注释，分析其基因组特征，并比较与其他毛竹种下分类群之间的叶绿体遗传信息差异和系统进化关系。

    • 1.   材料与方法

      1.1.   材料

    • 黄槽毛竹新鲜竹青采集于浙江农林大学东湖校区翠竹园(30°26′N, 119°72′E)。黄槽毛竹取植物枝条标本(编号LIUX202301)，保存在浙江农林大学林业与生物技术学院植物标本室。

      • 1.2.   DNA提取与测序

    • 取2 g新鲜竹青进行液氮研磨，随后转移到离心管，配合天根Plant Genomic DNA Kit (Cat.#DP305-03)试剂盒和其建议的操作流程进行基因组DNA提取。对检验合格后的DNA样品进行二代测序文库构建。在Illumina HiSeq 4000平台测序DNA测序，测序深度为60×。原始数据可经国家基因库生命大数据平台(CNGBdb)的样本编号CNS0314191获取。

      • 1.3.   叶绿体基因组组装和注释

    • 利用fastp[14]对二代测序数据进行过滤，去除接头和切除低质量的数据，具体参数设置为“-w 16 -5 25 -3 25 --length_required 50 --average_qual 25 -z 9”；通过GetOrganelle[15]对清洗后的数据进行叶绿体基因组进行组装；参数设置为“-F embplant_pt -o output -R 10 -t 10 -k 21,45,85,105”。原始数据上传至美国国家生物技术信息中心(NCBI) GenBank (https://www.ncbi.nlm.nih.gov)，登录号为OR597504。使用CPGAVAS2对叶绿体基因组进行注释。使用CHLOROPLOT在线软件(https://irscope.shinyapps.io/Chloroplot/)绘制基因组图谱。

      • 1.4.   叶绿体基因组密码子使用频率、简单重复序列(SSR)分析

    • 利用MISA[16]在线软件检测简单重复序列，相应参数设置为：单核苷酸重复≥10，二核苷酸≥5，三核苷酸重复≥4，四、五和六核苷酸重复≥3。采用REPuter在线软件(https://bibiserv.cebitec.uni-bielefeld.de/reputer)验证黄槽毛竹的重复序列，包括正向重复序列(F)、反向重复序列(R)、互补重复序列(C)以及回文重复序列(P)，设置参数为“Minimal repeat size = 15 bp，Edit distance ≥ 3 bp”。

      • 1.5.   叶绿体基因组序列变异比较分析

    • 边界收缩与扩张分析使用Genepioneer平台(http://cloud.genepioneer.com:9929/)的CPJSdraw-边界图绘制-v1.0.0功能进行绘制。利用mVISTA进行多序列变异分析，比对运算模式设置为Shuffle-LAGAN，可视化参数设置为Y轴起始坐标为0。核苷酸多样性分析使用JSHYCloud平台(http://cloud.genepioneer.com:9929)进行分析绘制。

      • 1.6.   系统进化分析

    • 序列比对使用软件Mafft v7.490[17]，参数设置为默认条件。系统发育进化关系重建分别采用了贝叶斯推理法和最大似然法。贝叶斯推理法所采用的软件为MrBayes v3.2.7 (https://mybiosoftware.com/mrbayes-3-1-2-bayesian-inference-phylogeny.html)，参数设置为“ngen=1 000 000, samplefreq=100, nchains = 4, temp = 0.1, burnin=2 500”。最大似然法采用了IQ-TREE v2.0.7[18]进行运算，最佳进化模型为程序自动选择。构建系统发育树所用分类群及其序列数据登录号：毛竹原变种Phyllostachys edulis f. pubescens (HQ337796)、黄槽毛竹(OR597504)、花毛竹(SRR6705383)、青龙竹(MW007169)、绿槽毛竹(OM084949)、黄皮毛竹(SRR6705382)、厚壁毛竹(MN537809)、淡竹Ph. nigra var. henonis (HQ154129)、筇竹Ph. glauca (MT657329)、红壳雷竹Ph. incarnata (OL457160)、桂竹Ph. reticulata (MN537808)、麻竹Dendrocalamus latifloru (FJ970916)、瓜多竹Guadua amplexifolia (KM365071)、巴西玉米竺Raddia brasiliensis (KJ870998)和莪莉竹Olyra latifolia (KF515509)。花毛竹和黄皮毛竹的基因组序列分别由NCBI获取的原始测序数据经GetOrganelle组装得到。

