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JIANG Zhuanzhuan, CHEN Hong, BAO Hongyan, DAI Yutong. Chloroplast genome characteristics and molecular marker development of 4 species of Pennisetum[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20240371
Citation: JIANG Zhuanzhuan, CHEN Hong, BAO Hongyan, DAI Yutong. Chloroplast genome characteristics and molecular marker development of 4 species of Pennisetum[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20240371
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Chloroplast genome characteristics and molecular marker development of 4 species of Pennisetum

doi: 10.11833/j.issn.2095-0756.20240371

  • College of Life Science/The Province Key Laboratory of the Biodiversity Study and Ecology Conservation, Anqing Normal University, Anqing 246133, Anhui, China

  • Received Date: 2024-05-30
  • Accepted Date: 2024-11-07
  • Rev Recd Date: 2024-10-22
  •   Objective  The objective is to analyze the chloroplast genome characteristics of four published plants in the genus Pennisetum, and to develop corresponding molecular markers. This will provide new insights into the evolutionary and phylogenetic relationships of Pennisetum.   Method  Bioinformatics methods were used to analyze the complete chloroplast genome sequences of four species of Pennisetum published in the NCBI database. The analysis included repeat sequences, codon preferences, polymorphisms, collinearity, and development of simple sequence repeat (SSR) molecular markers.   Result  The chloroplast genome length and GC content of the 4 species of Pennisetum are relatively similar, with a low degree of codon shift. Boundary expansion analysis indicates no significant contraction between different regions. Collinearity analysis shows strong inter-species collinearity among the 4 species of Pennisetum. Phylogenetic analysis based on chloroplast genome sequences reveals that P. flaccidum has the closest phylogenetic relationship with P. glaucum, P. purpureum, and P. alopecuroides. 5 pairs of SSR molecular markers have high practicality in plant species within the genus Pennisetum. Specifically, primer SSR2 is expressed in both P. flaccidium and P. alopecuroides; while primer SSR5 is specifically expressed in P. flaccidum. Additionally, SSR1, SSR3, and SSR4 show high conservatism among all four species in this genus.   Conclusion  This study concludes that the chloroplast genomes of these 4 species are relatively conserved; furthermore, it confirms that genetic relationship between P. flaccidium is closest to those found in P. flaccidum, P. purpureum as well as P. alopecuroides. SSR2 molecular markers are specifically expressed in both P. flaccidium and P. alopecuroides, while SSR5 molecular markers are specifically expressed in P. flaccidum. [Ch,7 fig. 2 teb. 28 ref.]
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Chloroplast genome characteristics and molecular marker development of 4 species of Pennisetum

doi: 10.11833/j.issn.2095-0756.20240371
  • College of Life Science/The Province Key Laboratory of the Biodiversity Study and Ecology Conservation, Anqing Normal University, Anqing 246133, Anhui, China
  • Received Date: 2024-05-30
  • Accepted Date: 2024-11-07
  • Rev Recd Date: 2024-10-22

Abstract:   Objective  The objective is to analyze the chloroplast genome characteristics of four published plants in the genus Pennisetum, and to develop corresponding molecular markers. This will provide new insights into the evolutionary and phylogenetic relationships of Pennisetum.   Method  Bioinformatics methods were used to analyze the complete chloroplast genome sequences of four species of Pennisetum published in the NCBI database. The analysis included repeat sequences, codon preferences, polymorphisms, collinearity, and development of simple sequence repeat (SSR) molecular markers.   Result  The chloroplast genome length and GC content of the 4 species of Pennisetum are relatively similar, with a low degree of codon shift. Boundary expansion analysis indicates no significant contraction between different regions. Collinearity analysis shows strong inter-species collinearity among the 4 species of Pennisetum. Phylogenetic analysis based on chloroplast genome sequences reveals that P. flaccidum has the closest phylogenetic relationship with P. glaucum, P. purpureum, and P. alopecuroides. 5 pairs of SSR molecular markers have high practicality in plant species within the genus Pennisetum. Specifically, primer SSR2 is expressed in both P. flaccidium and P. alopecuroides; while primer SSR5 is specifically expressed in P. flaccidum. Additionally, SSR1, SSR3, and SSR4 show high conservatism among all four species in this genus.   Conclusion  This study concludes that the chloroplast genomes of these 4 species are relatively conserved; furthermore, it confirms that genetic relationship between P. flaccidium is closest to those found in P. flaccidum, P. purpureum as well as P. alopecuroides. SSR2 molecular markers are specifically expressed in both P. flaccidium and P. alopecuroides, while SSR5 molecular markers are specifically expressed in P. flaccidum. [Ch,7 fig. 2 teb. 28 ref.]

