柑橘及其近缘属植物DNA条形码研究
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摘要
DNA条形码技术(DNA Barcoding)是一种利用短的DNA片段对物种进行识别和鉴定的分子生物学技术,它作为一种新兴的分类学研究技术,近年来引起了越来越多的生物学家的关注。DNA条形码技术的原理是根据基因序列的种间遗传变异大于种内遗传变异而实现准确快速的物种鉴定。自2003年加拿大科学家Paul Hebert等提出该技术以来,已在昆虫、鸟类、鱼类等动物及硅藻类和原核生物中成功应用。在动物的DNA条形码研究中,线粒体细胞色素c氧化酶亚基Ⅰ(COI)基因片段已被最成功地用于新种的发现和隐性种的鉴定。然而,COI基因在高等植物中进化速率相对较慢,并不适合作为植物DNA条形码。目前,国内外植物DNA条形码研究还处于寻找合适的基因片段的阶段,许多学者进行了积极的探索,报道了多种植物条形码候选片段或片段组合,但迄今仍没有找到能满足所有标准的DNA条形码特征片段。
柑橘属(Citrus L.)由林奈1753年订立,系芸香科(Rutaceae)、柑橘亚科(Aurantioideae)植物。根据雄蕊数目及汁胞构造的不同,Swingle和Reece(1967)将柑橘属及其近缘的五个属,枳属(Poncirus Raf.)、金柑属(Fortunella Swingle)、澳枳檬属(Microcitrus Swingle)、澳沙檬属(Eremocitrus Swingle)和多蕊橘属(Clymenia Swingle)一起划归为真正柑橘果树类。在过去250余年的时间里,国内外学者对柑桔及其近缘属植物的分类和系统发生问题从形态到分子开展了一系列研究。然而,由于研究方法和使用的证据的不同,不同学者得到了不同的研究结论,造成了柑橘分类在亚属划分和种的数目等关键问题上分歧依然很大。准确地弄清柑橘及其近缘属植物之间的系统进化关系,对有关的物种进行准确、快速的识别和鉴定,对未来柑橘属植物的遗传育种,种质资源的深入研究、保护和利用都具有重要的理论和实践价值。
本论文以真正柑橘果树类6属植物59个生物类型为研究材料,用matK、rpoB、rpoC1、trnG-trnS、psbH-petB、trnL-trnF等六个叶绿体片段和细胞核ITS序列作为候选基因片段,分别分析了这些候选片段在真正柑橘果树类6属间和柑橘属内物种的识别能力,试图为植物界的DNA条形码研究提供新信息。基于遗传距离分析了真正柑橘果树类属间及柑橘属内种间种内变异,讨论了各片段及片段组合作为该类群植物条形码的适应性。结果显示,在遗传距离方面,ITS序列表现出作为该类群植物条形码的潜力。我们还以九里香(Murraya paniculata L.)、蚝壳刺(Severinia buxifolia (Poir.) Tenore)和酒饼簕(Atalantia buxifolia (Poir.) Oliv.)作为外类群,利用PAUP计算机软件分别用MP法、NJ法和UPGMA法,使用七个单片段和三个组合(ITS+matK、matK+trnG-trnS+psbH-petB、matK+rpoB+rpoC1)分别构建了系统进化树,讨论了候选片段鉴定属的能力；以枳(P. trifoliata(L.)Raf.)、富民枳(P. polyandra S. Q. Ding, X. N. Zhang, Z. R.Bao & M. Q. Liang)和飞龙枳(P.trifoliata var. monstrosa (T. Ito) Swing.)为外类群,讨论各候选片段鉴定柑橘属内种的能力。结果显示,所有候选条码的属和属内种的物种鉴定率都较低,不能完成对该类群植物的准确鉴定。最后,我们重点讨论了ITS、matK和片段组合等作为柑橘及其近缘植物条形码的可能性。
DNA barcoding is a technique for identifying and characterizing species of organisms using a short DNA region. It is becoming more and more attractive to biologists as a new method to aid in species identification. DNA barcoding can identify species rapidly and accurately based on the principle of inter-specific divergence is greater than intra-specific divergence. Since 2003, Paul Hebert have suggested that we should choose one standard gene fragment as new "taxonomy character" to help traditional taxonomy, the mitochondrial cytochrome c oxidase subunit 1 (COI or coxl) sequence as DNA barcode has been successfully employed for species identification and revealing cryptic species or new species in diverse groups of animals such as insects, fishes, birds, diatoms and prokaryotes. However, COI gene is not suitable candidate for plant DNA barcoding because of its slow substitution rate in higher plant. Plant DNA barcode technique is still in the stage of searching for a suitable DNA region. Although different candidate single region or combination of regions from chloroplast genome were reported in the existing literature. we still lack an obvious well-characterized plant locus that meets all the necessary criteria of a DNA barcoding region.
