川西报春花资源及其遗传多样性与亲缘关系研究
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摘要
研究立足于对四川省西部部分地区报春花属植物资源实地调查的基础之上,通过RAPD技术和AFLP技术对其遗传多样性和亲缘关系进行研究,对不同生境、地理隔离等因素对报春花遗传变异、进化关系的影响进行了探讨,为报春花属植物的资源保护、核心种质构建以及开发利用提供基础理论依据和实践指导。
1、通过野外实地调查,川西高原地区分布有报春花植物31种(包括4亚种),分属11组,部分种类具有较高观赏价值,值得合理开发利用。
2、对川西13种报春花共85份材料进行了RAPD分析,28条引物共扩增出250条DNA带,其中多态性带达209条,占总数的83.6%。雅江报春扩增出173条DNA带,82条为多态性带,占47.4%;杂色钟花报春扩增出206条带,155条为多态性带,占75.2%;青城报春扩增出187条带,94条为多态性带,占50.3%。同时利用6对引物组合,对143个鹅黄灯台报春进行AFLP分析,得到546条大小为100～850bp扩增带,其中多态性条带218条。证明报春花具有较高的种间及种内遗传多样性。
3、基于RAPD标记结果进行UPGMA聚类分析,雅江报春17份材料的遗传距离变幅为0.0345～0.9523,材料间平均遗传距离为0.4584,木格措居群内平均遗传距离为0.6255,康定和理塘居群内遗传距离分别为0.2972和0.2239;木格措居群与康定居群之间的平均遗传距离为0.5352,表明木格措居群与康定居群之间的亲缘关系较近;而康定居群和理塘居群之间的平均遗传距离0.6134,表明康定居群和理塘居群之间差异较大,亲缘关系较远。居群间的遗传分化随居群间地理距离的增大而加大。
但是以单匹配相似系数(SM)为基础对143份鹅黄灯台报春进行UPGMA聚类分析,发现其居群间遗传距离和地理距离之间不存在明显的相关性。虽然康定折多山与丹巴党岭的距离比康定折多山与康定雅加埂的距离更大,但其亲缘关系却更近。康定雅加埂组单独聚为一类,康定折多山组和丹巴党岭组聚为一类,10个居群143个样品并未以各自居群为中心聚为一类,交叉较严重,说明居群内分化较严重,各居群间分化程度也不一样,且居群内存在一定程度的遗传漂变。
4、基于AFLP标记结果,进行鹅黄灯台报春的遗传结构分析,居群总的遗传多样性(Ht)为0.0961,居群内的遗传多样性(Hs)为0.0619,10个居群间的遗传分化系数(Gst)为0.3557,说明居群内个体间的分化水平较高;总的遗传变异主要来自于居群内个体间,其中康定雅加埂组Ht和Hs均高于丹巴党岭组。与分子方差变异分析(AMOVA)结果基本一致。
从总体上看,10个居群间的基因流(Nm)为0.9056<1,各居群间的基因交流较为缺乏,但在地区分组上,康定雅加埂组居群间Nm为1.3617>1,丹巴党岭组居群间Nm为1.0554>1,两个组组内各居群间的基因交流较为频繁,居群较稳定。
5、不同生境对报春花遗传多样性的影响较大,发育良好的生境有利于提高报春花的遗传多样性;同一区域内高水平、稳定的基因流有利于使报春花居群趋于一致。龙池的报春花材料基本聚为一类,明显区别于采自甘孜州的材料。
6、基于RAPD标记聚类分析与形态学分类存在一定的差异。在形态学分类上属于灯台报春组的粉被灯台报春没有与另外两种同组报春(桔红灯台报春、偏花报春)聚在一起,而是与同为龙池地区的材料聚类,说明生态地理类型对报春花遗传结构有影响,偏花报春与灯台报春组的材料聚为一类,表明其与灯台报春组具有更近的亲缘关系。
Based on the investigation of the primrose(Primula L.)germplasm resource in the western areas of Sichuan province,the genetic diversity and the phylogenetic relatives of different species was analyzed by using RAPD and AFLP makers. And the effect of influence factors (such as the different habitat and geographical barrier) on genetic variation and evolvement of Primula was discussed. Then the theoretical science guideline and production were proposed to the resource conservation, core idioplasm construction of primrose genus, as well as its development and utilization.The main results were as follows.
