建兰亚属植物种质资源分子分类及亲缘关系研究
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摘要
为了加快兰花的育种步伐,培育出优质的兰花品种。本文对建兰亚属兰花种质资源的形态及叶绿素含量进行比较分析,并利用RAPD、ERPAD、PCR-RFLP、ISSR、SRAP分子标记技术对建兰亚属植物资源进行分子分类及亲缘关系研究。
主要研究结果如下
1.对建兰亚属21个兰花品种的形态特征的观察和分析可以看出,除叶脉序特征均为平行脉序(弧形脉),叶片均为纸质外,不同品种间的一级与二级脉随品种也不相同,所有品种在形态上具一定的分化,表明其形态特征反映出不同品种间的相似性和差异性,从而为属下种间的界限和关系的探讨提供了重要的形态学证据。
2.应用SPAD-502叶绿素计和常规方法对21个建兰亚属兰花品种的叶绿素含量进行了比较分析。结果表明：大多数兰花在种间或种内的叶绿素a、叶绿素b、总叶绿素含量以及SPAD值均有所不同,但他们之间表现出优异的差异性；7个兰花种间的叶绿素含量与其SPAD值的相关性均达到了显著水平,表明利用SPAD值来衡量其叶绿素含量的可行性；通过聚类分析可以把21个兰花品种分为2大类群。
3.应用ERPAD(延长随机引物扩增DNA)标记对54个寒兰品种进行分析和鉴定。结果表明：8个多态性强引物的1个碱基延长,共扩增出2959条带,其中2527条带(85.40%)具有多态性,延长2个碱基引物ACTGAACGCCCG+ACTGAACGCCGG能获得一条2.5Kb RAPD特异性片段,此片段在RAPD引物(ACTGAACGC)和延长一个碱基的(ACTGAACGCC+ ACTGAACGCCC)引物具有相同的扩增片段。在无需克隆和测序的条件下,这种标记的稳定性和特异性均接近SCAR标记。通过聚类分析可以把54个寒兰品种分为2大类群。
4.应用PCR-RFLP标记对54个寒兰品种的叶绿体和线粒体基因组进行分析和鉴定。结果表明：在6个叶绿体基因组(cpDNA)的PCR-RFLP分析中,对4个(66.67%)可扩增出条带引物进行12种限制性内切酶消化,有19种(26.38%)引物酶组合能检测到116多态性带纹。在6个线粒体基因组中(mtDNA)的PCR-RFLP分析中,利用12种限制性内切酶对能扩增出条带的2种引物进行消化后,只有一种引物(Cox1)能扩增出55(53.49%)多态性带纹。通过聚类分析可以把54个寒兰品种分为4大类群。
5.应用ISSR标记对54个寒兰品种进行分析和鉴定。结果表明：8个ISSR引物共扩增出893条带,每对引物扩增条带数为2-6条,平均为3.22。其中有224个多态性条带,占总带数的24.72%,平均多态性条带数为2.73。扩增带纹片段多在200bp-2000bp之间。通过聚类分析可以把21个兰花品种分为6大类群。
6.应用SRAP标记对51个寒兰品种的基因组DNA进行分析。结果表明筛选出的11对SRAP引物组合对共扩增出586条带纹,其中504条为多态性(86.01%),平均每个引物扩增46条多态性带。聚类分析表明把51份材料根据起源和生物学特性划分为中国寒兰和日本寒兰两大类群。
综上所述,通过形态学标记、生理学标记以及分子标记对部分兰花进行比较分析,并探讨其亲缘关系,旨在为兰花品种的进一步研究和利用提供科学基础。
In order to speed up the pace of orchid breeding and to improve the high-quality orchids, the morphology and chlorophyll contents of Cymbidium kanran resources in Subgen Jensoa were compared and analysis, the molecular taxonomy and relative relationships research of Cymbidium kanran resources in Subgen Jensoa were studies by RAPD, ERPAD, PCR-RFLP, ISSR and SRAP molecular markers.
The study results were as the followings:
1. The venational morphology characters of 21 orchid's varieties were be observed and analyzed, which revealed that different first and secondary vein have different leaf venation characterization except parallel venation, and which could be provided the morphological evidence for their boundaries and relationships between species.
2. The chlorophyll contents of Twenty-one cultivars from Cymbidium species were measured and compared with chlorophyll meter SPAD-502 and conventional methods. The results showed that the chlorophyll a, chlorophyll b, total chlorophyll and SPAD value of most Cymbidium cultivars were different among inter-or intra-species. The correlative of segment cultivars between the chlorophyll content and SPAD value were obvious, which were proved that the chlorophyll content could be menstruated by SPAD value. Cluster analysis showed that all these Cymbidium species could be divided into 4 groups.