The genus Citrus L. established in 1753 by Carl Linnaeus belongs to the subfamily Aurantioideae of the family Rutaceae. Swingle and Reece (1967) classified the genus Citrus and its five close relatives, Poncirus, Fortunella, Microcitrus, Eremocitrus and Clymenia into the true citrus fruit trees group based mainly on the number of stamen and structure of pulp-vesicles of the genera. A series of study from morphology to molecular markers on the taxonomy and evolution of the genus Citrus and its close relatives has been carried out both in China and abroad in the past more than 250 years. The taxonomic and evolutionary relationships between Citrus and its close relatives have remained unresolved. A better understanding of the phylogenetic relationship between Citrus and its closely related genera is needed for future variety breeding, germplasm conservation and utilization of the genus Citrus.
In the present study, six candidate plant barcoding regions of chloroplast genome、rpoC1、trnG-trnS、psbH-petB and trnL-trnF, and nuclear ITS region of 59 biological types collected from the six genera of the true citrus fruit trees group were tested in an attempt to develop DNA barcoding makers for species identification and taxonomy of the genus Citrus and its closely related genera. To test the potential of the candidate makers being used as DNA barcoding of the true citrus fruit trees group, firstly, the inter-generic divergence of all genotypes and inter- and intra-specific divergence of Citrus L. were analyzed in present study based on genetic distances. The results of this study showed that none of the candidate markers proposed in the exsiting literature provided unique identifiers for all the species tested. The results of analysis of genetic distances show that ITS sequences have the potential as DNA barcoding of Citrus. And then, the maximum parsimony (MP), neighbor-joining (NJ) and unpaired group mean averages algorithm (UPGMA) trees of all genotypes were constructed with PAUP* version 4.10b software by using the DNA sequences obtained and Murraya paniculata (L.) Jack., Atalantia buxifolia Correa., and Severinia buxifolia (Poir.) Tenore as outgroups. In addition, the MP, NJ and UPGMA trees of 47 accessions of Citrus L. were constructed with PAUP* version 4.10b software by usingon the Citrus DNA sequences and P. trifoliata (L.) Raf., P. polyandra S. Q. Ding, X. N. Zhang, Z. R.Bao & M. Q. Liang, P. trifoliata var. monstrosa (T. Ito) Swingle as outgroups. Finally, the potential of using ITS, matK, or a combination of regions of cpDNA as the true citrus fruit trees group DNA barcoding was discussed in details.
柑橘属(Citrus L.)由林奈1753年订立,系芸香科(Rutaceae)、柑橘亚科(Aurantioideae)植物。根据雄蕊数目及汁胞构造的不同,Swingle和Reece(1967)将柑橘属及其近缘的五个属,枳属(Poncirus Raf.)、金柑属(Fortunella Swingle)、澳枳檬属(Microcitrus Swingle)、澳沙檬属(Eremocitrus Swingle)和多蕊橘属(Clymenia Swingle)一起划归为真正柑橘果树类。在过去250余年的时间里,国内外学者对柑桔及其近缘属植物的分类和系统发生问题从形态到分子开展了一系列研究。然而,由于研究方法和使用的证据的不同,不同学者得到了不同的研究结论,造成了柑橘分类在亚属划分和种的数目等关键问题上分歧依然很大。准确地弄清柑橘及其近缘属植物之间的系统进化关系,对有关的物种进行准确、快速的识别和鉴定,对未来柑橘属植物的遗传育种,种质资源的深入研究、保护和利用都具有重要的理论和实践价值。
本论文以真正柑橘果树类6属植物59个生物类型为研究材料,用matK、rpoB、rpoC1、trnG-trnS、psbH-petB、trnL-trnF等六个叶绿体片段和细胞核ITS序列作为候选基因片段,分别分析了这些候选片段在真正柑橘果树类6属间和柑橘属内物种的识别能力,试图为植物界的DNA条形码研究提供新信息。基于遗传距离分析了真正柑橘果树类属间及柑橘属内种间种内变异,讨论了各片段及片段组合作为该类群植物条形码的适应性。结果显示,在遗传距离方面,ITS序列表现出作为该类群植物条形码的潜力。我们还以九里香(Murraya paniculata L.)、蚝壳刺(Severinia buxifolia (Poir.) Tenore)和酒饼簕(Atalantia buxifolia (Poir.) Oliv.)作为外类群,利用PAUP计算机软件分别用MP法、NJ法和UPGMA法,使用七个单片段和三个组合(ITS+matK、matK+trnG-trnS+psbH-petB、matK+rpoB+rpoC1)分别构建了系统进化树,讨论了候选片段鉴定属的能力；以枳(P. trifoliata(L.)Raf.)、富民枳(P. polyandra S. Q. Ding, X. N. Zhang, Z. R.Bao & M. Q. Liang)和飞龙枳(P.trifoliata var. monstrosa (T. Ito) Swing.)为外类群,讨论各候选片段鉴定柑橘属内种的能力。结果显示,所有候选条码的属和属内种的物种鉴定率都较低,不能完成对该类群植物的准确鉴定。最后,我们重点讨论了ITS、matK和片段组合等作为柑橘及其近缘植物条形码的可能性。