    2.   结果与分析

      2.1.   黄槽毛竹叶绿体基因组基本特征

    • 黄槽毛竹绿色组织的二代测序获得42.78 Gb的原始数据，包含了285 198 507对读段。原始数据被清洗后，获得283 052 833对长度大于50 bp且平均碱基质量值大于25的读段。经GetOrganelle组装获得黄槽毛竹叶绿体基因组序列大小为139 678 bp。该叶绿体基因组具有典型的环状四分体结构，由大单拷贝区域(LSR)、小单拷贝区域(SSR)、2个反向互补重复区域(IRs)等4个部分组成，其中LSR长度为83 212 bp，SSR长度为12 870 bp，2个IRs (IRA和IRB)长度为43 596 bp (图1)。整个叶绿体基因组中GC含量为38.88%，LSC、SSC和IRs区域GC含量分别为36.97%、33.17%和44.22%，其中IRs区域GC含量明显高于2个单拷贝区域。

      Figure 1.  Genome map of the chloroplast of Ph. edulis f. luteosulcata

      • 2.2.   黄槽毛竹叶绿体基因组的组成及功能

    • 黄槽毛竹叶绿体基因组总共包含132个基因，包括85个蛋白质编码基因、39个tRNA、8个rRNA (表1)。根据生物学功能可以将85个蛋白质编码基因分为以下三大类：与光合作用相关基因共50个；与表达相关基因共31个；其他基因共4个。该基因组中共12个基因具有内含子结构[ndhA、ndhB(2)、petB、petD、atpF、rpl16、rpl2(2)、ycf3、rps16和rpoC2]，1个蛋白质编码基因含有反式剪接基因(rps12)。由于绿槽毛竹具有绿色的节间秆槽和黄色的节间秆壁，与黄槽毛竹的秆色表型相反，本研究比较了黄槽毛竹、绿槽毛竹和毛竹原变种的叶绿体基因组信息，发现黄槽毛竹叶绿体基因组的蛋白质编码基因、tRNA与毛竹原变种有所差异(表2)，相较于绿槽毛竹及毛竹原变种，黄槽毛竹的蛋白质编码基因缺少2个ycf68。黄槽毛竹叶绿体基因组的蛋白质编码基因数量也同毛竹原变种有所不同，前者比后者多1个编码基因rps12。

