湖南大戟科野桐属遗传多样性及其亲缘关系研究
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摘要
本研究结合ISSR和传统的形态分类学对湖南大戟科野桐属的12个种(南平野桐，毛桐，东南野桐，崖豆藤野桐，杠香藤，粗糠柴，白背叶，红叶野桐，小果野桐，野桐，野梧桐，桃源野桐)，以及假奓包叶(Discocleidion rufescens)和山麻杆(Alchornea hunanensis)的亲缘关系进行了分析。
本研究采用了CTAB法2提取各物种的植物基因组DNA，并对ISSR反应程序中的一些重要参数进行摸索和优化，建立了稳定的、适用于湖南大戟科野桐属遗传多样性和遗传分化研究的ISSR-PCR反应体系。经过优化后的反应体系为：10μlPCR体系中，0.5U TaqDNA聚合酶，0.2μmol/L引物，0.2mmol/L dNTPs，1.5mmol/LMgCl_2，10ng模板DNA。最佳扩增条件为：1)预变性：94℃，1.5分钟；2)变性：94℃，45秒；3)退火：52℃-55℃，45秒；4)延伸：72℃，1.5分钟；共35个循环后，最后延伸7分钟。
本研究用POPGENE软件对14个种进行了多态性比较和聚类分析，研究结果表明：1)野桐属具有相对较高的遗传多样性水平，这与其广泛的地理分布相符；2)作为参照的假奓包叶(Discocleidion rufescens)和山麻杆(Alchornea hunanensis)与野桐属(Mallotus)的12个种在遗传距离相差甚远，这与经典分类学的结果吻合；3)本研究聚类结果将崖豆藤野桐、杠香藤、粗糠柴聚在同一大类上是合理的；4)野桐与野梧桐不应合二为一，还是作为独立的种为好；5)桃源野桐是一新种，并非红叶野桐居群内部自然杂交产生的，且它与小果野桐的亲缘关系最近。此外，桃源野桐是否可另立一新组——桃源野桐组，还须通过实验进一步验证。
This research is about the relationship and genetic distance of 12 Mallotus species in Hunan (M. tenuifolius, M. Barbatus, M. Japonicus, M. millietii, M. Microcarpus, M. Apelta, M. repandus var. chrysocarpus, M. lianus, M. taoyuanensis, M. Dunnii, M. philippensis, M. paxii) as well as Discocleidion rufescens and Alchornea hunanensis, combining ISSR and classical Taxonomy.
Method CTAB2 is found to be effective in genomic DNA extraction. The optimized reaction condition of ISSR-PCR for Mallotus species in Hunan was: 10ngDNA, 0.5UTaq DNA polymerase(TAKARA), 0.2μmol/L primer, 0.2mmol/L dNTPs, 1.5mmol/L MgCl_2 in a total volume of 10 μl . The amplification condition was 1.5 min at 94℃, 35×45s at 94℃, 45s at 52℃-55℃, 1.5min at 72℃, 7min at 72℃. We analyzed ISSR-PCR results, using POPGENE software. The results show: 1) The genetic diversity of Mallotus is in high level, in accordance with its far-ranging geographic distribution. 2) There is an apparent difference between Mallotus species and Discocleidion rufescens, as well as Mallotus species and Alchornea hunanensis. 3) It is reasonable to cluster M. millietii, M. repandus var. chrysocarpus and M. philippensis. 4) M. tenuifolius and M. Japonicus are two different species. 5) The latest found M. taoyuanensis is a new species of mallotus. It is not a hybridized species of M. paxii population, but has a closest relationship with M. microcarpus. Still, it needs further research to testify whether M. taoyuanensis belongs to a new section of mallotus.
本研究采用了CTAB法2提取各物种的植物基因组DNA，并对ISSR反应程序中的一些重要参数进行摸索和优化，建立了稳定的、适用于湖南大戟科野桐属遗传多样性和遗传分化研究的ISSR-PCR反应体系。经过优化后的反应体系为：10μlPCR体系中，0.5U TaqDNA聚合酶，0.2μmol/L引物，0.2mmol/L dNTPs，1.5mmol/LMgCl_2，10ng模板DNA。最佳扩增条件为：1)预变性：94℃，1.5分钟；2)变性：94℃，45秒；3)退火：52℃-55℃，45秒；4)延伸：72℃，1.5分钟；共35个循环后，最后延伸7分钟。
本研究用POPGENE软件对14个种进行了多态性比较和聚类分析，研究结果表明：1)野桐属具有相对较高的遗传多样性水平，这与其广泛的地理分布相符；2)作为参照的假奓包叶(Discocleidion rufescens)和山麻杆(Alchornea hunanensis)与野桐属(Mallotus)的12个种在遗传距离相差甚远，这与经典分类学的结果吻合；3)本研究聚类结果将崖豆藤野桐、杠香藤、粗糠柴聚在同一大类上是合理的；4)野桐与野梧桐不应合二为一，还是作为独立的种为好；5)桃源野桐是一新种，并非红叶野桐居群内部自然杂交产生的，且它与小果野桐的亲缘关系最近。此外，桃源野桐是否可另立一新组——桃源野桐组，还须通过实验进一步验证。
This research is about the relationship and genetic distance of 12 Mallotus species in Hunan (M. tenuifolius, M. Barbatus, M. Japonicus, M. millietii, M. Microcarpus, M. Apelta, M. repandus var. chrysocarpus, M. lianus, M. taoyuanensis, M. Dunnii, M. philippensis, M. paxii) as well as Discocleidion rufescens and Alchornea hunanensis, combining ISSR and classical Taxonomy.
Method CTAB2 is found to be effective in genomic DNA extraction. The optimized reaction condition of ISSR-PCR for Mallotus species in Hunan was: 10ngDNA, 0.5UTaq DNA polymerase(TAKARA), 0.2μmol/L primer, 0.2mmol/L dNTPs, 1.5mmol/L MgCl_2 in a total volume of 10 μl . The amplification condition was 1.5 min at 94℃, 35×45s at 94℃, 45s at 52℃-55℃, 1.5min at 72℃, 7min at 72℃. We analyzed ISSR-PCR results, using POPGENE software. The results show: 1) The genetic diversity of Mallotus is in high level, in accordance with its far-ranging geographic distribution. 2) There is an apparent difference between Mallotus species and Discocleidion rufescens, as well as Mallotus species and Alchornea hunanensis. 3) It is reasonable to cluster M. millietii, M. repandus var. chrysocarpus and M. philippensis. 4) M. tenuifolius and M. Japonicus are two different species. 5) The latest found M. taoyuanensis is a new species of mallotus. It is not a hybridized species of M. paxii population, but has a closest relationship with M. microcarpus. Still, it needs further research to testify whether M. taoyuanensis belongs to a new section of mallotus.
引文
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