DNA barcoding is a technique for identifying and characterizing species of organisms using a short DNA region. It is becoming more and more attractive to biologists as a new method to aid in species identification. DNA barcoding can identify species rapidly and accurately based on the principle of inter-specific divergence is greater than intra-specific divergence. Since 2003, Paul Hebert have suggested that we should choose one standard gene fragment as new "taxonomy character" to help traditional taxonomy, the mitochondrial cytochrome c oxidase subunit 1 (COI or coxl) sequence as DNA barcode has been successfully employed for species identification and revealing cryptic species or new species in diverse groups of animals such as insects, fishes, birds, diatoms and prokaryotes. However, COI gene is not suitable candidate for plant DNA barcoding because of its slow substitution rate in higher plant. Plant DNA barcode technique is still in the stage of searching for a suitable DNA region. Although different candidate single region or combination of regions from chloroplast genome were reported in the existing literature. we still lack an obvious well-characterized plant locus that meets all the necessary criteria of a DNA barcoding region.
The genus Citrus L. established in 1753 by Carl Linnaeus belongs to the subfamily Aurantioideae of the family Rutaceae. Swingle and Reece (1967) classified the genus Citrus and its five close relatives, Poncirus, Fortunella, Microcitrus, Eremocitrus and Clymenia into the true citrus fruit trees group based mainly on the number of stamen and structure of pulp-vesicles of the genera. A series of study from morphology to molecular markers on the taxonomy and evolution of the genus Citrus and its close relatives has been carried out both in China and abroad in the past more than 250 years. The taxonomic and evolutionary relationships between Citrus and its close relatives have remained unresolved. A better understanding of the phylogenetic relationship between Citrus and its closely related genera is needed for future variety breeding, germplasm conservation and utilization of the genus Citrus.
In the present study, six candidate plant barcoding regions of chloroplast genome、rpoC1、trnG-trnS、psbH-petB and trnL-trnF, and nuclear ITS region of 59 biological types collected from the six genera of the true citrus fruit trees group were tested in an attempt to develop DNA barcoding makers for species identification and taxonomy of the genus Citrus and its closely related genera. To test the potential of the candidate makers being used as DNA barcoding of the true citrus fruit trees group, firstly, the inter-generic divergence of all genotypes and inter- and intra-specific divergence of Citrus L. were analyzed in present study based on genetic distances. The results of this study showed that none of the candidate markers proposed in the exsiting literature provided unique identifiers for all the species tested. The results of analysis of genetic distances show that ITS sequences have the potential as DNA barcoding of Citrus. And then, the maximum parsimony (MP), neighbor-joining (NJ) and unpaired group mean averages algorithm (UPGMA) trees of all genotypes were constructed with PAUP* version 4.10b software by using the DNA sequences obtained and Murraya paniculata (L.) Jack., Atalantia buxifolia Correa., and Severinia buxifolia (Poir.) Tenore as outgroups. In addition, the MP, NJ and UPGMA trees of 47 accessions of Citrus L. were constructed with PAUP* version 4.10b software by usingon the Citrus DNA sequences and P. trifoliata (L.) Raf., P. polyandra S. Q. Ding, X. N. Zhang, Z. R.Bao & M. Q. Liang, P. trifoliata var. monstrosa (T. Ito) Swingle as outgroups. Finally, the potential of using ITS, matK, or a combination of regions of cpDNA as the true citrus fruit trees group DNA barcoding was discussed in details.
引文
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