摘要
白木香为瑞香科沉香属植物,是我国特有的珍贵药用和香料植物,也是国产沉香唯一资源植物,现已濒临灭绝,被载入《中国保护植物红皮书》。然而有关其遗传资源总量和遗传多样性水平的研究几乎为空白。本研究系统调查了目前我国白木香自然种群的分布区域,采集到海南全岛和广东沿海地区的自然和保护地居群计11个群体的112份样本,并选择3份白木香近缘属植物腋生瑞香为参比外标,运用ISSR和SRAP标记系统检测了白木香自然群体的遗传多态性,借助NTSYS-pc、POPGENE32和Mega2软件,首次从分子水平上揭示白木香自然资源的遗传分类关系,自然居群的遗传多样性水平及其亲缘关系,基本上明确了白木香自然居群之间的遗传演化关系,并对其致濒原因做了讨论和提出保护策略。研究所获得的主要结果如下:
(1)通过对大量ISSR(95对)、SRAP(288对)引物的筛选及反应条件的优化,分别获得各20对优选扩增引物及其反应条件,首次建立了适宜的白木香ISSR和SRAP标记扩增体系,为相关理论研究提供了方法。
(2)通过ISSR和SRAP的对比分析,获得总基因扩增位点818个,平均每对引物20.45个,其中多态性位点567个,达到69.32%。丰富的多态性谱带,阐明白木香不同居群之间的遗传分类关系基本上与地理分布关系相符合,但是存在一些交叉现象,肯定了海南尖峰岭-毛感区系、霸王岭-南燕湾区系、五指山-黎母岭区系、吊罗山区系以及广东区系等5个区系存在的真实性。并通过二维向量分析揭示了白木香种群发生的海南地理群、广东地理群以及海南吊罗山过渡性地理类型。
(3)按照地理居群分组并统计遗传参数,阐明了主要地理居群内部及其总的遗传多样性水平。在7个地理群中以尖峰岭、霸王岭和广东群内部的遗传多样性水平相对较高,多样性指数Ⅰ分别达到0.2954、0.2859、0.2938,其余较低;综合估算总的基因杂合度(h)为0.2397,遗传多样性指数(Ⅰ)为0.3582,从而在理论上说明现有白木香遗传群体的基因多样性水平相对较低,属于濒危或者渐危状态,需要采取合理的措施加以保护。
(4)整合ISSR和SRAP标记的多态性信息,说明了白木香7个居群之间的遗传分化程度及其基因漂变水平,其总的遗传变异中有21.47%变异存在居群间(G_(ST)=0.2147),居群间存在中等程度的遗传分化,居群间基因流(Nm=1.8292)较小。其居群间关系为:白木香群体首先被划分为两个大类:尖峰岭Popl-JIANFENG和毛感Pop2-MAOGAN2个亚群聚为第一大类;而第二大类中包括了吊罗山Pop6-DIAOLUOSHAN和广东类型Pop7-GUANGDONG亚群,霸王岭Pop4-BAWANG和五指山Pop5-WUZHISHAN亚群,以及南燕湾Pop3-NANYANWAN三个亚群。
(5)白木香各个居群间的进化关系分析表明,白木香的11个自然群体中,海南吊罗山部分居群,海南五指山居群和黎母山的自然居群更接近进化树根,应为较原始类群;其次为来自广东的全部材料、海南吊罗山类群、陵水南燕湾和霸王岭自然居群,而海南尖峰岭和保亭县毛感乡的自然居群应为最年轻的类群。海南尖峰岭、吊罗山和广东类群其遗传多样性水平较高,可以作为目前我国白木香的遗传多样性热点区加以保护。
文章根据本研究所揭示的白木香资源遗传多样性水平及其分布,对自然群体致濒原因,可能采取的保护和利用策略等进行了讨论。但是有关分子水平遗传多样性与其经济性状之间的关系尚未涉及,需要在进一步在生理、生化和基因组水平阐释,以便促进白木香的利用和产业化。
Aquilaria sinensis(Lour.) Gilg,species in Thymelaeaceae,is one of the valuable medicinal plants uniquely native in China.But its natural resource has been over destroyed,and to be endangeried species listed in China Plants Red Book.Hardly any knowledge about the total amount of resources and population genetic diversity has been reported.In this study,total of 115 accessions include of 112 A.sinensis and 3 D.axillaris nearly relative species in 11 geographic natural populations have been collected basecd on full investigation to the distribution districts of A.sinensis.Integrating ISSR and SRAP marker systems were used to take examination and evaluation of the whole genetic diversity level and differention among the natural populations with NTSYS-pc,POPGENE32 and Mega2 softwares.Originally,it revealed that the genetic classification,diversity paramaters and the evolution relationship among different populatiuons in A.sinensis.It also has been discussed that the cause of endangering and the possible strategies to conserve.The main results got are as followings:
(1) Throughout selection and better large amount of ISSR and SRAP primers and its amplified conditions,each 20 pairs of primers have been choiced and set up the effective ISSR and SRAP analysis system in A.sinensis first timely,that will contribute to relative researches methodologilly.
