柑橘及其近缘属植物DNA条形码研制及其物种的鉴定研究
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摘要
中国是世界柑橘最重要的起源地之一,具有丰富的柑橘资源。柑橘属于芸香科(Rutaceae)柑橘亚科(Aurantioideae)植物。自1753年林奈创立柑橘属(Citrus L.)以来,国内外学者从形态学、细胞学到分子水平进行了一系列的研究,先后建立了多个柑橘属分类系统。其中,最著名的是美国的Swingle系统,包含16个种；日本的Tanaka系统,包含159个种和3个变种；我国的曾勉系统,包含30个种。由于柑橘属植物具有种间易杂交、无融合生殖及无性变异等特点,柑橘属植物物种的数目、种间亲缘关系和分类问题是迄今尚未解决的科学难题。
DNA条形码(DNA barcode)概念由加拿大动物学家Paul Hebert于2003年提出,是一种基于DNA分子进化原理,利用短的DNA片段和现代分子系统学的原理和方法对“传统物种”在分子水平进行身份鉴定的最新物种生物学技术。该技术与传统的物种鉴定方法相比,具有准确性高、效率高、不受被鉴定对象的环境、个体发育和鉴定专家个人因素影响等优点。DNA条形码技术自诞生以来,国内外学者运用线粒体细胞色素C氧化酶I(cytochrome c oxidase subunitⅠ, COⅠ)基因作为条形编码序列,对昆虫、鸟类、鱼类等动物进行物种鉴定和新种及隐存种的发现,取得很大的成功。但COⅠ基因在植物中的进化速率较慢,不适于植物条形码的研究。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。
本研究选用真正柑橘果树类植物6属59个生物类型为实验材料,采用叶绿体编码基因(matK、rpoB、rpoC1、rbcL)、叶绿体间隔序列(trnH-psbA、trnG-trnS、psbH-petB、trnL-trnF)和核基因内转录间隔序列(ITS)及其间隔区(ITS1、ITS2)共9个DNA序列作为条形码候选序列,使用MEGA5.05软件计算序列的碱基组成、序列间的碱基变异频率和序列间的转换颠换频率及其比率。使用SPSS17.0软件进行wilcoxon检验比较分析不同编码序列之间的差异。通过比较序列种内和种间差异的分布,运用单一片段和组合片段分别对柑橘属Swingle系统植物、Tanaka系统植物和柑橘及其近缘属6属植物进行鉴定,比较了各单一序列及片段组合鉴定率的大小。有关研究结果如下：
1.在单一序列对柑橘属Swingle系统、Tanaka系统28份材料鉴定中,以富民枳、飞龙枳、枳为外类群构建NJ距离树,比较各单一序列的鉴定率。结果表明：核基因内转录间隔序列(ITS)及其间隔区(ITS1、ITS2)序列的平均鉴定效率最高,叶绿体间隔序列(trnH-psbA、trnG-trnS、psbH-petB、trnL-trnF)次之,叶绿体编码基因(matK、rpoB、rpoC1、rbcL)的鉴定率最低。在Swingle系统中,ITS1鉴定能力最强为100%,psbH-petB、ITS序列鉴定能力次之,鉴定率均为93.8%,rpoC1序列鉴定率最低为12.5%。在Tanaka系统28份材料中psbH-petB、ITS鉴定率分别为100%,rpoC1序列最低为14.8%。
2.以九里香、酒饼筋和蚝壳刺为外类群,对柑橘及其近缘属植物6属进行鉴定,构建NJ距离树,比较各单一片段的鉴定率。结果表明：ITS的鉴定率最高,达到59.3%,psbH-petB次之为55.9%,matK为50.8%,其余片段均为50%以下。综合1、2结果,核基因ITS、叶绿体间隔序列psbH-petB、叶绿体编码基因matK在相同类型的DNA序列中鉴定率最高,是柑橘及其近缘属植物DNA条形码研究的重要片段。
3.结合第三届国际生物条形码的提议及前人研究组合方案和单一片段的鉴定结果,选用matK+rbcL、matK+trnH-psbA、matK+ITS、trnH-psbA+ITS、trnG-trnS+ITS2、matK+ITS2、matK+psbH-petB+trnG-trnS、matK+ITS+trnG-trnS、matK+rpoB+rpoC1片段组合为研究对象,以富民枳、飞龙枳、枳为为外类群对柑橘属Swingle、Tanaka系统28份植物构建NJ距离树,比较各组合片段的鉴定率。结果表明：在Swingle系统植物鉴定中,trnH-psbA+ITS的鉴定率最高为100%, matK+trnH-psbA、matK+ITS和trnG-trnS+ITS2次之为93.8%,matK+rpoB+rpoC1的鉴定率最低为81.3%。在Tanaka系统28份植物鉴定中,matK+ITS、trnG-trnS+ITS2及matK+ITS+trnG-trnS三者的鉴定率最高为96.3%,trnH-psbA+ITS、matK+trnH-psbA的鉴定能力均下降,分别为74.1%、51.9%。所以在利用组合片段对柑橘属Swingle、Tanaka系统28份植物鉴定中matK+ITS、trnG-trnS+ITS2的鉴定率最高。
4.以九里香、酒饼簕和蚝壳刺为外类群,利用组合片段对柑橘及其近缘属植物鉴定,构建NJ距离树,比较各片段组合的鉴定率。结果表明：matK+ITS+trnG-trnS组合鉴定率最高为83.1%, matK+ITS次之为81.4%。两者鉴定率相差不大,但后者少了trnG-trnS片段,序列长度更短,鉴定效率更高。