3. Fifty-four Cymbidium kanran cultivars were examined and analyzed by using the successive screening of 3'-end extended random primer amplified polymorphic DNA (ERPAD) markers to determine their molecular diversity and relationships. The results showed that 2959 bands including 2527 polymorphic bands (85.40%)were obtained by extended random primers,. ACTGAACGCCCG+ACTGAACGCCGG and ACTGAACGCCC+ACTGAACGCC were devel-oped from the original ACTGAACGC RAPD primer, which produced the same locus (2.5-kb), and the products of extended two basicity marker were more stable and specific than the original RAPD marker and were approach the SCAR marker. Unweighted pair-group mean analysis (UPGMA) grouped them into two clusters based upon geographical traits.
4. The Mitochondrial DNA and chloroplast DNA genome of fifty-four Cymbidium kanran cultivars were examined and analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) markers. The results showed that genetic differences were revealed in 4 of 6 primer sets (66.67%) and 19 of 72 primer-enzyme combinations (26.38%),116 polymorph--ic bands were detected in chloroplast (cp) DNA PCR-RFLP analyses, genetic differences were revealed in 2 of 6 primer sets (25%) and 55 polymorphic bands (53.49%) were detected with one restriction primer-enzyme combinations in mitochondrion (mt) DNA PCR-RFLP analyses. Unweighted pair-group mean analysis (UPGMA) grouped them into four clusters based upon geographical traits.
5. Fifty-four Cymbidium kanran cultivars were examined and analyzed using inter-simple sequence repeat (ISSR) markers. The result showed that 893 bands including 224 polymorphic bands (24.72%) were obtained, which were proved the average number of approximately3.22 DNA bands including 2.73 polymorphic DNA bands and the amplified bands from 2 to 6, all amplified bands located from 200bp to 2000bp. Unweighted pair-group mean analysis (UPGMA) grouped them into six clusters based upon geographical traits.
6. Fifty-four Cymbidium kanran cultivars were examined and analyzed by the sequence-related amplified polymorphism (SRAP) marker. A total of 586 DNA bands were amplified by 11 selective primers,504 of which (86.01%) were polymorphic. The average number of polymorphic DNA bands amplified by each prime was 46. Cluster analysis revealed that the cymbidium could be divided into two cluster (Chinese orchid and Japanese orchid) based on geographic and ecological traits.
In a word, Cymbidium cultivars were examined and analyzed using morphological markers, physiological markers and molecular markers, which could provide scientific basis for future study and utilization of Cymbidium.
主要研究结果如下
1.对建兰亚属21个兰花品种的形态特征的观察和分析可以看出,除叶脉序特征均为平行脉序(弧形脉),叶片均为纸质外,不同品种间的一级与二级脉随品种也不相同,所有品种在形态上具一定的分化,表明其形态特征反映出不同品种间的相似性和差异性,从而为属下种间的界限和关系的探讨提供了重要的形态学证据。
2.应用SPAD-502叶绿素计和常规方法对21个建兰亚属兰花品种的叶绿素含量进行了比较分析。结果表明：大多数兰花在种间或种内的叶绿素a、叶绿素b、总叶绿素含量以及SPAD值均有所不同,但他们之间表现出优异的差异性；7个兰花种间的叶绿素含量与其SPAD值的相关性均达到了显著水平,表明利用SPAD值来衡量其叶绿素含量的可行性；通过聚类分析可以把21个兰花品种分为2大类群。
3.应用ERPAD(延长随机引物扩增DNA)标记对54个寒兰品种进行分析和鉴定。结果表明：8个多态性强引物的1个碱基延长,共扩增出2959条带,其中2527条带(85.40%)具有多态性,延长2个碱基引物ACTGAACGCCCG+ACTGAACGCCGG能获得一条2.5Kb RAPD特异性片段,此片段在RAPD引物(ACTGAACGC)和延长一个碱基的(ACTGAACGCC+ ACTGAACGCCC)引物具有相同的扩增片段。在无需克隆和测序的条件下,这种标记的稳定性和特异性均接近SCAR标记。通过聚类分析可以把54个寒兰品种分为2大类群。
4.应用PCR-RFLP标记对54个寒兰品种的叶绿体和线粒体基因组进行分析和鉴定。结果表明：在6个叶绿体基因组(cpDNA)的PCR-RFLP分析中,对4个(66.67%)可扩增出条带引物进行12种限制性内切酶消化,有19种(26.38%)引物酶组合能检测到116多态性带纹。在6个线粒体基因组中(mtDNA)的PCR-RFLP分析中,利用12种限制性内切酶对能扩增出条带的2种引物进行消化后,只有一种引物(Cox1)能扩增出55(53.49%)多态性带纹。通过聚类分析可以把54个寒兰品种分为4大类群。
5.应用ISSR标记对54个寒兰品种进行分析和鉴定。结果表明：8个ISSR引物共扩增出893条带,每对引物扩增条带数为2-6条,平均为3.22。其中有224个多态性条带,占总带数的24.72%,平均多态性条带数为2.73。扩增带纹片段多在200bp-2000bp之间。通过聚类分析可以把21个兰花品种分为6大类群。
6.应用SRAP标记对51个寒兰品种的基因组DNA进行分析。结果表明筛选出的11对SRAP引物组合对共扩增出586条带纹,其中504条为多态性(86.01%),平均每个引物扩增46条多态性带。聚类分析表明把51份材料根据起源和生物学特性划分为中国寒兰和日本寒兰两大类群。
综上所述,通过形态学标记、生理学标记以及分子标记对部分兰花进行比较分析,并探讨其亲缘关系,旨在为兰花品种的进一步研究和利用提供科学基础。