1. Based on the investigation of the primrose(Primula L.)germplasm resource,31 species primrose (Primula L.)germplasm resource, including 4 subspecies were divided into 11 groups in the western plateau of Sichuan province. Some species with higher ornamental value are worthy to exploited.
2. By using RAPD maker, the results of 85 tested materials from 13 species of Primula indicated that 28 RPAD primers were amplified 250 DNA fragments, including 209 polymorphic fragments (83.6% in total) were detected. Therefore,173 DNA fragments were amplified from P. yargongens, including 82 polymorphic fragments (47.4% in total) were detected, 206 DNA fragments were amplified from P.alpicola, including 155 polymorphic fragments (75.2% in total) were detected,and 187 DNA fragments from P.chienii, 94 polymorphic fragments (50.3% in total).Meanwhile, 143 individuals of P.cockburniana were sampled and analyzed by using AFLP maker, which underlies selecting six primers combinations. 546 DNA fragments with 100-850bp were amplified,including 218 polymorphic fragments were detected.All of the results showed that the genetic diversity among the different species and in the same species of primrose was obvious.
3. Based on the tagged results of RAPD, UPGMA cluster analysis was carried out,the genetic distance of 17 tested materials of P. yargongens was between 0.0345～0.9523,and the mean of genetic distance among the materials was 0.4584. Within the population, The mean of genetic distance of Mugecuo population was 0.6255, the mean of genetic distance of Kangding population was 0.2972 and that of Litang population was 0.2239, the mean of genetic distance of Mugecuo population and Kanding population was 0.5352, which indicated relatives between the two population was closer. In contrast, the mean of genetic distance of Kangding population and Litang population was 0.6134, which indicated more genetic difference and less relatives exists between the two population.The genetic differentiation among populations increased along with the increase of geographical distance
However, 143 individuals of P.cockburniana were tested by UPGMA cluster analysis based on single match coefficient(SM). The results indicated that genetic distance among populations was not obviously related to geographical distance. The genetic relationship was more close though distance of Mt. Zheduo to Danba Dangling was more than that of Mt. Zheduo to Yajiageng of Kangding. Accordingly, that of Danba Tiantanggu-dangling groups clusters into one category in singular.That of Mt. Zheduo clusters with that of Danba Tiantanggu-dangling.143 individuals from 10 populations were not clustered in the core of respective population. Intercross was very serious, which indicates that differentiation within population was apparent, differentiation level was different among populations, and genetic drift exists within the populations.
4. The results of AFLP analysis showed that the total genetic diversity (Ht) of P.cockburniana population was 0.0961,and genetic diversity within the population was 0.0619. The coefficient of gene differentiation(Gst) in 10 populations was 0.3557. The results indicated that individual differentiation level was high within the population.All the genetic variation comes from individuals of population. Ht and Hs of that of Yajiageng of Kangding were higher than that of Danba Tiantanggu-dangling. The results was the same to that by AMOVA technique.
As a while, Gene flow (Nm) among the 10 populations was 0.9056<1. Genes transferring was lack among the population. But Nm in Yajiageng of Kangding population was 1.3617>1, and 1.0554>1 in Danba Tiantanggu-dangling population. It shows that frequency of Genes transferring was highly existed in populations of group so that it is moderate
5. The effects of different habitat and eco-geographic areas on genetic diversity of Primula were significant. Habitat with good condition was benefit to improve the genetic diversity of Primula. Gene flow with high quality and stability in the same district was benefit to the uniformity of Primula population. Individuals of Primula population in Longchi of western Sichuan were clustered together, and this was obviously different from that of Ganzi area.