JIANG Zhuanzhuan, CHEN Hong, BAO Hongyan, DAI Yutong. Chloroplast genome characteristics and molecular marker development of 4 species of Pennisetum[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20240371
Citation: JIANG Zhuanzhuan, CHEN Hong, BAO Hongyan, DAI Yutong. Chloroplast genome characteristics and molecular marker development of 4 species of Pennisetum[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20240371
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  • 狼尾草属Pennisetum是禾本科Poaceae中一类重要的牧草，其植物形态多样，包括1年生或多年生种类，集中分布于热带及亚热带地区，全球约140种，多数原产于非洲[1−2]。狼尾草属植物在畜牧业中具有重要地位，其种子可作为谷粒食用，具有潜在的农业价值。中国目前广泛利用的栽培种主要包括多年生象草P. purpureum [3]和一年生美洲狼尾草P. americanum以及它们的种间杂交种[4−9]。为了更好地开发这些植物资源，国内众多研究者对其进行了一系列研究，但尚未形成明确的分类结论。

    高等植物的叶绿体基因组是大小为72~217 kb的双链环DNA，含有约130个基因。叶绿体基因组是典型的四分体结构，在大部分植物中包含1个大单拷贝区(large single copy，LSC)、1个小单拷贝区(small single copy，SSC)以及1对反向重复序列(inverted repeat region，IR)[10−12]。相较于核基因组，叶绿体基因组为母系遗传，其体积较小、结构简单以及序列保守[13−14]。因此叶绿体基因组序列的研究对于揭示植物的遗传多样性、种间关系以及进化适应性具有重要意义。分子生物学的迅速发展已将分子标记成为研究生物分类和系统进化的关键工具。序列特异性扩增区(sequence characterized amplified regions，SCAR)[15]、随机扩增多态性DNA(random amplified polymorphic DNA，RAPD)[16]、简单重复序列标记(inter simple sequence repeat，ISSR)[17]等标记在植物中被广泛运用。其中，叶绿体简单重复序列(simple sequence repeat，SSR)标记是常用的遗传标记方法，通过标记叶绿体序列，进行物种识别、分析种间关系等研究，已经在许多植物物种中得到应用[18]。

    本研究拟选取4种狼尾草属植物为研究对象，采用比较基因组学方法，对狼尾草属叶绿体基因组的结构和组分特征进行系统性研究，并通过系统发育分析确定其亲缘关系，基于分析结果开发分子标记，为狼尾草属植物种间关系的鉴定及系统发育分析奠定理论基础。

    • 1.   材料与方法

      1.1.   材料

    • 材料来自于皖西南生物多样性研究与生态保护安徽省重点实验室大棚栽培的4种狼尾草属植物，分别为白草P. flaccidum、御谷P. glaucum、象草、狼尾草P. alopecuroides。

      • 1.2.   方法

      1.2.1.   叶绿体基因组注释及可视化

    • 通过查找美国国家生物技术信息中心(NCBI)数据库(www.ncbi.nlm. nih.gov\genome)中狼尾草属植物叶绿体基因组的信息，发现4种狼尾草属植物已发布完整的叶绿体基因组序列，即白草(NC_057588.1)、御谷(NC_057571.1)、象草(NC_036384.1)、狼尾草(MN180104.1)。使用CPGAVAS2 (http://47.96.249.172:16019/analyzer/home)对本研究选取的4种植物叶绿体基因组进行注释，通过OGDRAW (https://chlorobox.mpimp golm.mpg.de/OGDraw.html)可视化。