      基因类别基因分组基因列表
      光合作用基因ATP合酶亚基atpA、atpB、atpE 、 atpF*、atpH 、atpI
      依赖ATP的Clp蛋白酶蛋白水解亚基clpP
      光合系统Ⅱ亚基psbA、psbB、psbC、psbD、psbE 、psbF、psbH、psbI 、psbJ、psbK 、psbL、psbM 、psbN 、psbT 、psbZ
      NADH脱氢酶亚基ndhA*、ndhB*、ndhB*、ndhC 、ndhD 、ndhE 、ndhF 、ndhG、ndhH 、ndhI 、ndhJ 、ndhK
      细胞色b/f 复合物亚基petA、petB*、petD*、petG 、petL 、petN
      光合系统Ⅰ亚基psaA、psaB、psaC 、psaI 、psaJ
      光合系统Ⅰ组件ycf2、 pafI (ycf3)** 、ycf2
      光合系统Ⅱ组件pafⅡ (ycf4)
      二磷酸核酮糖羧化酶亚基rbcL
      表达相关基因核糖体大亚基rpl14、rpl16*、rpl20、rpl22、rpl23、rpl23 、rpl32 、rpl33 、rpl36、rpl2*、rpl2*
      依赖DNA的RNA 聚合酶rpoA、rpoB 、rpoC1 、rpoC2*
      核糖体小亚基rps2、rps3、rps4、rps7、rps7、rps8、rps11、rps12 、rps12、rps14、rps15、rps15、rps16*、rps18、rps19、rps19
      rRNA基因rrn16S、rrn23S 、rrn4.5S、rrn5S 、rrn5S、rrn4.5S、rrn23S、rrn16S
      tRNA基因trnA-UGC*、trnA-UGC*、trnC-GCA、trnD-GUC、trnE-UUC、trnF-GAA、trnG-GCC、trnH-GUG、trnH-GUG、trnI-CAU、trnI-CAU、trnI-GAU*、trnI-GAU*、trnK-UUU*、trnL-CAA、trnL-CAA、trnL-UAA*、trnL-UAG、trnM-CAU、trnM-CAU、trnM-CAU、trnN-GUU、trnN-GUU、trnP-UGG、trnQ-UUG、trnR-ACG、trnR-ACG、trnR-UCU、trnS-CGA*、trnS-GCU、trnS-GGA、trnS-UGA、trnT-GGU、trnT-UGU、trnV-GAC、trnV-GAC、trnV-UAC*、trnW-CCA、trnY-GUA
      其他基因C型细胞色素合成基因ccsA
      胞膜蛋白cemA
      成熟酶matK
      翻译起始因子infA
      　　说明：加粗代表多拷贝的基因；*表示带1个内含子的基因；**表示带2个内含子的基因。

      Table 1.  Gene of the Ph. edulis f. luteosulcata chloroplast genome

      项目黄槽毛竹毛竹绿槽毛竹
      蛋白编码基因数量858483
      转运RNA数量−−−
      核糖体RNA数量−−−
      蛋白质编码基因差异rps12 (2)、ycf2 (2)rps12、ycf68(2) rps12、ycf68
      转运RNA差异trnG-GCC、trnS-CGAtrnG-UCC、trnG-UCCtrnG-GCC、trnG-UCC
      　　说明：(2)代表拷贝数为2；−表示未检测到。

      Table 2.  Chloroplast gene differences among Ph. edulis f. luteosulcata, Ph. edulis f. bicolor, and Ph. edulis var. pubescens

      • 2.3.   黄槽毛竹的相对同义密码子使用频率(RSCU)、重复序列和SSR分析

    • 根据黄槽毛竹叶绿体基因组RSCU(图2A)显示：共有64种密码子编码20种氨基酸，密码子中UUA的RSCU为最高值(1.97)，CUG为最低值(0.33)。RSCU＞1的有32个，其中以A/U结尾的密码子有30个，提示密码子偏好使用以A/U结尾的密码子。重复序列按照不同的排列方式可分为正向重复序列、反向重复序列、回文重复序列和互补重复序列4种类型。黄槽毛竹叶绿体基因组中共检测出49个重复序列，其中40个为正向重复序列，9个为回文重复序列，未发现反向和互补重复序列。所有重复序列长度为30~100 bp，其中序列长度在30~60 bp的重复序列占比较高，为85.7%。此外，在基因组中共检测出55个SSR位点(图2B)，包括34个单核苷酸重复序列、4个二核苷酸重复序列、3个三核苷酸重复序列、13个四核苷酸重复序列和1个五核苷酸重复序列，其中简单重复序列最多的类型为A/T，共23个。