综上所述,在单一序列鉴定柑橘属Swingle系统、Tanaka系统28份材料和柑橘及其近缘属植物中,psbH-petB、ITS具有最高的鉴定率,并且psbH-petB、ITS、matK序列在相同类别的DNA序列中鉴定率最高,三者是柑橘条形码研究的重要片段；组合片段与单一片段相比,鉴定率均有较大幅度提高,matK+ITS与其它组合片段相比,具有较高的鉴定率、较短的序列长度等特点,可用于柑橘及其近缘属植物DNA条形码研究。DNA条形码技术作为一种新型的物种鉴定手段,可用于柑橘及其近缘属植物的鉴定。
China is one of the most important origin centers of the genus Citrus and is rich in citrus germplasm resources in the world. The genus Citrus belongs to the subfamily Aurantioideae of Rutaceae family. The genus Citrus L. was established by Carl Linnaeus in 1753. Since then, scholars both in China and abroad have carried out a series of studies on the taxonomy of Citrus using morphology, cytology, and molecular data. In the current literature several classification systems for the genus Citrus have been suggested, of which the well-recognized are Swingle's 16 species system and Tanaka's 159 species system. In addition, Chinese scholar Tseng has also suggested a system composed of 30 species. Up to now, however, because of hybridization, apomixes and asexual variation, the number of species, inter-specific relationship and classification of the genus Citrus L. are still unresolved scientific questions.
DNA barcoding is a technique for identifying and characterizing species of organisms using a short DNA region, which was proposed by Canadian zoologist Paul Hebert in 2003. DNA barcodes compared with the traditional morphological data can rapidly identify species based on DNA molecular evolution principles, and have many advantages in species identification, such as high accuracy, high efficiency, and not influenced by environmental factors and development stage of materials. Since the birth of DNA barcoding technology, the mitochondrial cytochrome c oxidase subunitⅠ(COⅠ) fragment as the barcoding sequence has been successfully used to identify species, find new species and cryptic species in insects, birds, fish and other animals. Unfortunately, CO I gene has a very slow evolutionary rate in plants, which makes it unsuitable for DNA barcoding in plants. Until now, plant barcoding is still at the stage of searching for a suitable locus and kinds of candidates. Many researchers propose a single barcoding region or combinations of regions from the chloroplast genome. At present, a well-characterized plant locus that meets all necessary criteria is lacking.