      • 1.2.2.   叶绿体基因组简单与长重复序列分析

    • 运用Misa (https://webblast.ipk-gatersleben.de/misa/index.php)检测SSR，单核苷酸SSR (mono-nucleotide SSR)、双核苷酸SSR(di-nucleotide SSR)、三核苷酸SSR (tri-nucleotide SSR)。运用Reputer (https://bibiserv.cebitec.uni-bielefeld.de/reputer )[19]分析长重复序列(long sequence repeat，LSR)，反向重复(reverse repeats)、正向重复(forward repeats)、回文重复(palindromic repeats)，设置运行参数最大重复为1 000，最小重复为30，Hamming距离为3。

      • 1.2.3.   叶绿体基因组密码子偏倚分析

    • 运用CodonW和EMBOSS Explorer[20] (http://emboss.toulouse.inra.fr/cgi-bin/emboss/cusp)进行密码子偏倚性分析，计算出同义密码子使用频率(relative synonymous codon usage，RSCU)，密码子适应性指数(codon adaption index，CAI)，密码子偏好性指数(Codon bias index，CBI)，有效密码子数(effective number of codon，Enc)，最优密码子使用频率(frequency of optical codons，FOP)，第三位同义密码子GC含量(GC3s)。

      • 1.2.4.   狼尾草属植物序列差异分析

    • 运用软件Launch Dnasp 6.0[21]，滑动窗法分析4种狼尾草属植物叶绿体全基因组，分析叶绿体基因组之间的核苷酸变异度(nucleotide diversity，Pi)，设置步长为200 bp，窗口长度为600 bp。再利用VISTA (https://genome.lbl.gov/vista/index.shtml)进行序列比对，筛选出变异度较高位点的基因。

      • 1.2.5.   叶绿体基因组LSC、SSC和IR边界扩张分析

    • 利用Rstudio将在线程序IRscope[22]本地化后使用，使4种狼尾草属植物叶绿体基因组的LSC、SSC和IR区域边界可视化，显示不同植物间各区域的收缩与扩张。

      • 1.2.6.   叶绿体基因组共线性分析与系统发育树的构建

    • 运用Circoletto[23] (http://tools.batinfspire.org/cirroletto)分析4种狼尾草属植物叶绿体基因组的共线性关系，以白草的叶绿体基因组作为参考序列。使用在线工具Mulan (https://mulan.dcode.org)对4种狼尾草属序列，以及从NCBI数据库获得的12种禾本科植物叶绿体基因组[大麦Hordeum vulgare (MW017635.1)、高粱Sorghum bicolor (NC_008602.1)、黑麦Secale cereale (NC_021761.1)、梁Setaria italica (KJ001642.1)、柳枝稷Panicum virgatum (NC_015990.1)、芒草Miscanthus transmorrisonensis (NC_035752.1)、粟草Milium effusum (NC_058911.1)、五节芒Miscanthus floridulus (NC_035750.1)、小麦Triticum aestivum (KC912694.1)、燕麦Avena sativa (MG687313.1)、玉米Zea mays (NC_001666.2)、玉山竹Yushania brevipaniculata (NC_043894.1)]进行多序列比对，校正对齐得到可信序列后运用软件MEGA11构建系统进化树。

      • 1.2.7.   分子标记的开发

    • 基于上述简单重复序列分析获得的结果，针对4种狼尾草属叶绿体基因组的SSR位点利用Primer 3.0 (https://primer3.ut.ee/)设计引物。将SSR位点处序列上传，设置参数，获得引物后，以Tm值不超过60 ℃，引物长度在20~25 bp作为筛选条件，选用5种SSR引物(表1)。

      引物
      名称      		 正向引物(5′→3′) 反向引物(5′→3′)
      SSR1 GAATGCAAGTCCTCCCCTTAAA AATCATTTTGGCTGGCTGTTTT
      SSR2 TGCTTCAATCGAGATCCTTCAA CTACTAGAAGTGGCGAGTAAAA
      SSR3 ACACCGCTGCTTAATCCCTTAG AAGGAAAGACACTTCACGAGAA
      SSR4 AAGTCGAATCGTGAGCCTATTA GGAGCCTTGGAATGGTCTTAAC
      SSR5 AGACTACTTCTTCTGGATCCAA CCCGGGCCTATTCGAGAAC