In order to speed up the pace of orchid breeding and to improve the high-quality orchids, the morphology and chlorophyll contents of Cymbidium kanran resources in Subgen Jensoa were compared and analysis, the molecular taxonomy and relative relationships research of Cymbidium kanran resources in Subgen Jensoa were studies by RAPD, ERPAD, PCR-RFLP, ISSR and SRAP molecular markers.
The study results were as the followings:
1. The venational morphology characters of 21 orchid's varieties were be observed and analyzed, which revealed that different first and secondary vein have different leaf venation characterization except parallel venation, and which could be provided the morphological evidence for their boundaries and relationships between species.
2. The chlorophyll contents of Twenty-one cultivars from Cymbidium species were measured and compared with chlorophyll meter SPAD-502 and conventional methods. The results showed that the chlorophyll a, chlorophyll b, total chlorophyll and SPAD value of most Cymbidium cultivars were different among inter-or intra-species. The correlative of segment cultivars between the chlorophyll content and SPAD value were obvious, which were proved that the chlorophyll content could be menstruated by SPAD value. Cluster analysis showed that all these Cymbidium species could be divided into 4 groups.
3. Fifty-four Cymbidium kanran cultivars were examined and analyzed by using the successive screening of 3'-end extended random primer amplified polymorphic DNA (ERPAD) markers to determine their molecular diversity and relationships. The results showed that 2959 bands including 2527 polymorphic bands (85.40%)were obtained by extended random primers,. ACTGAACGCCCG+ACTGAACGCCGG and ACTGAACGCCC+ACTGAACGCC were devel-oped from the original ACTGAACGC RAPD primer, which produced the same locus (2.5-kb), and the products of extended two basicity marker were more stable and specific than the original RAPD marker and were approach the SCAR marker. Unweighted pair-group mean analysis (UPGMA) grouped them into two clusters based upon geographical traits.
4. The Mitochondrial DNA and chloroplast DNA genome of fifty-four Cymbidium kanran cultivars were examined and analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) markers. The results showed that genetic differences were revealed in 4 of 6 primer sets (66.67%) and 19 of 72 primer-enzyme combinations (26.38%),116 polymorph--ic bands were detected in chloroplast (cp) DNA PCR-RFLP analyses, genetic differences were revealed in 2 of 6 primer sets (25%) and 55 polymorphic bands (53.49%) were detected with one restriction primer-enzyme combinations in mitochondrion (mt) DNA PCR-RFLP analyses. Unweighted pair-group mean analysis (UPGMA) grouped them into four clusters based upon geographical traits.
5. Fifty-four Cymbidium kanran cultivars were examined and analyzed using inter-simple sequence repeat (ISSR) markers. The result showed that 893 bands including 224 polymorphic bands (24.72%) were obtained, which were proved the average number of approximately3.22 DNA bands including 2.73 polymorphic DNA bands and the amplified bands from 2 to 6, all amplified bands located from 200bp to 2000bp. Unweighted pair-group mean analysis (UPGMA) grouped them into six clusters based upon geographical traits.
6. Fifty-four Cymbidium kanran cultivars were examined and analyzed by the sequence-related amplified polymorphism (SRAP) marker. A total of 586 DNA bands were amplified by 11 selective primers,504 of which (86.01%) were polymorphic. The average number of polymorphic DNA bands amplified by each prime was 46. Cluster analysis revealed that the cymbidium could be divided into two cluster (Chinese orchid and Japanese orchid) based on geographic and ecological traits.
In a word, Cymbidium cultivars were examined and analyzed using morphological markers, physiological markers and molecular markers, which could provide scientific basis for future study and utilization of Cymbidium.
引文
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