山西省华北落叶松天然种群遗传多样性的AFLP分析
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摘要
华北落叶松(Larix principis-rupprechtii Mayr.)是我国华北地区寒温性针叶林中的主要树种之一。其天然林主要分布在河北和山西两省,具有改良土壤,涵养水源等作用。本研究以山西省的6个华北落叶松天然种群为研究对象,采用AFLP分子标记技术,对其遗传多样性和空间遗传结构特征进行研究。主要研究结果如下：
1)建立了华北落叶松AFLP反应体系和程序,并优化了影响AFLP反应的5个主要因素(酶切时间、内切酶用量、DNA用量、连接产物和预扩增产物的稀释倍数),即在反应体系均为20μL的条件下,酶切时间为7h (37℃3.5h; 65℃3.5h), Eco I内切酶和MseⅠ内切酶用量均为1U, DNA用量为3μL (50ng/μL),连接产物稀释10倍取5μL用于预扩增,预扩增产物稀释70倍取4gL用于选择性扩增。
2)用筛选出的8对引物组合对6个华北落叶松天然种群的144个个体进行AFLP分析,共扩增出402条谱带,其中289条为多态性带,多态性带占71.89%。恒山张家洼种群(HS)的多态位点百分率和遗传多样性指数最高(PPB=55.97%,h=0.2065,I=0.3047)；关帝山庞泉沟种群(GD)的多态位点百分率和遗传多样性指数最低(PPB=44.53%,h=0.1519,I=0.2284)。
3)华北落叶松总基因多样性(Ht)为0.2245,种群内基因多样性(Hs)为0.1810。遗传分化系数(Gst)为0.1936,表明在总的遗传变异中有19.36%的变异存在于种群间,80.64%的变异存在于种群内,说明华北落叶松的遗传变异主要存在于种群内,且种群内个体间的遗传分化程度较高。基因流(Nm)为2.0821,表明种群间基因交流较频繁。
4)6个华北落叶松种群间遗传一致度(I)在0.9269-0.9677之间,平均为0.9409；遗传距离(D)在0.0328-0.0759之间,平均为0.0610。表明这6个华北落叶松天然种群间的亲缘关系很近,但又不是完全一致,各种群间存在着一定的遗传变异。其中关帝山庞泉沟种群(GD)和芦芽山荷叶坪种群(LY)的亲缘关系最近(I=0.9677；D=0.0328),而六棱山桦林背种群(LL)和恒山张家洼种群(HS)的亲缘关系最远(I=0.9269；D=0.0759)。用UPGMA法进行聚类分析,可将6个种群分为三组：五台山茂材林种群(WT)和六棱山桦林背种群(LL)为一组；芦芽山荷叶坪种群(LY)和关帝山庞泉沟种群(GD)为一组；恒山张家洼种群(HS)和太岳山好地方种群(TY)为一组。
Prince Rupprecht's larch (Larix principis-rupprechtii Mayr.), one of cold temperate coniferous trees in north China, is important for soil improvement and water conservation. The natural forest of this species is distributed mainly in Hebei and Shanxi with the elevation ranged from 1500m to 2700m. The object of this study is to estimate genetic diversity and spatial genetic structure characteristics of six natural populations of L. principis-rupprechtii in Shanxi Province by AFLP. The main results are as follows:
1) AFLP reaction system and procedures was established. The influencing factors (the time of DNA digestion, the amount of endonucleases EcoR I and Mse I, the amount of DNA, the dilution times for ligated products and pre-amplification products) were optimized with 20μL reaction system. The reaction system was established as follows:7 hours (37℃3.5h; 65℃3.5h) of DNA digestion; 1U of EcoR I and Mse I enzymes, respectively; 3μL (50ng/μL) of DNA; 5μL of ligated products diluted by 10 times; and 4μL of pre-amplification products diluted by 70 times.
2) 8 primer combinations were taken to generate 402 clearly identifiable bands of 144 individuals from 6 populations, and 289 bands were polymorphic (PPB=71.89%). Population HS (PPB=55.97%,h=0.2065, I=0.3047) had the highest genetic variation, whereas population GD (PPB=44.53%, h=0.1519,I=0.2284) had the lowest genetic variation.
3) The six populations of Larix principis-rupprechtii Mayr. had a high level of gene diversity (Ht=0.2245,Hs=0.1810, Gst=0.1936), distributed mainly within (80.64%) rather than among (19.36%) populations. The gene flow among populations was at a high probability (Nm=2.0821).
4) The Nei's unbiased measures (1978) of genetic identity (I) was ranged from 0.9269 to 0.9677, and the average was 0.9409. The genetic distance (D) was in the range of 0.0328-0.0759, and the average was 0.0610. While closely related, the six populations were not completely consistent. The highest genetic identity (I=0.9677) and lowest genetic distance (D=0.0328) was detected between populations GD and LY, while the lowest genetic identity (I=0.9269) and highest genetic distance (D=0.0759) was detected between population LL and HS. The result of UPGMA cluster analysis showed that six populations could be divided into three groups:WT and LL; LY and GD; HS and TY.