      Table 1.  Primer used in this study

      • 1.2.8.   DNA提取与引物的扩增检测

    • 利用CTAB法提取4种狼尾草属植物DNA，再利用50 µL的扩增体系进行PCR反应，DNA模板2 µL，Taq DNA聚合酶预混液25 µL，超纯水18 µL，上下游引物各2.5 µL。在100 V恒电压下，将3 µL PCR产物用质量浓度为0.8%的琼脂糖凝胶电泳40 min，然后用核酸凝胶成像仪进行成像，最后送至奥科(武汉)生物科技有限公司测序。

    2.   结果与分析

      2.1.   叶绿体基因组的组成与特征

    • 4种狼尾草属植物的叶绿体基因组均为典型的四分体结构，长度为138 119~1381 16 bp，GC含量均为38.6%。其中，SSC区较短，为12 384~12 421 bp，IR区为44 576~44 676 bp，LSC区为81 034~81 329 bp，4种植物叶绿体之间各区域长度相差不大，但御谷的编码区是4种植物之间最大，为129 176 bp，其非编码区长度与其他3种植物有较大差异，为最小值9 160 bp(表2)。

      植物 基因组
      长度/bp      		 GC含
      量/%      		 LSC长度/
      bp      		 SSC长度/
      bp      		 IR长度/
      bp      		 蛋白质编码
      数/个      		 RNA
      数/个      		 编码序列
      长度/bp      		 非编码序列
      长度/bp
      白草 138 294 38.6 81 299 12 419 44 576 86 52 126 240 12 054
      御谷 138 336 38.6 81 329 12 421 44 586 85 45 129 176 9 160
      象草 138 199 38.6 81 149 12 384 44 666 88 49 125 881 12 318
      狼尾草 138 119 38.6 81 034 12 409 44 676 84 47 126 031 12 088

      Table 2.  Chloroplast genome characteristics of 4 species of Pennisetum

      • 2.2.   叶绿体基因组的重复序列分析

    • SSR序列分析结果表明(图1A和1B)：4种植物均只存在单核苷酸重复。御谷、白草、狼尾草和象草的SSR位点分别为36、34、34和32个，御谷的SSR位点最多。LSC序列分析结果(图1C和1D)显示：白草中有42个正向重复和32个回文重复；御谷中有44个正向重复和40个回文重复；象草中有48个正向重复和42个回文重复；而在狼尾草中则检测到50个正向重复和34个回文重复。4个物种中只有白草和狼尾草存在反向重复且数量均为6个，均未检测到互补重复。

      Figure 1.  Number and length of short repeats and long repeats of the chloroplast genome sequence in 4 species of Pennisetum

      • 2.3.   密码子偏好性分析

    • 由图2A所示：密码子的适应性指数为0.156~0.160，密码子的偏好指标为0.363~0.368，表明适应性不高，有效的密码子数量为55.77~56.18个，密码子偏好性不高，每种植物的同义密码子使用频率大于1的数量占据少数，如AGA、UCU、GGA、CAA (图2B)。可见4种植物密码子使用偏好性不高，基因组较为保守。

      Figure 2.  Statistics of codon usage bias (A) and relative synonymous codon usage (B) in chloroplast genomes sequence of 4 species of Pennisetum

      • 2.4.   多态性分析

    • 核苷酸多态性分析结果(图3)表明：存在6个明显序列变异的高变区域(Pi＞0.016)，分别为rps16、trnT-GUU、rps4、ndhJ、petA/pbsJ以及ndhF。

      Figure 3.  Nucleotide variability values (Pi) and the number of site discrepancy of the 4 species of Pennisetum

      • 2.5.   叶绿体基因组边界扩张

    • 由图4可明显发现：IR区的收缩数值相差较小。4个物种的LSC-IRA 连接点都位于psbA和rps19基因之间，基因rps19距离LSC-IRA连接点的距离为36~42 bp，而psbA位于LSC-IRA交界上。对于所有所研究的物种，rps15位于IRA-SSC的交界处，而IRA-SSC交界处位于ndhH的边界。