      Figure 2.  Repeat analysis of chloroplast genomes of Ph. edulis f. luteosulcata

      • 2.4.   黄槽毛竹与近缘分类群的叶绿体基因组序列分析

    • 高等植物的叶绿体通常为典型的四分体结构，但是常常发生SSC和LSC的扩张和收缩，导致四分体的边界发生变化。对黄槽毛竹与其他毛竹变型等7个种下分类群的叶绿体基因组四分体边界进行了分析(图3A)显示：黄槽毛竹叶绿体基因组四分体边界的扩张和收缩与其他分类群的高度一致，并且前者边界基因也与其他的高度统一。然而，黄槽毛竹、青龙竹和黄皮毛竹与其他毛竹种下分类群之间存在着边界编码基因编码长度不一致的情况，如前三者的rps19编码长度为216 bp，其他毛竹种下分类群的该基因编码282 bp。mVISTA分析显示(图3B)：对比毛竹原变种，黄槽毛竹以及其他毛竹种下分类群的叶绿体基因组变异程度较低，仅在psbC、ycf3基因编码区与非编码区之间(psbA附近)具有一定程度的序列差异。此外，基于CPGview鉴定黄槽毛竹、毛竹原变种和绿槽毛竹之间的顺式剪切基因，黄槽毛竹有12个，后两者有11个。其中黄槽毛竹较后两者多获得rpoC2的顺式剪切形式(图4A)。将核苷酸多样性(Pi)截断点设定为Pi≥0.04，在7个毛竹种下分类群的基因组序列比对中发现了3个高变异区域(图4B)。这些高变异区均位于蛋白编码基因ndhF附近，主要表现在叶绿体基因组ndhF和rpl32之间的SSC区。这些变异区域将可以用于毛竹种下分类群的分子鉴定依据。上述序列数据表明：毛竹种下分类群之间的序列变异较低，但不同分类群之间仍具有一些共有和特异的变异特征。

      Figure 3.  Comparison of chloroplast genome sequence variation between Ph. edulis f. luteosulcata and other subspecies in moso bamboo

      Figure 4.  Comparison of the cis-splicing genes and analysis of nucleotide diversity (Pi) of cp genomes between Ph. edulis f. luteosulcata and other subspecies in moso bamboo

      • 2.5.   系统进化分析

    • 为了进一步了解黄槽毛竹在竹子分类群内进化关系，利用毛竹种下分类群(毛竹原变种、绿槽毛竹、厚壁毛竹、青龙竹、黄皮毛竹、花毛竹和黄槽毛竹)，4个刚竹属物种(淡竹、筇竹、红壳雷竹和桂竹)，麻竹、瓜多竹、外类群草本竹巴西玉米竺和莪莉竹的叶绿体全基因组进行系统进化树的重建。贝叶斯推理法重建的系统进化树(图5)显示：毛竹种下分类群为单系且毛竹分类群与淡竹为姐妹。毛竹分类群之中，黄槽毛竹与毛竹原变种为姐妹关系，两者与花毛竹和青龙竹组成的分支为姐妹关系。秆色变异表型与黄槽毛竹呈“反转”状态的绿槽毛竹和黄槽毛竹以及毛竹原变种之间，并没有组成一支，而是与黄皮毛竹组成姐妹类群。厚壁毛竹与本研究分析的其他6个毛竹种下分类群的为姐妹关系。

      Figure 5.  Phylogenetic relationship of Ph. edulis f. luteosulcata based on whole-chloroplast genome sequences

    3.   讨论

    • 植物叶绿体基因组已被广泛应用于植物分类、分子进化以及系统发育等相关研究。竹亚科作为禾本科中唯一木质化结构的类群，其种群数量较多、分布较广且有性繁殖周期长，因此在系统分类学上造成分类困难[19]。在分子水平上，黄槽毛竹所属的木本竹进化相对缓慢，较低的分子进化速度可能会使系统发育研究复杂化，因此对于这些较低阶元分类群，叶绿体系统发育基因组学是较好的解决方案[20]。叶绿体作为来源于蓝藻菌的独立细胞器[21]，具有较为保守的特性，叶绿体基因组在包含大量遗传信息的同时相对于核基因组和线粒体基因组序列大小适中，便于测序；其次，核酸取代率相对较慢，进化速率保守，为深层研究植物系统发育和物种鉴定提供基础[22]。

