重庆市不同居群野百合的遗传多样性研究
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摘要
野百合(Lilium brownii F.E.Brown ex Miellez)为百合科百合属多年生草本,具有较高的观赏价值和食用价值,而且是百合育种中常用的杂交亲本之一。本实验以重庆市野百合为研究对象,首先选择了巫山县和巫溪县两地野百合为实验材料,建立并优化了野百合AFLP反应体系,然后对巫山县、巫溪县、城口县、江津区、金佛山和三泉镇六个地方的野百合居群进行了遗传多样性分析。得到如下结论:
(1)建立并优化了野百合AFLP反应体系。DNA提取时CTAB的最佳用量为4ml/g。粗提和纯化过程中抽提3次,冰上沉淀20分钟后采用低速离心(6000rpm左右)获得的野百合基因组DNA符合AFLP实验要求。酶切采用分步酶切各4小时,加入浓度为50pmol/ml的接头于16℃下连接过夜。连接产物稀释20倍作为预扩增模板,50ul体系加入1.0ul的dNTP,以72℃开始进行预扩增。扩增产物稀释30倍作为选择扩增模板。扩增体系和预扩增体系相同。最终扩增产物符合AFLP实验要求。
(2)从64对引物组合中,筛选出了适合野百合AFLP反应的20对引物组合,他们分别是:(1)E-AAC和M-CAC,(2)E-AAG和M-CAC,(3)E-AAG和M-CAG,(4)E-ACA和M-CAA,(5)E-ACA和M-CAG,(6)E-ACT和M-CAG,(7)E-ACC和M-CAA,(8)E-ACC和M-CAC,(9)E-ACC和M-CAG,(10)E-ACC和M-CTG,(11)E-ACG和M-CAA,(12)E-ACG和M-CAC,(13)E-ACG和M-CTG,(14)E-AGC和M-CAA,(15)E-AGC和M-CAC,(16)E-AGC和M-CAG,(17)E-AGC和M-CTC,(18)E-AGG和M-CAA,(19)E-AGG和M-CAC,(20)E-AGG和M-CAG。
(3)采用12对引物组合共获得了982条AFLP带,平均每对引物组合获得81.83条;其中6个居群所有个体共有带344条,平均每对引物组合共有带为28.66条,占总带数的35.03%;获得多态性带638条,平均每对引物组合为53.17条,占总带数的64.97%。从DNA水平揭示了同种之间的遗传共性和不同居群之间的遗传多样性。
(4)虽然本试验采用混合取样的方法,但是最终结果依然能发现居群内4个重复之间存在差异,它们的遗传距离在0.06—0.09之间,居群间所有重复之间的遗传距离在0.06—0.15之间,这从侧面反映了野百合个体之间存在着丰富的遗传多样性。
(5)通过遗传距离和相似性比较,最终得到各居群间的简单匹配系数均大于78%。用聚类分析方法最终将重庆市6个野百合居群分为东北和西南两支。其中分布于重庆市东北部的巫山、巫溪居群和城口县居群被归为东北支,分布于重庆市西南部的江津居群、三泉镇居群和金佛山居群被归为西南支。在两大支系内,地理分布距离较近的居群又先聚为一类。这都说明了重庆市野百合居群间遗传差异受地理分布影响较大。
Lilium brownii belongs to Lilium of Liliaceae in plant taxonomy.It is one of commonly—used cross breeding parents.And it was provided that it has great ornamental worth and edible worth. This experiment materials come from Wushan town,Wuxi town,Chengkou town,Jiangjin town, Jinfo mountain,Sanquan town in Chongqing city,China.First,select two populations which come from Wushan town and Wuxi town as experiment materials.Establish and optimize AFLP reaction system for Lilium brownii.Then,compare the genetic diversity between the 6 populations of Lilium brownii.The results were list as following:
(1)The AFLP reaction system for Lilium brownii was established and optimized.In DNA extraction process,the best ratio of CTAB and material was 4ml/g.the DNA should be extracted 3 times by the mixtion of trichlorpromazine and isoamylacohol(24:1).Incubate at 0℃for 20 minutes. Then,centrifuge in low speed(approximately 10,000rpm)for sever seconds.Discard the flow-through.The DNA was fit for AFLP.The DNA should be digested by EcorⅠand MselⅠ, every enzyme for 4 hours.The best adaptor concentration was 50 pmol/μl.The ligation products should be diluted 20 folds.1.0ul dNTP should be added into 50μl PCR reaction solution.And the pre—amplify reaction began at 72℃is better than 94℃.The pre—amplification products should be diluted 30 folds.The protocol of select-amplification was as same as pre—amplification.The products were fit for AFLP.
