米心水青冈（Fagus engleriana）生态及遗传多样性研究
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摘要
本文以浙江、湖北、四川、贵州等省份9个居群的米心水青冈为研究材料,采用RAPD分子标记,主要探讨了米心水青冈的遗传变异,同时还研究了米心水青冈生态和水青冈属5种植物的遗传变异,提出了米心水青冈的保护措施,研究结果如下:
1.米心水青冈在我国分布范围广,一般喜湿润和阴凉的环境。四川南江的米心水青冈群落中占优势的科为壳斗科、槭树科和桦木科。乔木层一般分为3层:在第一层,落叶阔叶树种占优势,米心水青冈的重要值为47.53,是优势种,其次为锐齿槲栎和短柄枹栎，重要值分别为11.43和10.85;第二及第三层以常绿树种为主,主要为青冈、小叶青冈和刺叶栎。四川南江米心水青冈群落的建群种为米心水青冈。
2.本研究采用四种DNA提取方法,通过比较得出改良CTAB法提出的DNA纯度较高,能够达到扩增要求,因此采用此方法用于正式DNA的提取。适合米心水青冈RAPD反应体系:反应体积为25μl,模板DNA40ng,引物0.8μM,Taq聚合酶1.25u,Mg2+浓度2.0 mM,dNTP浓度0.16 mM。适合米心水青冈RAPD扩增程序: 94℃预变性3min,一个循环, 94℃变性30s,37℃退火1min,72℃延伸2min,45个循环。
3.20条随机引物共扩出260条带,平均每条引物扩增出13个条带,其DNA带的分子量在200-3000bp之间。其中多态性条带有180条,多态位点比率(PPL)为69.2%。种内的Shannon多样性指数为0.3021,群体内占的比例为72.10%,群体间占的比例为27.90%。种内的Nei's基因多样性0.2045,群体内的平均基因多样性为0.1502,基因分化系数GST为0.2631。
4.巴山水青冈、浙江水青冈和亮叶水青冈之间的亲缘关系较近,长柄水青冈和米心水青冈之间的亲缘关系较近,它们之间的遗传分化较大。
5.米心水青冈的遗传多样性水平处于中等,遗传分化也处于中等。本文同意将巴山水青冈和浙江水青冈并到台湾水青冈。
6.建议,水青冈以就地保护为主,同时进行人工育苗和造林。米心水青冈群体间已经出现了一定程度的遗传分化,建议在天然群体间进行植株和种子幼苗相互移栽,以增强群体间的基因流,最大限度地保护米心水青冈的遗传多样性。
In this paper, nine populations of F.engleriana from four provinces were analyzed by random amplified polymorphic DNA (RAPD) markers to determine genetic diversity and genetic variations. In addition, ecology of F.engleriana and genetic variations among five species of Fagus were studied. The main results were as follows:
1.F.engleriana ,growing best on loamy soils, was sensitive to reduced soil moisture. In the community of Nan jiang town, the dominant families were Fagaceae, Aceraceae and Betulaceae. The tree crown could be divided into three layers. In the first layer, deciduous trees were dominant, and dominant species was F. engleriana. Its important value was 47.43. Relatively dominant species were Quercus aliena var.acuteserrata and Quercus glandulifera.Their important values were 11.43 and 10.85. In the second and third layers, evergreen species were dominant species. They were Cyclobalanopsis. myrsinaefolia, Cyclobalanopsis glauca and Quercus spinosa. So F.engleriana was the key species of this community.
2.Total DNA in leaf tissue of F. engleriana was extracted by four methods. Improved CTAB method was proven to be the best one. The DNA sample obtained by this method was fit for RAPD experiment. RAPD reaction system(25μl):40ngDNA template, 0.8μM primer,Taq DNA polymerase 1.25u,2.0 mM Mg2+, 0.16 mM each dNTP. PCR amplification program was denaturation at 94℃in advance for 3min,then followed 45 cycles,every cycle: denaturation at 94℃for 30s,anneal at 37℃for 1 min, extention at 72℃for 2 min.
3.260 bands were amplified by 20 primers in Fagus engleriana,180 bands were polymorphic and percentage of polymorphic loci was 69.2%. The average Shannon index within species of F. engleriana was 0.3021,and percentages that among and within nine populations were respectively 72.10% and 27.90%.The average genetic diversity within species of F. engleriana was 0.2045, and 0.1502 for that within population.The coefficient of genetic differentiation was 0.2631.
4.F. hayatae var. zhengjiangensis, F. pashanica and F.lucida had closer relationship, and F. longipetiolata and F. engleriana had closer relationship.There was much gene differentiation between them.
5. Compared with other species,the genetic diversity of F. engleriana was at a moderate level.And the great mass of genetic variance was within populations.F. hayatae var. zhengjiangensis , F. pashanica should be clustered into Fagus hayatae.
6.Natural populations of F. engleriana protected in situ was advised. Forestation and seading cultivation of F. engleriana were necessary. In addition,base on the certain level of genetic differentiation of among the natural populations,it was advised that adult plants or seedlings of F. engleriana be transPlanted in order to enhance the gene fiow among them and preserve the genetic diversity of F. engleriana.