6. Cluster analysis based on RAPD marker was different from the category of morphological type. P. pulverulenta Duthie didn’t cluster with P.bulleyana and P.secundiflora, which were belong to Sect. Proliferae, originated from the same group in category of morphology. The P.pulverulenta Duthie was clustered with materials that originated from the same district, Longchi. It indicated that the genetic structure of Primula was affected by the type of Eco-geography. Likewise, systematic positioning to P.secundiflora was studied. The result indicated that P.secundiflora was clustered with Sect. Proliferae. So, it showed that P.secundiflora was more close to Sect. Proliferae
1、通过野外实地调查,川西高原地区分布有报春花植物31种(包括4亚种),分属11组,部分种类具有较高观赏价值,值得合理开发利用。
2、对川西13种报春花共85份材料进行了RAPD分析,28条引物共扩增出250条DNA带,其中多态性带达209条,占总数的83.6%。雅江报春扩增出173条DNA带,82条为多态性带,占47.4%;杂色钟花报春扩增出206条带,155条为多态性带,占75.2%;青城报春扩增出187条带,94条为多态性带,占50.3%。同时利用6对引物组合,对143个鹅黄灯台报春进行AFLP分析,得到546条大小为100～850bp扩增带,其中多态性条带218条。证明报春花具有较高的种间及种内遗传多样性。
3、基于RAPD标记结果进行UPGMA聚类分析,雅江报春17份材料的遗传距离变幅为0.0345～0.9523,材料间平均遗传距离为0.4584,木格措居群内平均遗传距离为0.6255,康定和理塘居群内遗传距离分别为0.2972和0.2239;木格措居群与康定居群之间的平均遗传距离为0.5352,表明木格措居群与康定居群之间的亲缘关系较近;而康定居群和理塘居群之间的平均遗传距离0.6134,表明康定居群和理塘居群之间差异较大,亲缘关系较远。居群间的遗传分化随居群间地理距离的增大而加大。
但是以单匹配相似系数(SM)为基础对143份鹅黄灯台报春进行UPGMA聚类分析,发现其居群间遗传距离和地理距离之间不存在明显的相关性。虽然康定折多山与丹巴党岭的距离比康定折多山与康定雅加埂的距离更大,但其亲缘关系却更近。康定雅加埂组单独聚为一类,康定折多山组和丹巴党岭组聚为一类,10个居群143个样品并未以各自居群为中心聚为一类,交叉较严重,说明居群内分化较严重,各居群间分化程度也不一样,且居群内存在一定程度的遗传漂变。
4、基于AFLP标记结果,进行鹅黄灯台报春的遗传结构分析,居群总的遗传多样性(Ht)为0.0961,居群内的遗传多样性(Hs)为0.0619,10个居群间的遗传分化系数(Gst)为0.3557,说明居群内个体间的分化水平较高;总的遗传变异主要来自于居群内个体间,其中康定雅加埂组Ht和Hs均高于丹巴党岭组。与分子方差变异分析(AMOVA)结果基本一致。
从总体上看,10个居群间的基因流(Nm)为0.9056<1,各居群间的基因交流较为缺乏,但在地区分组上,康定雅加埂组居群间Nm为1.3617>1,丹巴党岭组居群间Nm为1.0554>1,两个组组内各居群间的基因交流较为频繁,居群较稳定。
5、不同生境对报春花遗传多样性的影响较大,发育良好的生境有利于提高报春花的遗传多样性;同一区域内高水平、稳定的基因流有利于使报春花居群趋于一致。龙池的报春花材料基本聚为一类,明显区别于采自甘孜州的材料。
6、基于RAPD标记聚类分析与形态学分类存在一定的差异。在形态学分类上属于灯台报春组的粉被灯台报春没有与另外两种同组报春(桔红灯台报春、偏花报春)聚在一起,而是与同为龙池地区的材料聚类,说明生态地理类型对报春花遗传结构有影响,偏花报春与灯台报春组的材料聚为一类,表明其与灯台报春组具有更近的亲缘关系。
Based on the investigation of the primrose(Primula L.)germplasm resource in the western areas of Sichuan province,the genetic diversity and the phylogenetic relatives of different species was analyzed by using RAPD and AFLP makers. And the effect of influence factors (such as the different habitat and geographical barrier) on genetic variation and evolvement of Primula was discussed. Then the theoretical science guideline and production were proposed to the resource conservation, core idioplasm construction of primrose genus, as well as its development and utilization.The main results were as follows.