(2)20 pairs of primer combinations were screened from 64 EcoR1/Mse1 primer combinations. They were:(1)E-AAC/M-CAC,(2)E-AAG/M-CAC,(3)E-AAG/M-CAG,(4)E-ACA/M-CAA,(5) E-ACA/M-CAG,(6)E-ACT/M-CAG,(7)E-ACC/M-CAA,(8)E-ACC/M-CAC,(9)E-ACC/M-CAG, (10)E-ACC/M-CTG,(11)E-ACG/M-CAA,(12)E-ACG/M-CAC,(13)E-ACG/M-CTG,(14)E-AGC/M-CAA, (15)E-AGC/M-CAC,(16)E-AGC/M-CAG,(17)E-AGC/M-CTC,(18)E-AGG/M-CAA,(19)E-AGG/M-CAC, (20)E-AGG/M-CAG.
(3)In the AFLP experiment with 12 pairs of primer combinations,we've got 982 AFLP bands.81.83 bands per primer combination on average.In the 6 populations examineted,344 bands were obtained,28.66 bands per primer combination on average,and accounts for 35.03%in 982 total bands.638 polymorphic bands were obtained,53.17 per primer combination on average,and accounts for 64.97%.These results proved that the 6 Lilium brownii populations have the general character and genetic diversity.
(4)Although every sample contain several plants.There are still some differences existing in 4 samples of the same population.The genetic distance of the same population's Lilium brownii is between 0.06-0.09.All the Lilium brownii's genetic distance were between 0.06-0.15.The results proved that the Lilium brownii show great genetic diversity form different individual.
(5)Though the comparetion of genetic distance and similarity,we found that all the simple matching coefficients were more than 0.78.The 6 populations were divided into two parts.Wushan population,Wuxi population and Chengkou population belonged to the northeast part;Jiangjin population,Jinfo mountain population and Sanquan population belonged to southwest part.In the two parts,the two populations which were near in geographical position were considered as a new part first.Then,it was compared with the other population.All the results reported that the genetic distance was influenced by geography distance.
(1)建立并优化了野百合AFLP反应体系。DNA提取时CTAB的最佳用量为4ml/g。粗提和纯化过程中抽提3次,冰上沉淀20分钟后采用低速离心(6000rpm左右)获得的野百合基因组DNA符合AFLP实验要求。酶切采用分步酶切各4小时,加入浓度为50pmol/ml的接头于16℃下连接过夜。连接产物稀释20倍作为预扩增模板,50ul体系加入1.0ul的dNTP,以72℃开始进行预扩增。扩增产物稀释30倍作为选择扩增模板。扩增体系和预扩增体系相同。最终扩增产物符合AFLP实验要求。
(2)从64对引物组合中,筛选出了适合野百合AFLP反应的20对引物组合,他们分别是:(1)E-AAC和M-CAC,(2)E-AAG和M-CAC,(3)E-AAG和M-CAG,(4)E-ACA和M-CAA,(5)E-ACA和M-CAG,(6)E-ACT和M-CAG,(7)E-ACC和M-CAA,(8)E-ACC和M-CAC,(9)E-ACC和M-CAG,(10)E-ACC和M-CTG,(11)E-ACG和M-CAA,(12)E-ACG和M-CAC,(13)E-ACG和M-CTG,(14)E-AGC和M-CAA,(15)E-AGC和M-CAC,(16)E-AGC和M-CAG,(17)E-AGC和M-CTC,(18)E-AGG和M-CAA,(19)E-AGG和M-CAC,(20)E-AGG和M-CAG。
(3)采用12对引物组合共获得了982条AFLP带,平均每对引物组合获得81.83条;其中6个居群所有个体共有带344条,平均每对引物组合共有带为28.66条,占总带数的35.03%;获得多态性带638条,平均每对引物组合为53.17条,占总带数的64.97%。从DNA水平揭示了同种之间的遗传共性和不同居群之间的遗传多样性。
(4)虽然本试验采用混合取样的方法,但是最终结果依然能发现居群内4个重复之间存在差异,它们的遗传距离在0.06—0.09之间,居群间所有重复之间的遗传距离在0.06—0.15之间,这从侧面反映了野百合个体之间存在着丰富的遗传多样性。
(5)通过遗传距离和相似性比较,最终得到各居群间的简单匹配系数均大于78%。用聚类分析方法最终将重庆市6个野百合居群分为东北和西南两支。其中分布于重庆市东北部的巫山、巫溪居群和城口县居群被归为东北支,分布于重庆市西南部的江津居群、三泉镇居群和金佛山居群被归为西南支。在两大支系内,地理分布距离较近的居群又先聚为一类。这都说明了重庆市野百合居群间遗传差异受地理分布影响较大。