1.米心水青冈在我国分布范围广,一般喜湿润和阴凉的环境。四川南江的米心水青冈群落中占优势的科为壳斗科、槭树科和桦木科。乔木层一般分为3层:在第一层,落叶阔叶树种占优势,米心水青冈的重要值为47.53,是优势种,其次为锐齿槲栎和短柄枹栎，重要值分别为11.43和10.85;第二及第三层以常绿树种为主,主要为青冈、小叶青冈和刺叶栎。四川南江米心水青冈群落的建群种为米心水青冈。
2.本研究采用四种DNA提取方法,通过比较得出改良CTAB法提出的DNA纯度较高,能够达到扩增要求,因此采用此方法用于正式DNA的提取。适合米心水青冈RAPD反应体系:反应体积为25μl,模板DNA40ng,引物0.8μM,Taq聚合酶1.25u,Mg2+浓度2.0 mM,dNTP浓度0.16 mM。适合米心水青冈RAPD扩增程序: 94℃预变性3min,一个循环, 94℃变性30s,37℃退火1min,72℃延伸2min,45个循环。
3.20条随机引物共扩出260条带,平均每条引物扩增出13个条带,其DNA带的分子量在200-3000bp之间。其中多态性条带有180条,多态位点比率(PPL)为69.2%。种内的Shannon多样性指数为0.3021,群体内占的比例为72.10%,群体间占的比例为27.90%。种内的Nei's基因多样性0.2045,群体内的平均基因多样性为0.1502,基因分化系数GST为0.2631。
4.巴山水青冈、浙江水青冈和亮叶水青冈之间的亲缘关系较近,长柄水青冈和米心水青冈之间的亲缘关系较近,它们之间的遗传分化较大。
5.米心水青冈的遗传多样性水平处于中等,遗传分化也处于中等。本文同意将巴山水青冈和浙江水青冈并到台湾水青冈。
6.建议,水青冈以就地保护为主,同时进行人工育苗和造林。米心水青冈群体间已经出现了一定程度的遗传分化,建议在天然群体间进行植株和种子幼苗相互移栽,以增强群体间的基因流,最大限度地保护米心水青冈的遗传多样性。
In this paper, nine populations of F.engleriana from four provinces were analyzed by random amplified polymorphic DNA (RAPD) markers to determine genetic diversity and genetic variations. In addition, ecology of F.engleriana and genetic variations among five species of Fagus were studied. The main results were as follows:
1.F.engleriana ,growing best on loamy soils, was sensitive to reduced soil moisture. In the community of Nan jiang town, the dominant families were Fagaceae, Aceraceae and Betulaceae. The tree crown could be divided into three layers. In the first layer, deciduous trees were dominant, and dominant species was F. engleriana. Its important value was 47.43. Relatively dominant species were Quercus aliena var.acuteserrata and Quercus glandulifera.Their important values were 11.43 and 10.85. In the second and third layers, evergreen species were dominant species. They were Cyclobalanopsis. myrsinaefolia, Cyclobalanopsis glauca and Quercus spinosa. So F.engleriana was the key species of this community.
2.Total DNA in leaf tissue of F. engleriana was extracted by four methods. Improved CTAB method was proven to be the best one. The DNA sample obtained by this method was fit for RAPD experiment. RAPD reaction system(25μl):40ngDNA template, 0.8μM primer,Taq DNA polymerase 1.25u,2.0 mM Mg2+, 0.16 mM each dNTP. PCR amplification program was denaturation at 94℃in advance for 3min,then followed 45 cycles,every cycle: denaturation at 94℃for 30s,anneal at 37℃for 1 min, extention at 72℃for 2 min.
3.260 bands were amplified by 20 primers in Fagus engleriana,180 bands were polymorphic and percentage of polymorphic loci was 69.2%. The average Shannon index within species of F. engleriana was 0.3021,and percentages that among and within nine populations were respectively 72.10% and 27.90%.The average genetic diversity within species of F. engleriana was 0.2045, and 0.1502 for that within population.The coefficient of genetic differentiation was 0.2631.
4.F. hayatae var. zhengjiangensis, F. pashanica and F.lucida had closer relationship, and F. longipetiolata and F. engleriana had closer relationship.There was much gene differentiation between them.
5. Compared with other species,the genetic diversity of F. engleriana was at a moderate level.And the great mass of genetic variance was within populations.F. hayatae var. zhengjiangensis , F. pashanica should be clustered into Fagus hayatae.
6.Natural populations of F. engleriana protected in situ was advised. Forestation and seading cultivation of F. engleriana were necessary. In addition,base on the certain level of genetic differentiation of among the natural populations,it was advised that adult plants or seedlings of F. engleriana be transPlanted in order to enhance the gene fiow among them and preserve the genetic diversity of F. engleriana.
引文
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2.陈灵芝.中国的生物多样性—现状及其保护对策.北京: 科学出版社,1993.
3.崔艳秋.神农架米心水青冈林的结构特点.信阳师范学院学报(自然科学 版),1999,12(1).
4.范复生主编.浙江森林.北京:中国林业出版社,1993.
5.方建初主编。湖北森林.北京:中国林业出版社,1991
6.方精云等.浙江水青冈属植物的解剖特征及分类学意义.北京大学学报(自然 科学版),2000,36(4):509-515.
7 葛颂等.马尾松 GOT,LDH 和 MDH 同工酶的遗传方式和连锁关系.植物学报, 1987,14(6):428-435.
8.葛颂.植物群体遗传结构研究的回顾和展望. 见:李承森(主编).植物科学 进展(第一卷).北京:高等教育出版社,1997.1-15
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