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Analysis of Complete Chloroplast Genome Sequences and Phylogenetic Evolution of Zanthoxylum armatum ‘Jiuyeqing’

  • Corresponding author: CHEN Ze-xiong, chenzexiong1979@163.com
  • Received Date: 2022-05-30
    Accepted Date: 2022-08-29
  • Objective To reveal the structural characteristics and phylogenetic relationships of the chloroplast genomes of Zanthoxylum armatum ‘Jiuyeqing’, and provide reference for identifying germplasm resources, breeding new varieties and the genetic analysis among varieties of the genus Zanthoxylum. Method The total DNA of Z. armatum ‘Jiuyeqing’ was extracted using a modified CTAB method. High-throughput sequencing was performed using the BGISeq-500 platform, the chloroplast genome was assembled by SPAdes v3.13.0 software, the whole chloroplast genome information of Z. armatum ‘Jiuyeqing’ was annotated by GeSeq software, and the structural characteristics, repetitive sequences, codon preference and phylogenetic relationship were analyzed. Result Z. armatum ‘Jiuyeqing’ was a typical tetrad structure with a full-length sequence of 158 579 bp, encoding 133 genes. 19 tandem repeats were detected, 49 long repeat sequences, 70 simple sequence repeat (SSR) were detected. A total of 26 398 codons (excluding stop codons) were detected in the protein-coding gene of the chloroplast genome of Z. armatum ‘Jiuyeqing’, and there was a strong A/T base preference at the third base of the codon. Conclusion The complete chloroplast genome sequences of Z. armatum 'Jiuyeqing' was firstly assembled for the study. The results of phylogenetic analysis show that the genus Zanthoxylum is a monophyletic group, and the Z. armatum 'Jiuyeqing' is closely related to Z. simulans. The results of the study enrich the genetic information of Zanthoxylum and improve important references for Zanthoxylum germplasm resource evaluation, molecular breeding, development of cpSSR molecular markers, and genetic diversity research.
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Analysis of Complete Chloroplast Genome Sequences and Phylogenetic Evolution of Zanthoxylum armatum ‘Jiuyeqing’

    Corresponding author: CHEN Ze-xiong, chenzexiong1979@163.com
  • 1. College of Landscape Architecture and Life Science, Chongqing University of Arts and Sciences, Chongqing 402160, China
  • 2. Spice Crops Research Institute, College of Horticulture and Gardening, Yangtze University, Wuhan, 434025, Hubei, China
  • 3. College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing, 404000, China

Abstract:  Objective To reveal the structural characteristics and phylogenetic relationships of the chloroplast genomes of Zanthoxylum armatum ‘Jiuyeqing’, and provide reference for identifying germplasm resources, breeding new varieties and the genetic analysis among varieties of the genus Zanthoxylum. Method The total DNA of Z. armatum ‘Jiuyeqing’ was extracted using a modified CTAB method. High-throughput sequencing was performed using the BGISeq-500 platform, the chloroplast genome was assembled by SPAdes v3.13.0 software, the whole chloroplast genome information of Z. armatum ‘Jiuyeqing’ was annotated by GeSeq software, and the structural characteristics, repetitive sequences, codon preference and phylogenetic relationship were analyzed. Result Z. armatum ‘Jiuyeqing’ was a typical tetrad structure with a full-length sequence of 158 579 bp, encoding 133 genes. 19 tandem repeats were detected, 49 long repeat sequences, 70 simple sequence repeat (SSR) were detected. A total of 26 398 codons (excluding stop codons) were detected in the protein-coding gene of the chloroplast genome of Z. armatum ‘Jiuyeqing’, and there was a strong A/T base preference at the third base of the codon. Conclusion The complete chloroplast genome sequences of Z. armatum 'Jiuyeqing' was firstly assembled for the study. The results of phylogenetic analysis show that the genus Zanthoxylum is a monophyletic group, and the Z. armatum 'Jiuyeqing' is closely related to Z. simulans. The results of the study enrich the genetic information of Zanthoxylum and improve important references for Zanthoxylum germplasm resource evaluation, molecular breeding, development of cpSSR molecular markers, and genetic diversity research.