      高等植物的叶绿体基因组通常为闭合的环形四分体结构，长度为108~165 kb，约包含80个编码基因[23]。本研究测序结果显示：黄槽毛竹的叶绿体基因组核苷酸序列为139 678 bp，为典型的四分体结构；这与竹子叶绿体基因组的长度接近且结构形式一致[9, 24]。黄槽毛竹叶绿体基因组共注释132个基因，包含85个蛋白质编码基因。这与系统发育分析所显示关系最近的毛竹原变种的编码基因数量差异较小，后者84个[19]。但最近报道的绿槽毛竹叶绿体基因组包含了85个蛋白编码基因[9]。本研究对黄槽毛竹、毛竹原变种和绿槽毛竹的叶绿体蛋白编码基因的结构分析发现：三者之间的rpoC2存在顺式剪切结构的差异；并且三者之间的tRNA和蛋白质编码基因类型也有所差异。虽然毛竹种下分类群的叶绿体基因组的四分体边界保持一致，但是边界的蛋白编码基因rps19的编码长度分为216和282 bp两大类。此外，叶绿体基因组的mVISTA分析也支持毛竹种下分类群之间也存在序列变异的区域，并且核苷酸多样性分析显示高变异区均位于叶绿体基因组SSC区的ndhF和rpl32之间。综合上述序列比较的信息，rpoC2的顺式剪切、rps19编码区长度以及高核酸多样性的ndhF和ndhF-rpl32区间等，可以用于毛竹种下分类群鉴定的潜在DNA片段。这些结果显示毛竹种下分类群的叶绿体基因组序列特征具有多样性。

      重复序列广泛分布于叶绿体基因组中，能够通过特异结合蛋白质促使核酸形成复杂结构[7, 21]。本研究黄槽毛竹叶绿体基因组中的SSR分析显示：相对于二、三、四、五和六核苷酸重复相比，单核苷酸重复出现的频次更高，AT/TA以及TC是最常见的二核苷酸重复基序，而GC/CG较少或并不存在。这与VIEIRA等[25]研究的20种热带木本竹结果相同。密码子在叶绿体基因组中也起着关键作用，本研究相对同义密码子使用频率显示：RSCU值＞1的有32个，其中以A/U结尾的密码子有30个，提示密码子偏好使用以A/U结尾的密码子，这与其他竹类叶绿体基因组的分析一致[26]。

      叶绿体全基因组能够较好地鉴定种间及以上分类群，但偶见利用于种内群体的鉴定，如朴树Celtis sinensis [27]。本研究利用全基因组序列重建了7个毛竹种下分类群的系统发育关系。结果显示：毛竹种下分类群为单系(100/90，后验概率/最大似然法自展值）。然而，JING等[9]研究显示：毛竹种下分类群与淡竹、筇竹和桂竹形成并系。本研究的系统发育分析显示：这3个物种并不与毛竹分类群形成并系。这可能是由于采样类群不一致甚至序列比对的结果差异所导致。因此，毛竹种下分类群具争议性的系统发育关系仍待更为全面和深入的研究。本研究重建的系统发育关系表明：黄槽毛竹与毛竹原变种亲缘关系最近，暗示两者有共同的起源；与黄槽毛竹表型相反的绿槽毛竹与黄皮毛竹为姐妹关系，表明两者有共同的最近祖先。目前，通过叶绿体全基因组序列重建的进化树在毛竹种下分类群之间的系统发育关系鉴定分辨率较低，仅能有效鉴定厚壁毛竹(厚壁毛竹+6个分类群)和黄皮毛竹(黄皮毛竹+绿槽毛竹)的分支。

    4.   结论

    • 黄槽毛竹的叶绿体基因组是长度为139 678 bp的双环DNA，包含132个基因。这些基因包括85个蛋白质编码基因、8个核糖体RNA(rRNA)以及39个转运RNA(tRNA)。该基因组偏好使用以A/U碱基结尾的密码子，且简单重复序列最多的类型为A/T。在系统发育分析方面，黄槽毛竹与其他毛竹种下分类群共同构成了单系分支，且与毛竹原变种具有最近的亲缘关系。种下分类群的叶绿体基因组比较分析发现毛竹种下分类群之间存在序列差异。

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