芫花居群遗传多样性分析及保护策略
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摘要
本文采用标本查阅、电话访问、电子邮件、实地调查等方式研究了芫花的分布现状；采用ISSR分子标记对芫花6个居群进行了遗传多样性和遗传结构研究；利用TTC法测定了芫花花粉生活力及花粉寿命,采用不同浓度蔗糖培养基测定了花粉萌发率,采用联苯胺-过氧化氢法测定了柱头可授性,以及芫花种子萌发试验。结果如下：
1.北到陕西省眉县(33°N),南到江西省赣州市(24°N),东至浙江省的舟山群岛(121°E),西至四川省木里藏族自治区(101°E左右),均有芫花的分布记录。芫花的伴生植物多为草本,少数为乔灌木和藤本；芫花的居群结构不合理,芫花依靠种子繁殖和自身萌蘖两种方式进行居群更新。芫花生境遭到严重破坏,居群数量及个体数量急剧下降。
2.芫花最佳ISSR-PCR反应体系及扩增条件为：20μl反应体系中含Mg2+: 2μl(2.5mmol/L), dNTP:1.6μl(0.2mmol/L), TaqDNA:0.2μl(1.0u), Primer:1.6μl(0.8μmol/L),模版DNA:2μl(40ng/μl), Buffer:2μl,双蒸水：10.6μl。利用ISSR分子标记对芫花6个居群进行遗传多样性水平和遗传结构分析,11条引物对6个居群的180份样品扩增到229个位点。POPGENE32软件计算芫花的多态位点百分率(PPB)、等位基因观察值(Na)、平均有效等位基因数(Ne)、Nei's基因多样性指数(H)和Shannon多样性指数(Ⅰ)分别为40.17%、1.9869、1.3767、0.2355和0.3724,可见,芫花的遗传多样性比较低。芫花6个居群的居群间平均遗传分化系数Gst=0.4409,居群间的基因流Nm=0.6340。并构建了UPGMA聚类树状图。
3.芫花花粉为单粒型花粉,呈球形或近球形,直径在15.6-21.6μm之间,平均直径为19.1μm。花粉粒表面有萌发孔10-16个,近均匀分布于花粉粒表面,萌发孔呈不规则圆形。花粉粒表面为粗网状纹饰,网格呈近四边形至七边形(多为五至六边形)。当天散粉的成熟花粉,生活力为48%；开花第2天,芫花柱头可授性最强,为50%；在开花第2天,花粉在柱头上萌发,花粉管深入到柱头。芫花的果实较小,呈黑色。离体胚及去种壳的种子可正常萌发,而完整种子几乎不萌发,这说明芫花种子具有休眠性。不同质量蔗糖浓度对芫花花粉萌发影响显著,以50g/L时,花粉萌发率最高,约为27%,当蔗糖浓度高于250g/L时,花粉不再萌发。
4.芫花濒危的主要原因可能有：一是芫花种子少,自然更新差；二是生境破坏和人为利用使其在受破坏后短时间内难以恢复。因此,需要对其采取合理有效的保护措施。一是就地保护,二是迁地保护,三是加强芫花的人工繁殖,四是建立濒危植物信息系统,五是加强立法和宣传教育。
Preliminary investigation and analysis on the distribution of Daphne genkwa were studied by sample inspection, consulting literature, field investigations and telephone interview. Genetic diversity and genetic structure between 6 populations of Daphne genkwa were analyzed by ISSR markers. Pollen morphology was observed by scanning electronic microscope(SEM), and its viability was measured by TTC and sucrose in vitro culture methods. Stigma receptivity by benzidine testing, H2O2 testing, and seed germination was observerd. The results were showed as follows:
1. Daphne genkwa is distributed throughout the country, from Ganzhou City, Jiangxi province(24°N) to Meixian County, Shanxi province(33°N); from Tibetan autonomous region, sichuan province (101°E or so) to the zhoushan islands, zhejiang province(121°E). Daphne genkwa populations represent a patch-shaped distribution, most of companion species are culinary herbs, only a handful of them are trees, shrubs or vine. Population structure of Daphne genkwa is unreasonable and degenerate. It reproduced by seeds or sprouted buds. The growing environment were destroyed seriously, the distribution reduced, amount of population and individual declined quickly.