1)建立了华北落叶松AFLP反应体系和程序,并优化了影响AFLP反应的5个主要因素(酶切时间、内切酶用量、DNA用量、连接产物和预扩增产物的稀释倍数),即在反应体系均为20μL的条件下,酶切时间为7h (37℃3.5h; 65℃3.5h), Eco I内切酶和MseⅠ内切酶用量均为1U, DNA用量为3μL (50ng/μL),连接产物稀释10倍取5μL用于预扩增,预扩增产物稀释70倍取4gL用于选择性扩增。
2)用筛选出的8对引物组合对6个华北落叶松天然种群的144个个体进行AFLP分析,共扩增出402条谱带,其中289条为多态性带,多态性带占71.89%。恒山张家洼种群(HS)的多态位点百分率和遗传多样性指数最高(PPB=55.97%,h=0.2065,I=0.3047)；关帝山庞泉沟种群(GD)的多态位点百分率和遗传多样性指数最低(PPB=44.53%,h=0.1519,I=0.2284)。
3)华北落叶松总基因多样性(Ht)为0.2245,种群内基因多样性(Hs)为0.1810。遗传分化系数(Gst)为0.1936,表明在总的遗传变异中有19.36%的变异存在于种群间,80.64%的变异存在于种群内,说明华北落叶松的遗传变异主要存在于种群内,且种群内个体间的遗传分化程度较高。基因流(Nm)为2.0821,表明种群间基因交流较频繁。
4)6个华北落叶松种群间遗传一致度(I)在0.9269-0.9677之间,平均为0.9409；遗传距离(D)在0.0328-0.0759之间,平均为0.0610。表明这6个华北落叶松天然种群间的亲缘关系很近,但又不是完全一致,各种群间存在着一定的遗传变异。其中关帝山庞泉沟种群(GD)和芦芽山荷叶坪种群(LY)的亲缘关系最近(I=0.9677；D=0.0328),而六棱山桦林背种群(LL)和恒山张家洼种群(HS)的亲缘关系最远(I=0.9269；D=0.0759)。用UPGMA法进行聚类分析,可将6个种群分为三组：五台山茂材林种群(WT)和六棱山桦林背种群(LL)为一组；芦芽山荷叶坪种群(LY)和关帝山庞泉沟种群(GD)为一组；恒山张家洼种群(HS)和太岳山好地方种群(TY)为一组。
Prince Rupprecht's larch (Larix principis-rupprechtii Mayr.), one of cold temperate coniferous trees in north China, is important for soil improvement and water conservation. The natural forest of this species is distributed mainly in Hebei and Shanxi with the elevation ranged from 1500m to 2700m. The object of this study is to estimate genetic diversity and spatial genetic structure characteristics of six natural populations of L. principis-rupprechtii in Shanxi Province by AFLP. The main results are as follows:
1) AFLP reaction system and procedures was established. The influencing factors (the time of DNA digestion, the amount of endonucleases EcoR I and Mse I, the amount of DNA, the dilution times for ligated products and pre-amplification products) were optimized with 20μL reaction system. The reaction system was established as follows:7 hours (37℃3.5h; 65℃3.5h) of DNA digestion; 1U of EcoR I and Mse I enzymes, respectively; 3μL (50ng/μL) of DNA; 5μL of ligated products diluted by 10 times; and 4μL of pre-amplification products diluted by 70 times.
2) 8 primer combinations were taken to generate 402 clearly identifiable bands of 144 individuals from 6 populations, and 289 bands were polymorphic (PPB=71.89%). Population HS (PPB=55.97%,h=0.2065, I=0.3047) had the highest genetic variation, whereas population GD (PPB=44.53%, h=0.1519,I=0.2284) had the lowest genetic variation.
3) The six populations of Larix principis-rupprechtii Mayr. had a high level of gene diversity (Ht=0.2245,Hs=0.1810, Gst=0.1936), distributed mainly within (80.64%) rather than among (19.36%) populations. The gene flow among populations was at a high probability (Nm=2.0821).
4) The Nei's unbiased measures (1978) of genetic identity (I) was ranged from 0.9269 to 0.9677, and the average was 0.9409. The genetic distance (D) was in the range of 0.0328-0.0759, and the average was 0.0610. While closely related, the six populations were not completely consistent. The highest genetic identity (I=0.9677) and lowest genetic distance (D=0.0328) was detected between populations GD and LY, while the lowest genetic identity (I=0.9269) and highest genetic distance (D=0.0759) was detected between population LL and HS. The result of UPGMA cluster analysis showed that six populations could be divided into three groups:WT and LL; LY and GD; HS and TY.
引文
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