  • 九叶青花椒(Zanthoxylum armatum ‘Jiuyeqing’)为芸香科(Rutaceae)花椒属(Zanthoxylum L.)竹叶花椒的主要栽培品种,具有矮化、丰产、质优、抗逆等优良特性[1],其果实清香,麻味纯正,备受消费者喜爱,市场需求旺盛[2]。我国花椒属植物的基础研究十分薄弱,花椒属物种间的系统发育关系尚待进一步解析。

    长期以来,国内外有关花椒属植物的研究大都集中在其化学成分的提取和分离[3-4],以及提取物种所具有的生物活性[5]和相应的药用价值方面[6],仅有少数学者基于少量的核基因(ETS 和 ITS)[7-8]、叶绿体基因(rps16trnL-trnF[8]、分子标记(SSR、RAPD 和 SRAP)[9-10]开展了花椒属内几个物种间的亲缘关系研究。但基于单个片段或分子标记其有效信息位点不足,花椒属内物种间的系统位置支持率不高,花椒属内种间系统进化关系未能很好的解析。因此,解析九叶青花椒的叶绿体基因组结构特征与系统进化关系,为花椒种质资源的分子鉴定、品种选育和农艺经济性状的遗传分析具有重要意义,亦为花椒属系统进化研究提供依据。

    叶绿体是植物进行光合作用的重要细胞器,在植物生长发育过程中具有重要作用[11]。叶绿体基因组一般具有保守的四分体结构,即包括大单拷贝区(Large Single Copy, LSC),小单拷贝区(Small Single Copy, SSC),2个反向重复区(Inverted Repeat Region, IRs)。大多数被子植物叶绿体基因组为母系遗传,且叶绿体基因组的核苷酸替换率适中[12],因此,叶绿体基因组是进行系统进化和分化时间分析的理想标记[13]。近年来,随着叶绿体基因组测序成本降低,使其在植物系统进化分析中广泛应用[14]。本研究利用高通量测序技术进行主栽品种九叶青花椒叶绿体全基因组测序,通过组装、拼接和注释得到其全长叶绿体基因组序列,并对其叶绿体基因组结构、组成及系统进化关系进行分析,旨在丰富花椒的遗传信息,为未来研究花椒属植物间的系统进化、亲缘关系及品种鉴定提供理论依据。

    • 九叶青花椒叶片采于重庆文理学院特色植物研究院花椒示范基地,植物标本(2021023jyq)放置于重庆文理学院园林与生命科学学院标本馆。采用改良CTAB法对九叶青花椒提取基因组DNA,-20 ℃冰箱保存,备用。

    • 利用提取的DNA构建shotgun文库,并在BGISEQ-500平台(中国,广州)进行测序。PE150双末端测序得到raw data,过滤掉低质量序列后得到reads进行拼接组装。使用SOAPnuke软件[15]进行质量控制后,以花椒(Zanthouxylum bungeanum Maxim.)的叶绿体基因组[16]作为参考,用SPAdes v3.13.0软件[17]组装叶绿体基因组。基因组注释采用GeSeq(https://chlorobox.mpimp-golm.mpg.de/geseq.html)软件[18]。注释后的叶绿体基因组序列提交至GenBank数据库中。

    • 九叶青叶绿体基因组序列的GC含量采用Editseq v7.1.0软件[19]计算,OGDRAW软件[20]制作九叶青花椒叶绿体基因组图谱。CodonW v.1.4.2 软件[21]分析相对密码子使用度(Relative Synonymous Codon Usage, RSCU),研究其偏好性。

    • 通过Tandem Repeats Finder(https://tandem.bu.edu/trf/trf.html)搜寻基因组序列中的串联重复序列,用REPuter(https://bibiserv.cebitec.uni-bielefeld.de/reputer/)在线服务分析叶绿体基因组中的重复序列(正向、反向、回文和互补重复),采用MISA (https://webblast.ipk-gatersleben.de/misa/)在线预测九叶青花椒叶绿体基因组包含的简单重复序列(Simple Sequence Reapeat, SSR),其中,一、二、三、四、五、六碱基重复的检测标准分别设置为10,6,5,5,5,5。2个SSR之间的最小距离设置为100 bp,小于100 bp时,2个微卫星组成1个复合微卫星。

    • 从NCBI上下载得到12个芸香科物种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九叶青花椒的完整叶绿体基因组序列通过MAFFT软件[22 ] 进行比对分析,其中黄檗(Phellodendron amurense Rupr.)和川黄檗(Phellodendron chinense Schneid.)的叶绿体基因组序列作为外类群。采用RAxML软件[23]构建ML (Maximum Likelihood)树,建树过程中通过GTR模型确定最优树。MrBayes3.1.2软件[24]构建BI(Bayesian)树,采用GTR替代模型和伽马分布率,每1 000代进行1次计算。