2. The optimal PCR system(20μl) for ISSR analysis was Mg2+:2μl(2.5mmol/L), dNTP: 1.6μl(0.2mmol/L), TaqDNA:0.2μl(1.0u), Primer:1.6μl(0.8μmol/L), template DNA:2μl(40ng/μl), Buffer:2μl, H2O2:10.6μl. Genetic diversity and genetic structure among 6 populations of Daphne genkwa were analyzed by ISSR markers.229 loci were observed among the 180 samples from 6 populations using 11 primers. As analyzed by POPGENE32, the Daphne genkwa's value of observed PPB, number of alleles, effective number of alleles, Nei's gene diversity and Shannon information index were 40.17%,0.3724,1.9869,1.3767 and 0.2355, respectively. The level of genetic diversity of Daphne genkwa was in low level. Genetic differentiation coefficient (Gst) and gene flow (Nm) of Daphne genkwa were 0.4409 and 0.6340. Dendrogram was built by UPGMA based on Nei's genetic distances.
3. Pollen is stenopalynous type with a diamer of 15.6-21.6μm. Per pollen has 10-16 apertures which is irregular circular. Surface ornamentation of pollen is rough reticulate pattern which is circular polygon (tetragon-heptaong, mostly pentagon-hexagon). Pollen viability is 48%, the highest value of stigma receptivity was 50% at 2 days flowering. Pollen germinated on stigma. Fruit is black and small. Embryo and decorticated seed germinated normally, However, intact seed hardly germinated, which indicated that the seed possesses the character of dormancy. Sucrose of different concentrations has a significant effect on pollen germination rate during pollen culture. And pollen germination rate is the highest with a percentage of 27.0% in medium containing 50g/L sucrose, while pollen could not germinate in medium containing sucrose over 250 g/L.
4. The reasons for Daphne genkwa being endangered may be as below descript:First, seeds were too few to reproduce normally; second, it is difficult to restore for this kind of resources during a short time after its environment destroyed and excessive utilizing of human beings. For protecting Daphne genkwa and effective utilization, this paper proposes effective approaches which include in situ conservation, ex situ conservation, artificial propagation, information system of endangered species.
1.北到陕西省眉县(33°N),南到江西省赣州市(24°N),东至浙江省的舟山群岛(121°E),西至四川省木里藏族自治区(101°E左右),均有芫花的分布记录。芫花的伴生植物多为草本,少数为乔灌木和藤本；芫花的居群结构不合理,芫花依靠种子繁殖和自身萌蘖两种方式进行居群更新。芫花生境遭到严重破坏,居群数量及个体数量急剧下降。
2.芫花最佳ISSR-PCR反应体系及扩增条件为：20μl反应体系中含Mg2+: 2μl(2.5mmol/L), dNTP:1.6μl(0.2mmol/L), TaqDNA:0.2μl(1.0u), Primer:1.6μl(0.8μmol/L),模版DNA:2μl(40ng/μl), Buffer:2μl,双蒸水：10.6μl。利用ISSR分子标记对芫花6个居群进行遗传多样性水平和遗传结构分析,11条引物对6个居群的180份样品扩增到229个位点。POPGENE32软件计算芫花的多态位点百分率(PPB)、等位基因观察值(Na)、平均有效等位基因数(Ne)、Nei's基因多样性指数(H)和Shannon多样性指数(Ⅰ)分别为40.17%、1.9869、1.3767、0.2355和0.3724,可见,芫花的遗传多样性比较低。芫花6个居群的居群间平均遗传分化系数Gst=0.4409,居群间的基因流Nm=0.6340。并构建了UPGMA聚类树状图。
3.芫花花粉为单粒型花粉,呈球形或近球形,直径在15.6-21.6μm之间,平均直径为19.1μm。花粉粒表面有萌发孔10-16个,近均匀分布于花粉粒表面,萌发孔呈不规则圆形。花粉粒表面为粗网状纹饰,网格呈近四边形至七边形(多为五至六边形)。当天散粉的成熟花粉,生活力为48%；开花第2天,芫花柱头可授性最强,为50%；在开花第2天,花粉在柱头上萌发,花粉管深入到柱头。芫花的果实较小,呈黑色。离体胚及去种壳的种子可正常萌发,而完整种子几乎不萌发,这说明芫花种子具有休眠性。不同质量蔗糖浓度对芫花花粉萌发影响显著,以50g/L时,花粉萌发率最高,约为27%,当蔗糖浓度高于250g/L时,花粉不再萌发。
4.芫花濒危的主要原因可能有：一是芫花种子少,自然更新差；二是生境破坏和人为利用使其在受破坏后短时间内难以恢复。因此,需要对其采取合理有效的保护措施。一是就地保护,二是迁地保护,三是加强芫花的人工繁殖,四是建立濒危植物信息系统,五是加强立法和宣传教育。