In the present study, the 59 biological types of 6 genera belonged to the true citrus fruit plants were selected as the experimental materials. The chloroplast encoded genes (matK, rpoB, rpoC1, rbcL) and chloroplast intergenic sequences (trnH-psbA, trnG-trnS, psbH-petB, trnL-trnF), as well as nuclear ITS and its first and second space sequence (ITS 1, ITS2) were analysed as the candidate plant barcoding regions. The MEGA 5.05 software was used to calculate the basic composition, the frequency of DNA mutation and the ratio of transition/transversion. Additionally, SPSS17.0 software was used to analyse the sequence divergence through wilcoxon test. In our study, the distribution of intra-specific and inter-specific genetic distance was compared to identify the species of Citrus Swingle, Tanaka systems and the species of the Citrus and its related genera using the single fragment and the combined fragments respectively. The results of this study are as follows:
1. Using Poncirus polyandra, P.trifoliata var. monstrosa, P.trifoliata as outgroups, the NJ trees of the Swingle system and Tanaka system with a single fragment were constructed to compare the identification rate. The results showed that the average identification ratio of nuclear ITS and its spacer sequence (ITS1, ITS2) was the highest, followed by the chloroplast intergenic sequence (trhH-psbA, trnG-trnS, psbH-petB, trnL-trnF), and the chloroplast encoded genes (matK, rpoB, rpoC1, rbcL) were third. In Swingle system, the strongest capability of identification was ITS 1 with a ratio of 100%; psbH-petB and ITS were second, and the rate was 93.8%; the rpoC1 was lowest, only 12.5%. In Tanaka system, the identification rate of psbH-petB and ITS were 100%, and the rate of rpoC1 was 14.8%.
2. Using Murraya paniculata, Atalantia buxifolia, and Severinia buxifolia as outgroups, the NJ trees of the Citrus and its related genera with each fragments were constructed to compare the identification rate. The results showed that the identification rates of ITS, psbH-petB and matK were 59.3%,55.9% and 50.8% respectively, and the remaining fragments were below 50%. ITS was the highest, rpoC1 was lowest with only 23.7%. So because of the highest identification rate in their alike type, the nuclear gene ITS, chloroplast intergenic sequence psbH-petB, chloroplast encode gene matK were the important fragments in the DNA barcode of the Citrus and its related genera.
3. Following the proposal of the third international barcode conference and the combined schemes of the predecessors, as well as the the identification results of the single fragment, matK+rbcL, matK+trnH-psbA, matK+ITS, trnH-psbA+ITS, trnG-trnS+ITS2, matK+ITS2, matK+psbH-petB+trnG-trnS, matK+ITS+trnG-trnS, matK+rpoB+rpoC1 fragments were selected together as the objects in our research. Using Poncirus polyandra, P.trifoliata var. monstrosa, P.trifoliata as outgroups, the phylogenies NJ trees of the Swingle system and Tanaka system with the combined fragments were constructed to compare the identification rate. The results showed that, in Swingle system, the rate of trnH-psbA+ITS was highest, reached 100%. The matK+trnH-psbA、matK+ITS and trnG-trnS+ITS2 were second with 93.8%, and matK+rpoB+rpoC1 was lowest with 81.3%. In Tanaka system, the rate of matK+ITS、trnG-trnS+ITS2 and matK+ITS+trnG-trnS were highest, reached 96.3%. However, the rate of trnH-psbA+ITS and matK+trnH-psbA was 74.1% and 51.9% respectively. So the rate of matK+ITS and trnG-trnS+ITS2 were higher than the other combined fragments to identify the speciese of Swingle system and Tanaka system.
4. Using Murraya paniculata, Atalantia buxifolia, and Severinia buxifolia as outgroups, the NJ trees of 59 species of the Citrus and its related genera with combined fragments were constructed to compare the identification rate. The results showed that:the rate of matK+ITS+trnG-trnS was the highest, reached 83.1%, and matK+ITS was second with 81.4%. There was little difference of the identification rate between matK+ITS and marK+ITS+trnG-trnS, but there wasn't a fragment of trnG-trnS in matK+ITS, which made it more efficient in the identification because of its shorter sequence length.