    2.   结果与分析
    • 注释组装获得九叶青花椒完整的叶绿体基因组图谱见图1。该叶绿体基因组序列全长158 579 bp,包括大单拷贝区域(LSC)长85 780 bp,小单拷贝区域(SSC)长17 603 bp,2个反向重复区(IRa和IRb)相等长度为27 598 bp。共注释了133个基因,其中,88个蛋白编码基因,37个tRNA基因和8个 rRNA基因。叶绿体基因组总的GC含量为38.5%,IR区域(42.5%)的GC含量明显高于LSC 区域(36.8%)和SSC 区域(33.6%),这可能主要是IR区域具有高GC含量的rRNA基因造成的。在注释的九叶青花椒叶绿体基因组中18个特异基因具有内含子,包括12个特异编码基因和6个特异tRNA基因。除pafI具有2个内含子外,其余均具有1个内含子。rps12基因被截断为2个片段,其中,1个外显子位于LSC区域,另外2个外显子位于IR区域。最长内含子位于trnK-UUU基因,长2 503 bp,主要是因为内部包含matK基因;rps12基因内含子最短(538 bp)。

      Figure 1.  Gene map of the chloroplast genome of Z. armatum‘Jiuyeqing’

    • 通过软件测算在九叶青花椒叶绿体基因组中存在19个串联重复序列,其中,长度最大为25 bp,最小为14 bp,长度为18 bp的重复序列最多,有6个。该叶绿体基因组中共检测到长片段重复序列49个,其中,21个正向重复,8个反向重复,19个回文重复和1个互补重复(图2)。重复长度范围为19~50 bp,其中,长度为20 bp的重复最多(10个),其次是19 bp(9个),而重复长度为26、31、48、50 bp的最少,均1个。在九叶青花椒叶绿体基因组中共预测到70个SSR,包括60个单核苷酸,1个二核苷酸,9个复合型SSR(表1)。75.7%的SSR位于IGS和内含子等非编码区域且A/T碱基在SSR中出现频率较高,在60个单核苷酸SSR中,包含A碱基SSR有26个,包含T碱基SSR有32个。从表1可以看出,SSR在叶绿体基因组中分布随机,表明SSR数量丰富,遗传信息含量高,可筛选具有多态性的引物,为未来研究花椒种群遗传多样性和遗传结构提供参考。

      Figure 2.  Types of repeat sequences in the chloroplast genome of Z. armatum‘Jiuyeqing’