Preliminary investigation and analysis on the distribution of Daphne genkwa were studied by sample inspection, consulting literature, field investigations and telephone interview. Genetic diversity and genetic structure between 6 populations of Daphne genkwa were analyzed by ISSR markers. Pollen morphology was observed by scanning electronic microscope(SEM), and its viability was measured by TTC and sucrose in vitro culture methods. Stigma receptivity by benzidine testing, H2O2 testing, and seed germination was observerd. The results were showed as follows:
1. Daphne genkwa is distributed throughout the country, from Ganzhou City, Jiangxi province(24°N) to Meixian County, Shanxi province(33°N); from Tibetan autonomous region, sichuan province (101°E or so) to the zhoushan islands, zhejiang province(121°E). Daphne genkwa populations represent a patch-shaped distribution, most of companion species are culinary herbs, only a handful of them are trees, shrubs or vine. Population structure of Daphne genkwa is unreasonable and degenerate. It reproduced by seeds or sprouted buds. The growing environment were destroyed seriously, the distribution reduced, amount of population and individual declined quickly.
2. The optimal PCR system(20μl) for ISSR analysis was Mg2+:2μl(2.5mmol/L), dNTP: 1.6μl(0.2mmol/L), TaqDNA:0.2μl(1.0u), Primer:1.6μl(0.8μmol/L), template DNA:2μl(40ng/μl), Buffer:2μl, H2O2:10.6μl. Genetic diversity and genetic structure among 6 populations of Daphne genkwa were analyzed by ISSR markers.229 loci were observed among the 180 samples from 6 populations using 11 primers. As analyzed by POPGENE32, the Daphne genkwa's value of observed PPB, number of alleles, effective number of alleles, Nei's gene diversity and Shannon information index were 40.17%,0.3724,1.9869,1.3767 and 0.2355, respectively. The level of genetic diversity of Daphne genkwa was in low level. Genetic differentiation coefficient (Gst) and gene flow (Nm) of Daphne genkwa were 0.4409 and 0.6340. Dendrogram was built by UPGMA based on Nei's genetic distances.
3. Pollen is stenopalynous type with a diamer of 15.6-21.6μm. Per pollen has 10-16 apertures which is irregular circular. Surface ornamentation of pollen is rough reticulate pattern which is circular polygon (tetragon-heptaong, mostly pentagon-hexagon). Pollen viability is 48%, the highest value of stigma receptivity was 50% at 2 days flowering. Pollen germinated on stigma. Fruit is black and small. Embryo and decorticated seed germinated normally, However, intact seed hardly germinated, which indicated that the seed possesses the character of dormancy. Sucrose of different concentrations has a significant effect on pollen germination rate during pollen culture. And pollen germination rate is the highest with a percentage of 27.0% in medium containing 50g/L sucrose, while pollen could not germinate in medium containing sucrose over 250 g/L.
4. The reasons for Daphne genkwa being endangered may be as below descript:First, seeds were too few to reproduce normally; second, it is difficult to restore for this kind of resources during a short time after its environment destroyed and excessive utilizing of human beings. For protecting Daphne genkwa and effective utilization, this paper proposes effective approaches which include in situ conservation, ex situ conservation, artificial propagation, information system of endangered species.
引文
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