(2) Combining to ISSR and SRAP marker data,total of 818 alleles have been found,average 20.45 alleles per primer and there are 567 polymorphic alleles with ratio of 69.32%.With the enrich diversity loci,it revealed that the genetic classification of population is coincide to the geography distribution with some of the crosses,such as the 5 genetic and geograpgic populations of A.sinensis:Jianfengling-Maogan clade,Bawang-nanyanwan clade, Wuzhishan-limuling clade,Diaoluoshan clade and Guangdong clade.PCA analysis further showed that there are three large scale level geographic distribution zones of A.sinensis,they are Hainan group,Guangdong group and among them of Hainan Diaoluoshan group.
(3) The intra-group and total genetic diversity parameters have been estimated based on geographic population classification.Among the 7 populations,the genetic diversity index(Ⅰ) of Jianfengling,Bawang and Guangdong are little higher(0.2954、0.2859、0.2938) than others. The current genetic diversity of whole population is low(h=0.2397,I=0.3582),indicating that less genetic diversity in population A.sinensis means it was reaching to or in the endangered state theoretically,and need pay attentation to conservation.
(4) Integrated ISSR and SRAP allele imformation described the genetic differentiation and gene flow among the 7 populations.Coefficient of gene differentiation among populations was 0.2147(Gst),which means 21.47%of total genetic variation residented among populations.Gene flow among populations was relatively restricted(Nm=1.8292).The relationship of the geographic groups:Primaryly,there are two bigger groups,Jiangfengling (Pop1) and Maogan(Pop2) sub-group constructed the first group;the second group consist of three sub-groups,DIAOLUOSHAN(Pop6) and GUANGDONG(Pop7) sub-group,BAWANG (Pop4) and WUZHISHAN(Pop5) sub-group,NANYANWAN(Pop3) sub-group.
(5) According to evolutionary analysis using D.axillaris to be root,it is indicated that among the 11 natural populations,some of the DIAOLUOSHAN,WUZHISHAN and LIMUSHAN accessions were first branched,and they will be the originally types;The others alphbitically were GUANGDONG accessions,Hainan DIAOLUOSHAN accessions,NANYANWAN and BAWANG accessions,and then JIANFENGLING and MAOGAN accessions.The latest branched Hainan Jianfengling and Maogan types are the younger developed populations with higher diversity.They could be the hot spot to take the in situ conservation.
In the paper,the rationale of endangering and the available conservative strategies have been discussed according to revealed genetic diversity level and distribution of whole resources in A.sinensis.However,what about the relationship between molecular genetic diversity to the economic traits has not been studied,and it need take test and evaluation in physiology,biochemistry and genomics level in the further work,to benefit to use and industrialization of A.sinensis species.
引文
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