In conclusion, in the identification of Swingle and Tanaka system of the Citrus and its related genera, ITS、psbH-petB as single fragment had the highest identification rate, and ITS、psbH-petB、matK were important fragments in the research of Citrus DNA barcode, which had a higher identification rate in the same category. Compared the combined fragments with the single fragments, the identification rate of combined fragments were greatly improved. Compared matK+ITS with other combined fragments, matK+ITS was more suitable for the research of Citrus and its related genera of plant, which had the characteristics of high identification rate and shorter sequence length. From what we have studied above, we can draw a conclusion that the DNA Barcode technique can be used to identify the species of Citrus and its related genera as a new species-identification method.
DNA条形码(DNA barcode)概念由加拿大动物学家Paul Hebert于2003年提出,是一种基于DNA分子进化原理,利用短的DNA片段和现代分子系统学的原理和方法对“传统物种”在分子水平进行身份鉴定的最新物种生物学技术。该技术与传统的物种鉴定方法相比,具有准确性高、效率高、不受被鉴定对象的环境、个体发育和鉴定专家个人因素影响等优点。DNA条形码技术自诞生以来,国内外学者运用线粒体细胞色素C氧化酶I(cytochrome c oxidase subunitⅠ, COⅠ)基因作为条形编码序列,对昆虫、鸟类、鱼类等动物进行物种鉴定和新种及隐存种的发现,取得很大的成功。但COⅠ基因在植物中的进化速率较慢,不适于植物条形码的研究。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。
本研究选用真正柑橘果树类植物6属59个生物类型为实验材料,采用叶绿体编码基因(matK、rpoB、rpoC1、rbcL)、叶绿体间隔序列(trnH-psbA、trnG-trnS、psbH-petB、trnL-trnF)和核基因内转录间隔序列(ITS)及其间隔区(ITS1、ITS2)共9个DNA序列作为条形码候选序列,使用MEGA5.05软件计算序列的碱基组成、序列间的碱基变异频率和序列间的转换颠换频率及其比率。使用SPSS17.0软件进行wilcoxon检验比较分析不同编码序列之间的差异。通过比较序列种内和种间差异的分布,运用单一片段和组合片段分别对柑橘属Swingle系统植物、Tanaka系统植物和柑橘及其近缘属6属植物进行鉴定,比较了各单一序列及片段组合鉴定率的大小。有关研究结果如下：
1.在单一序列对柑橘属Swingle系统、Tanaka系统28份材料鉴定中,以富民枳、飞龙枳、枳为外类群构建NJ距离树,比较各单一序列的鉴定率。结果表明：核基因内转录间隔序列(ITS)及其间隔区(ITS1、ITS2)序列的平均鉴定效率最高,叶绿体间隔序列(trnH-psbA、trnG-trnS、psbH-petB、trnL-trnF)次之,叶绿体编码基因(matK、rpoB、rpoC1、rbcL)的鉴定率最低。在Swingle系统中,ITS1鉴定能力最强为100%,psbH-petB、ITS序列鉴定能力次之,鉴定率均为93.8%,rpoC1序列鉴定率最低为12.5%。在Tanaka系统28份材料中psbH-petB、ITS鉴定率分别为100%,rpoC1序列最低为14.8%。
2.以九里香、酒饼筋和蚝壳刺为外类群,对柑橘及其近缘属植物6属进行鉴定,构建NJ距离树,比较各单一片段的鉴定率。结果表明：ITS的鉴定率最高,达到59.3%,psbH-petB次之为55.9%,matK为50.8%,其余片段均为50%以下。综合1、2结果,核基因ITS、叶绿体间隔序列psbH-petB、叶绿体编码基因matK在相同类型的DNA序列中鉴定率最高,是柑橘及其近缘属植物DNA条形码研究的重要片段。
3.结合第三届国际生物条形码的提议及前人研究组合方案和单一片段的鉴定结果,选用matK+rbcL、matK+trnH-psbA、matK+ITS、trnH-psbA+ITS、trnG-trnS+ITS2、matK+ITS2、matK+psbH-petB+trnG-trnS、matK+ITS+trnG-trnS、matK+rpoB+rpoC1片段组合为研究对象,以富民枳、飞龙枳、枳为为外类群对柑橘属Swingle、Tanaka系统28份植物构建NJ距离树,比较各组合片段的鉴定率。结果表明：在Swingle系统植物鉴定中,trnH-psbA+ITS的鉴定率最高为100%, matK+trnH-psbA、matK+ITS和trnG-trnS+ITS2次之为93.8%,matK+rpoB+rpoC1的鉴定率最低为81.3%。在Tanaka系统28份植物鉴定中,matK+ITS、trnG-trnS+ITS2及matK+ITS+trnG-trnS三者的鉴定率最高为96.3%,trnH-psbA+ITS、matK+trnH-psbA的鉴定能力均下降,分别为74.1%、51.9%。所以在利用组合片段对柑橘属Swingle、Tanaka系统28份植物鉴定中matK+ITS、trnG-trnS+ITS2的鉴定率最高。