      Figure 4.  Boundary analysis of chloroplast genome in 4 species of Pennisetum

      • 2.6.   共线性分析与系统发育树的构建

    • 共线性分析(图5A)得出狼尾草与白草的基因组相似比最高，达99.94%，象草次之，为99.02%，御谷最低，为98.91%。根据进化树的结果(图5B)可以发现：白草和御谷的亲缘关系最近，而狼尾草和象草关系较近，表明基于叶绿体基因组分析的亲缘关系与基于植物形态学的分类较为一致。

      Figure 5.  Collinearity analysis of 4 species of Pennisetum(A) and the phylogenetic tree of 16 plant species (B)

      • 2.7.   SSR分子标记开发

    • 开发5对引物的扩增效果(图6)表明：5对引物在4种狼尾草属植物中仅有SSR1、SSR3和SSR4可扩增出比较明显的条带，而SSR2在御谷和狼尾草中扩增出清晰条带，SSR5在御谷中扩增出清晰条带。

      Figure 6.  Amplification results of primers in 4 species of Pennisetum

      通过单端测序并对测序结果进行序列比对(图7)，发现3对引物：SSR1、SSR3和SSR4在4种狼尾草属植物中显示出较高的保守性，特别是SSR1，在4种植物中的保守程度最高，并且只有象草存在2个SNP位点，而SSR3和SSR4在4个物种中均存在多个单核苷酸多态性位点(single nucleotide polymorphism, SNP)。

      Figure 7.  Comparative results of SSR sequence in 4 species of Pennisetum

    3.   讨论

    • 本研究结果表明：所选用的4种狼尾草属植物的叶绿体基因组均是典型的四分体结构，其中SSC区为基因变异较多的编码区，而狼尾草属叶绿体基因组的SSC区较短，其叶绿体基因组基因变异的概率较低，表现出较高的保守性，同已有研究表明的叶绿体基因组结构特征保持一致[24 − 25]。较长的IR区可提供更多的重复序列增加叶绿体基因组的稳定性，可以有效减少叶绿体基因组中结构重组的危险[26]。尽管IR区的基因突变率较低，但在大多数被子植物中，IR区的收缩和扩张会造成基因及内含子的丢失，使其突变率变高。这可能是导致狼尾草属植物IR区以及LSC区和SSC区边界位置多样性的原因之一。

      密码子偏好性结果显示：4种狼尾草属植物的叶绿体基因组Enc值均高于55.7，且大部分RSCU值低于1，这说明4种植物密码子使用偏好性不强[27]。此外，4种植物多态性较低，暗示着它们可能在遗传上相对保守，或者受到一定程度的遗传漂变的限制。同时通过共线性分析与系统发育树的构建，得出4种植物基因组相似度较高，这与前期多态性分析结果保持一致。

      SSR标记所获得的多态性条带数量较多，在物种间的遗传多样性研究方面更具优越性，这一点与已有研究结果[28]较为吻合。本研究根据4种植物叶绿体基因组的SSR位点，结合生物信息学手段开发了SSR分子标记。由于基因组中全为单核苷酸重复位点，所以本研究的SSR标记都是基于单核苷酸重复位点开发。其中引物SSR5表现出了极高的区分能力，仅在御谷中特异性表达，引物SSR2在白草和象草中特异性表达，为物种鉴别提供了可靠的分子工具。但是受扩增检测技术的限制，目前还不能精确地判定不同类群间的差别。

    4.   结论

    • 本研究对4种狼尾草属植物叶绿体基因型中的SSR序列进行分析，仅存在单核苷酸重复；非简单序列重复分析检测到正文重复、回文重复和反向重复，均未检测到互补重复。狼尾草属植物不同位置GC含量相差不大，叶绿体基因组密码子使用偏好性不强。多态性分析结果表明狼尾草属植物遗传多态性较低，其基因组可能具有较高的保守性。后续研究叶绿体基因组边界扩张、共线性分析与系统发育树的构建以及SSR分子标记的开发，均佐证了这一结论，且狼尾草属之间的亲缘关系较为密切。

Reference (28)

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