      序号
      Number
      重复类型
      Repeat type
      简单重复序列
      SSR
      起始
      Start
      终止
      End
      所在位置
      Position
      1 p1 (T)19 1 771 1 789 IGS
      2 p1 (T)11 2 418 2 428 matK
      3 p1 (A)10 3 085 3 094 matK
      4 p1 (A)10 4 684 4 693 IGS
      5 p1 (A)10 6 287 6 296 IGS
      6 p1 (T)13 6 873 6 885 IGS
      7 c (A)12gacgtacattttatcaaaaattatacttaatagagttgctcaaa
      ggggggaattc(T)12
      7 281 7 359 IGS
      8 p1 (A)14 7 770 7 783 IGS
      9 p1 (T)14 8 334 8 347 IGS
      10 c (A)11(AAT)5 8 717 8 740 IGS
      11 p1 (T)11 9 319 9 329 IGS
      12 p1 (A)13 9 834 9 846 intron
      13 p1 (A)15 13 273 13 287 intron
      14 p1 (A)11 14 122 14 132 IGS
      15 p1 (T)10 16 570 16 579 IGS
      16 p1 (T)11 19 493 19 503 rpoC2
      17 p1 (A)12 23 671 23 682 intron
      18 p1 (T)20 24 067 24 086 intron
      19 p1 (T)10 27 372 27 381 rpoB
      20 p1 (A)18 28 352 28 369 IGS
      21 p1 (C)10 28 617 28 626 IGS
      22 c (T)12caatagaaaaaaagaaagagaaaagagagggattaagatattgtgtgttttgagatactataaaatcaattgcagaaatggagttgctccaaaaaaag(A)10 29 037 29 156 IGS
      23 p1 (A)10 29 784 29 793 IGS
      24 p1 (T)10 31 390 31 399 IGS
      25 p1 (T)10 31 563 31 572 IGS
      26 p1 (T)11 32 418 32 428 IGS
      27 p1 (T)13 33 368 33 380 IGS
      28 p1 (G)11 36 561 36 571 psbC
      29 p1 (T)10 45 777 45 786 intron
      30 p1 (A)12 47 071 47 082 IGS
      31 p1 (T)15 51 184 51 198 IGS
      32 p1 (T)10 52 626 52 635 IGS
      33 p1 (T)10 53 551 53 560 intron
      34 p1 (T)19 56 537 56 555 IGS
      35 p1 (A)12 57 034 57 045 IGS
      36 p1 (T)12 61 599 61 610 IGS
      37 c (T)14cattttttcaaaatattcgagagtttcttttttaatag(A)10 62 628 62 689 IGS
      38 p1 (A)10 65 486 65 495 IGS
      39 p1 (A)10 67 533 67 542 IGS
      40 p1 (A)10 68 248 68 257 IGS
      41 p1 (A)10 68 735 68 744 IGS
      42 p1 (A)11 69 053 69 063 IGS
      43 p1 (T)10 69 446 69 455 IGS
      44 p1 (A)10 72 760 72 769 clpP
      45 p1 (T)10 74 477 74 486 IGS
      46 p1 (T)10 76 170 76 179 IGS
      47 p1 (T)12 83 898 83 909 rpl16
      48 p1 (T)16 85 546 85 561 IGS
      49 p1 (T)11 101 363 101 373 IGS
      50 p1 (A)16 110 363 110 378 IGS
      51 p1 (T)10 110 534 110 543 IGS
      52 c (T)11atttaccattaatggtaaat(A)11 110 781 110 822 IGS
      53 p1 (A)12 112 609 112 620 ycf1
      54 c (A)10ttcttttgctattcatttttcttttgctattcatagttattttgaatctttcccaac
      aactt (A)10
      115 675 115 756 IGS
      55 c (A)17cgtac(T)11 116 867 116 899 IGS
      56 p1 (A)11 117 100 117 110 IGS
      57 p1 (T)12 117 541 117 552 IGS
      58 c (T)10atttaactgaaactgaagagaaggaaaaagacttccttgttcattggctaacgaac(T)12 118 691 118 768 IGS
      59 p1 (A)10 120 421 120 430 IGS
      60 p1 (T)10 122 121 122 130 IGS
      61 p2 (TA)6 122 278 122 289 IGS
      62 p1 (A)13 124 680 124 692 intron
      63 p1 (T)11 126 547 126 557 IGS
      64 p1 (A)10 127 040 127 049 IGS
      65 p1 (T)11 129 898 129 908 ycf1
      66 p1 (T)12 131 740 131 751 ycf1
      67 c (T)11atttaccattaatggtaaat(A)11 133 538 133 579 IGS
      68 p1 (A)10 133 817 133 826 IGS
      69 p1 (T)16 133 982 133 997 IGS
      70 p1 (A)11 142 987 142 997 IGS
      注:p单个SSR类型;p1/p2/p3中数字分别表示构成基序的碱基个数;c复合SSR类型;IGS代表基因内间隔区
        Notes: p is a single SSR type; the numbers in p1/p2/p3 indicate the number of bases constituting the motif, respectively;c is the compound SSR type; IGS represents the intergenic spacer

      Table 1.  Chloroplast genome SSR prediction

    • 在九叶青花椒叶绿体基因组中的蛋白编码基因共有26 398个密码子(不包括终止密码子),其中,亮氨酸Leu的密码子最丰富(2 821个),占总数的10.69%,编码Leu的6种同义密码子中TTA数量最多;编码半胱氨酸Cys的数量最少(310个),仅占密码子总数的1.17%。通过对密码子使用频率的计算结果表明:编码异亮氨酸Ile的ATT(1 078)使用次数最多,编码甲硫氨酸Met的ATT(1)和GTG(2)的使用次数最少。相对同义密码子使用率值(RSCU)受影响密码子使用模式的突变因子的影响,因此,RSCU<1表示密码子使用率较低,RSCU >1表示密码子使用率较高,RSCU=1表示无偏倚[25]。经计算分析,相对同义密码子使用值(RSCU)的范围为0.005(ATT)到2.986(ATG),分析还发现RSCU>1的密码子有31种,说明这些密码子有一定的偏好性。在这些密码子中,29个以A或T结尾,仅2个以G结尾,这说明九叶青花椒在密码子第三位碱基上有较强的A/T碱基偏好性。由图3可以看出:编码精氨酸Arg、亮氨酸Leu和丝氨酸Ser密码子种类最多,有6种密码子编码。色氨酸Trp不存在密码子偏好性(RSCU=1),只有1个密码子(TGG)编码。

      Figure 3.  Amino acid RSCU values of the chloroplast genome of Z. armatum‘Jiuyeqing’