4.以九里香、酒饼簕和蚝壳刺为外类群,利用组合片段对柑橘及其近缘属植物鉴定,构建NJ距离树,比较各片段组合的鉴定率。结果表明：matK+ITS+trnG-trnS组合鉴定率最高为83.1%, matK+ITS次之为81.4%。两者鉴定率相差不大,但后者少了trnG-trnS片段,序列长度更短,鉴定效率更高。
综上所述,在单一序列鉴定柑橘属Swingle系统、Tanaka系统28份材料和柑橘及其近缘属植物中,psbH-petB、ITS具有最高的鉴定率,并且psbH-petB、ITS、matK序列在相同类别的DNA序列中鉴定率最高,三者是柑橘条形码研究的重要片段；组合片段与单一片段相比,鉴定率均有较大幅度提高,matK+ITS与其它组合片段相比,具有较高的鉴定率、较短的序列长度等特点,可用于柑橘及其近缘属植物DNA条形码研究。DNA条形码技术作为一种新型的物种鉴定手段,可用于柑橘及其近缘属植物的鉴定。
China is one of the most important origin centers of the genus Citrus and is rich in citrus germplasm resources in the world. The genus Citrus belongs to the subfamily Aurantioideae of Rutaceae family. The genus Citrus L. was established by Carl Linnaeus in 1753. Since then, scholars both in China and abroad have carried out a series of studies on the taxonomy of Citrus using morphology, cytology, and molecular data. In the current literature several classification systems for the genus Citrus have been suggested, of which the well-recognized are Swingle's 16 species system and Tanaka's 159 species system. In addition, Chinese scholar Tseng has also suggested a system composed of 30 species. Up to now, however, because of hybridization, apomixes and asexual variation, the number of species, inter-specific relationship and classification of the genus Citrus L. are still unresolved scientific questions.
DNA barcoding is a technique for identifying and characterizing species of organisms using a short DNA region, which was proposed by Canadian zoologist Paul Hebert in 2003. DNA barcodes compared with the traditional morphological data can rapidly identify species based on DNA molecular evolution principles, and have many advantages in species identification, such as high accuracy, high efficiency, and not influenced by environmental factors and development stage of materials. Since the birth of DNA barcoding technology, the mitochondrial cytochrome c oxidase subunitⅠ(COⅠ) fragment as the barcoding sequence has been successfully used to identify species, find new species and cryptic species in insects, birds, fish and other animals. Unfortunately, CO I gene has a very slow evolutionary rate in plants, which makes it unsuitable for DNA barcoding in plants. Until now, plant barcoding is still at the stage of searching for a suitable locus and kinds of candidates. Many researchers propose a single barcoding region or combinations of regions from the chloroplast genome. At present, a well-characterized plant locus that meets all necessary criteria is lacking.