Lilium brownii belongs to Lilium of Liliaceae in plant taxonomy.It is one of commonly—used cross breeding parents.And it was provided that it has great ornamental worth and edible worth. This experiment materials come from Wushan town,Wuxi town,Chengkou town,Jiangjin town, Jinfo mountain,Sanquan town in Chongqing city,China.First,select two populations which come from Wushan town and Wuxi town as experiment materials.Establish and optimize AFLP reaction system for Lilium brownii.Then,compare the genetic diversity between the 6 populations of Lilium brownii.The results were list as following:
(1)The AFLP reaction system for Lilium brownii was established and optimized.In DNA extraction process,the best ratio of CTAB and material was 4ml/g.the DNA should be extracted 3 times by the mixtion of trichlorpromazine and isoamylacohol(24:1).Incubate at 0℃for 20 minutes. Then,centrifuge in low speed(approximately 10,000rpm)for sever seconds.Discard the flow-through.The DNA was fit for AFLP.The DNA should be digested by EcorⅠand MselⅠ, every enzyme for 4 hours.The best adaptor concentration was 50 pmol/μl.The ligation products should be diluted 20 folds.1.0ul dNTP should be added into 50μl PCR reaction solution.And the pre—amplify reaction began at 72℃is better than 94℃.The pre—amplification products should be diluted 30 folds.The protocol of select-amplification was as same as pre—amplification.The products were fit for AFLP.
(2)20 pairs of primer combinations were screened from 64 EcoR1/Mse1 primer combinations. They were:(1)E-AAC/M-CAC,(2)E-AAG/M-CAC,(3)E-AAG/M-CAG,(4)E-ACA/M-CAA,(5) E-ACA/M-CAG,(6)E-ACT/M-CAG,(7)E-ACC/M-CAA,(8)E-ACC/M-CAC,(9)E-ACC/M-CAG, (10)E-ACC/M-CTG,(11)E-ACG/M-CAA,(12)E-ACG/M-CAC,(13)E-ACG/M-CTG,(14)E-AGC/M-CAA, (15)E-AGC/M-CAC,(16)E-AGC/M-CAG,(17)E-AGC/M-CTC,(18)E-AGG/M-CAA,(19)E-AGG/M-CAC, (20)E-AGG/M-CAG.
(3)In the AFLP experiment with 12 pairs of primer combinations,we've got 982 AFLP bands.81.83 bands per primer combination on average.In the 6 populations examineted,344 bands were obtained,28.66 bands per primer combination on average,and accounts for 35.03%in 982 total bands.638 polymorphic bands were obtained,53.17 per primer combination on average,and accounts for 64.97%.These results proved that the 6 Lilium brownii populations have the general character and genetic diversity.
(4)Although every sample contain several plants.There are still some differences existing in 4 samples of the same population.The genetic distance of the same population's Lilium brownii is between 0.06-0.09.All the Lilium brownii's genetic distance were between 0.06-0.15.The results proved that the Lilium brownii show great genetic diversity form different individual.
(5)Though the comparetion of genetic distance and similarity,we found that all the simple matching coefficients were more than 0.78.The 6 populations were divided into two parts.Wushan population,Wuxi population and Chengkou population belonged to the northeast part;Jiangjin population,Jinfo mountain population and Sanquan population belonged to southwest part.In the two parts,the two populations which were near in geographical position were considered as a new part first.Then,it was compared with the other population.All the results reported that the genetic distance was influenced by geography distance.
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