1. Based on the investigation of the primrose(Primula L.)germplasm resource,31 species primrose (Primula L.)germplasm resource, including 4 subspecies were divided into 11 groups in the western plateau of Sichuan province. Some species with higher ornamental value are worthy to exploited.
2. By using RAPD maker, the results of 85 tested materials from 13 species of Primula indicated that 28 RPAD primers were amplified 250 DNA fragments, including 209 polymorphic fragments (83.6% in total) were detected. Therefore,173 DNA fragments were amplified from P. yargongens, including 82 polymorphic fragments (47.4% in total) were detected, 206 DNA fragments were amplified from P.alpicola, including 155 polymorphic fragments (75.2% in total) were detected,and 187 DNA fragments from P.chienii, 94 polymorphic fragments (50.3% in total).Meanwhile, 143 individuals of P.cockburniana were sampled and analyzed by using AFLP maker, which underlies selecting six primers combinations. 546 DNA fragments with 100-850bp were amplified,including 218 polymorphic fragments were detected.All of the results showed that the genetic diversity among the different species and in the same species of primrose was obvious.
3. Based on the tagged results of RAPD, UPGMA cluster analysis was carried out,the genetic distance of 17 tested materials of P. yargongens was between 0.0345～0.9523,and the mean of genetic distance among the materials was 0.4584. Within the population, The mean of genetic distance of Mugecuo population was 0.6255, the mean of genetic distance of Kangding population was 0.2972 and that of Litang population was 0.2239, the mean of genetic distance of Mugecuo population and Kanding population was 0.5352, which indicated relatives between the two population was closer. In contrast, the mean of genetic distance of Kangding population and Litang population was 0.6134, which indicated more genetic difference and less relatives exists between the two population.The genetic differentiation among populations increased along with the increase of geographical distance
However, 143 individuals of P.cockburniana were tested by UPGMA cluster analysis based on single match coefficient(SM). The results indicated that genetic distance among populations was not obviously related to geographical distance. The genetic relationship was more close though distance of Mt. Zheduo to Danba Dangling was more than that of Mt. Zheduo to Yajiageng of Kangding. Accordingly, that of Danba Tiantanggu-dangling groups clusters into one category in singular.That of Mt. Zheduo clusters with that of Danba Tiantanggu-dangling.143 individuals from 10 populations were not clustered in the core of respective population. Intercross was very serious, which indicates that differentiation within population was apparent, differentiation level was different among populations, and genetic drift exists within the populations.
4. The results of AFLP analysis showed that the total genetic diversity (Ht) of P.cockburniana population was 0.0961,and genetic diversity within the population was 0.0619. The coefficient of gene differentiation(Gst) in 10 populations was 0.3557. The results indicated that individual differentiation level was high within the population.All the genetic variation comes from individuals of population. Ht and Hs of that of Yajiageng of Kangding were higher than that of Danba Tiantanggu-dangling. The results was the same to that by AMOVA technique.
As a while, Gene flow (Nm) among the 10 populations was 0.9056<1. Genes transferring was lack among the population. But Nm in Yajiageng of Kangding population was 1.3617>1, and 1.0554>1 in Danba Tiantanggu-dangling population. It shows that frequency of Genes transferring was highly existed in populations of group so that it is moderate
5. The effects of different habitat and eco-geographic areas on genetic diversity of Primula were significant. Habitat with good condition was benefit to improve the genetic diversity of Primula. Gene flow with high quality and stability in the same district was benefit to the uniformity of Primula population. Individuals of Primula population in Longchi of western Sichuan were clustered together, and this was obviously different from that of Ganzi area.
6. Cluster analysis based on RAPD marker was different from the category of morphological type. P. pulverulenta Duthie didn’t cluster with P.bulleyana and P.secundiflora, which were belong to Sect. Proliferae, originated from the same group in category of morphology. The P.pulverulenta Duthie was clustered with materials that originated from the same district, Longchi. It indicated that the genetic structure of Primula was affected by the type of Eco-geography. Likewise, systematic positioning to P.secundiflora was studied. The result indicated that P.secundiflora was clustered with Sect. Proliferae. So, it showed that P.secundiflora was more close to Sect. Proliferae
引文
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