In the present study, the 59 biological types of 6 genera belonged to the true citrus fruit plants were selected as the experimental materials. The chloroplast encoded genes (matK, rpoB, rpoC1, rbcL) and chloroplast intergenic sequences (trnH-psbA, trnG-trnS, psbH-petB, trnL-trnF), as well as nuclear ITS and its first and second space sequence (ITS 1, ITS2) were analysed as the candidate plant barcoding regions. The MEGA 5.05 software was used to calculate the basic composition, the frequency of DNA mutation and the ratio of transition/transversion. Additionally, SPSS17.0 software was used to analyse the sequence divergence through wilcoxon test. In our study, the distribution of intra-specific and inter-specific genetic distance was compared to identify the species of Citrus Swingle, Tanaka systems and the species of the Citrus and its related genera using the single fragment and the combined fragments respectively. The results of this study are as follows:
1. Using Poncirus polyandra, P.trifoliata var. monstrosa, P.trifoliata as outgroups, the NJ trees of the Swingle system and Tanaka system with a single fragment were constructed to compare the identification rate. The results showed that the average identification ratio of nuclear ITS and its spacer sequence (ITS1, ITS2) was the highest, followed by the chloroplast intergenic sequence (trhH-psbA, trnG-trnS, psbH-petB, trnL-trnF), and the chloroplast encoded genes (matK, rpoB, rpoC1, rbcL) were third. In Swingle system, the strongest capability of identification was ITS 1 with a ratio of 100%; psbH-petB and ITS were second, and the rate was 93.8%; the rpoC1 was lowest, only 12.5%. In Tanaka system, the identification rate of psbH-petB and ITS were 100%, and the rate of rpoC1 was 14.8%.
2. Using Murraya paniculata, Atalantia buxifolia, and Severinia buxifolia as outgroups, the NJ trees of the Citrus and its related genera with each fragments were constructed to compare the identification rate. The results showed that the identification rates of ITS, psbH-petB and matK were 59.3%,55.9% and 50.8% respectively, and the remaining fragments were below 50%. ITS was the highest, rpoC1 was lowest with only 23.7%. So because of the highest identification rate in their alike type, the nuclear gene ITS, chloroplast intergenic sequence psbH-petB, chloroplast encode gene matK were the important fragments in the DNA barcode of the Citrus and its related genera.
3. Following the proposal of the third international barcode conference and the combined schemes of the predecessors, as well as the the identification results of the single fragment, matK+rbcL, matK+trnH-psbA, matK+ITS, trnH-psbA+ITS, trnG-trnS+ITS2, matK+ITS2, matK+psbH-petB+trnG-trnS, matK+ITS+trnG-trnS, matK+rpoB+rpoC1 fragments were selected together as the objects in our research. Using Poncirus polyandra, P.trifoliata var. monstrosa, P.trifoliata as outgroups, the phylogenies NJ trees of the Swingle system and Tanaka system with the combined fragments were constructed to compare the identification rate. The results showed that, in Swingle system, the rate of trnH-psbA+ITS was highest, reached 100%. The matK+trnH-psbA、matK+ITS and trnG-trnS+ITS2 were second with 93.8%, and matK+rpoB+rpoC1 was lowest with 81.3%. In Tanaka system, the rate of matK+ITS、trnG-trnS+ITS2 and matK+ITS+trnG-trnS were highest, reached 96.3%. However, the rate of trnH-psbA+ITS and matK+trnH-psbA was 74.1% and 51.9% respectively. So the rate of matK+ITS and trnG-trnS+ITS2 were higher than the other combined fragments to identify the speciese of Swingle system and Tanaka system.
4. Using Murraya paniculata, Atalantia buxifolia, and Severinia buxifolia as outgroups, the NJ trees of 59 species of the Citrus and its related genera with combined fragments were constructed to compare the identification rate. The results showed that:the rate of matK+ITS+trnG-trnS was the highest, reached 83.1%, and matK+ITS was second with 81.4%. There was little difference of the identification rate between matK+ITS and marK+ITS+trnG-trnS, but there wasn't a fragment of trnG-trnS in matK+ITS, which made it more efficient in the identification because of its shorter sequence length.
In conclusion, in the identification of Swingle and Tanaka system of the Citrus and its related genera, ITS、psbH-petB as single fragment had the highest identification rate, and ITS、psbH-petB、matK were important fragments in the research of Citrus DNA barcode, which had a higher identification rate in the same category. Compared the combined fragments with the single fragments, the identification rate of combined fragments were greatly improved. Compared matK+ITS with other combined fragments, matK+ITS was more suitable for the research of Citrus and its related genera of plant, which had the characteristics of high identification rate and shorter sequence length. From what we have studied above, we can draw a conclusion that the DNA Barcode technique can be used to identify the species of Citrus and its related genera as a new species-identification method.
引文
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