    • 以黄檗和川黄檗2个黄檗属植物为外类群,连同花椒属11个植物的全叶绿体基因组序列构建ML和BI系统进化树,2种方法得到的拓扑结构基本一致,仅在个别分支节点支持率上略有差别。从图4可看出:花椒属物种聚为一支,2种方法在分支上的支持率均为100%,为单系类群。Z. paniculatumZ. madagascariense 位于分支基部与其他花椒物种构成姊妹群关系,且分支支持率为100%和1(ML和BI)。狭叶花椒、青花椒和Z. pinnatum聚为一个分支与Z. tragodes、胡椒木、花椒、Z. sp. NH-2018、野花椒和九叶青花椒形成的分支构成姊妹群关系,分支支持率均为100%,其中,九叶青花椒与野花椒呈姊妹关系,亲缘关系最近,与花椒(Z. bungeanum)亲缘关系相对较远。

      Figure 4.  Phylogenetic tree based on complete chloroplast genomes sequences of 13 species

    3.   讨论
    • 在大多数的被子植物中,叶绿体基因组具有典型的四分体结构[26],序列长度一般为120~160 kb。九叶青花椒叶绿体基因组同样具有相同的序列结构特征,其叶绿体基因组总长度为158 579 bp,其中,LSC长85 780 bp,SSC长17 603 bp,IRs长度为27 598 bp,总的GC含量为38.5%,IR区域(42.5%)的GC含量明显高于LSC 区域(36.8%)和SSC 区域(33.6%),这主要是因为IR区域具有高GC含量的rRNA基因造成的。

      九叶青花椒叶绿体基因组中的蛋白编码基因共有26 398个密码子,在31种RSCU>1的密码子中,除了TTG和ATG外,其余密码子均以A或T结尾,这说明了九叶青花椒在密码子的使用偏向于第三个密码子位置为A和T,有较强的A/T碱基偏好性。研究结果与蒺藜苜蓿(Medicago truncatula Gaertn.)[27]、杓兰(Cypripedium calceolus L.)[28]、和马尾松(Pinus massoniana Lamb.)[29]等植物叶绿体基因的第3位密码子偏好性趋势相吻合,但与酸枣(Ziziphus jujuba var. Spinosa (Bunge) Hu ex H. F. Chow)[30]、陆地棉(Gossypium hirsutum L.)[31]和樟树(Cinnamomum camphora (L.) Presl)[32]等植物的叶绿体基因分析结果不同。这表明密码子使用偏好性在不同物种中存在一定的差异。

      叶绿体SSR是研究群体遗传学、生物地理学和系统进化等高效的分子标记[33],在大量研究中广泛应用。在九叶青花椒叶绿体基因组SSR预测中,绝大多数为单碱基重复(60/70),这与肖蒲桃(Syzygium acuminatissimum (Blume) Candolle)[34]、胡桃(Juglans regia L.)[35]和金钱槭(Dipteronia sinensis Oliv.)[36]等大多数植物研究结果一致。重复序列的分析为将来青花椒品种及其他植物的物种鉴定和个体水平的遗传差异分析提供了丰富的遗传信息支撑。

      我国花椒属物种繁多,品种较多,为该属的物种、品种鉴定带来极大困难。本研究基于叶绿体基因组序列得到的花椒属系统进化结果与Appelhans 等[8]利用叶绿体基因片段和核基因片段构建的进化树结果一致,花椒属物种均聚为一个分支,形成单系类群,这与Feng等[37]的研究结果一致。花椒属物种形成2大分支,即崖椒亚属和花椒亚属2大分支,这与传统的分类学结果一致。本研究表明,九叶青花椒与野花椒亲缘关系较近,与北方主栽花椒亲缘关系相对较远,这与Feng等[37]的研究结果一致。但由于花椒属物种较多,目前提交到数据库中的叶绿体基因组序列有限,基于叶绿体基因组序列构建花椒属的系统进化关系还不够全面,将来需获取更多花椒属物种的数据来全面分析物种间的系统进化关系。

    4.   结论
    • 本研究利用高通量测序技术,组装和注释了九叶青花椒完整的叶绿体基因组,并解析了该基因组的结构特征和系统进化关系,结果表明,九叶青花椒叶绿体基因组的结构特征与其他花椒属植物相似,为典型的四分体组成结构,全长序列为158 579 bp,编码133个基因,26 398个密码子,检测到19个串联重复序列,49个长片段重复、70个简单重复序列。系统进化关系分析表明,花椒属为单系类群,九叶青花椒与野花椒关系密切。研究结果可为未来研究花椒种群遗传多样性和遗传结构提供参考。

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