中国苎麻属植物遗传多样性及其系统发育关系研究
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摘要
本论文以nrDNA的ITS序列为研究对象,分析了野生苎麻属植物的系统发育关系;同时还利用ISSR(简单重复序列间区)分子标记技术,探讨苎麻栽培品种间的遗传多样性。通过苎麻属植物亲缘关系的研究和苎麻栽培品种遗传多样性的分析,对于指导苎麻种质资源的搜集、保存、遗传育种与种质创新等均有十分重要的理论意义和应用价值。实验主要结论如下:
1.通过PCR扩增、测序、序列同源性比对及聚类分析,得到了苎麻14个种和8个变种植物以及1个外类群种的ITS序列。并进行了相应的系统发育分析。在苎麻14个种和8个变种植物以及1个外类群种的ITS序列中,ITS转录间隔区(包括ITS1和ITS2)内信息位点达28.9%。由此可见,整个苎麻属内,ITS区序列信息位点比较丰富。从分支支持率来看,属内各类群的划分均具有较高的支持率,这证明ITS区可以为解决苎麻属植物的系统发育问题提供较强的证据。
2.利用MP法和NJ法构建的系统发育树表明:外类群冷水花属的植物首先与苎麻属植物分开。而苎麻属14个种和8个变种的植物共可被分为四大类。其中,大叶苎麻组和序叶苎麻组植物聚成一类;帚序苎麻组植物和越南苎麻形成了第二个分支;苎麻组植物形成第三大组;而腋球苎麻单独成为一个分支,并与苎麻组植物具有较近的亲缘关系。它们的分支支持率都达到了50%以上。
3.苎麻ISSR分析的技术体系建立:25ul反应体系中,100ng模板DNA,0.4μmol/L的引物,1×PCR反应缓冲液,0.2mmol/LdNTP,1U的TaqDNA聚合酶,1.5mmol/LMgCl_2。反应程序为:94℃预变性5min;94℃变性45s,56℃退火45s,72℃延伸1min,35个循环;最后72℃保持7min。15个ISSR引物共扩增出258条带,其中多态性带247条,多态性比率(PPB)为95.82%。平均每个引物扩增出17.2条带。研究说明苎麻栽培品种间遗传差异较大。
4.依据ISSR标记谱带对供试苎麻栽培品种进行了UPGMA聚类分析,利用两条结合线可将56个苎麻品种划分成二个聚合等级。在遗传距离为0.635处的结合线可将56个苎麻品种分为5类。其中,湖南与湖北的51个苎麻品种聚为一类;贵州的2个苎麻品种聚为另一类;来自江西的2个苎麻品种和来自四川的1个品种各自成为一类。从分类结果可以看出,基本上相邻或相近地区品种的遗传距离较近,说明品种间的亲缘关系与地理位置密切相关。
Based on the difference in nrDNA ITS, we analyzed the phylogenetic relationships of Boehmeria; In the second part, by the usage of ISSR analyzing technique, we discussed the genetic diversities among 56 varieties of B.nivea. The thesis provide theoretical foundation for the evaluation, classification, selection of good rermplasm resources, utilization and protection. Major results were as follows:
1. The nrDNA ITS sequences of the 14 species and 8 varieties of Boehmeria and one outgroup were obtained by PCR and sequencing, which subsequently were used to analyze the phylogenetic relationships in the genus by homologous blast and clade anlaysis. In the data set of 14 species and 8 varieties of Boehmeria, the percentage of phylogenetically informative is 28.9% in ITS regions (including two spacers, without 5.8S subunit). According to the above statistics, there are relatively abundant informative sites in the ITS sequences of genus Boehmeria. According to the well bootstrap support among the most of clades, which proved that the ITS regions are valuable to resolve the phylogenetic relationships in Boehmeria.
2. According to the two ITS-based trees employing NJ and MP analyses: Pilea notata formed a single group, it has the most distant to the other species of Boehmeria. The 14 species and 8 varieties of Boehmeria formed four groups. The plants in Sect. Duretia and Sect. Phyllostachys formed the first monophyletic group; The plants in Sect. Zollingetrianae and B.tonkinensis formed the second monophyletic group; The plants in Sect. Tilocnide formed the third monophyletic group; B.malabarica formed a single monophyletic group, but it has the nearest distance to the plants of Sect. Tilocnide. Each clades has a well bootstrap support.
3. The establishment of an optimized PCR reaction system for ISSR analysis: PCR was performed in a 25ul reaction mixture with 100ng DNA, 0.4μmol/L primer, 0.2mmol/LdNTP, 1U Taq DNA polymerase, 1.5mmol/L Mg~(2+), 1×buffer. The reaction process went as following: the temperature profile used for PCR was 94℃for 5 minutes, followed by 35 cycles of 94℃for 45 seconds, 56℃for 45 seconds, 72℃for 1 second, and was terminated with a 7 minute DNA extension step at 72℃.
4. The primer sieving of B.nivea ISSR and amplification of PCR: in 56 experimental varieties, 258 DNA bands were amplified by 15 ISSR primers, in which 247 DNA bands were polymorphic, polymorphic ratio (PPB) was 95.82%. 17.2 bands were amplified by every primer. According to the widely distributing and adapting to various climate and soil condition of varieties of B.nivea, we can prove B.nivea heve strong adaption and genetic ability, potential of breeding amending. Based on the ISSR mark bands, the UPGMA cluster analysis was carried on, the results indicated that: the 56 varieties were divided into five groups by first combined line (D1=0.635). The fifty-one varieties in Hunan and Hubei province formed a group; the two varieties in Guizhou province formed the second group; the two varieties in Jiangxi and one variety in Sichuan formed three groups. The result was shown that there was relation of genetic relationship with geographical position.
1.通过PCR扩增、测序、序列同源性比对及聚类分析,得到了苎麻14个种和8个变种植物以及1个外类群种的ITS序列。并进行了相应的系统发育分析。在苎麻14个种和8个变种植物以及1个外类群种的ITS序列中,ITS转录间隔区(包括ITS1和ITS2)内信息位点达28.9%。由此可见,整个苎麻属内,ITS区序列信息位点比较丰富。从分支支持率来看,属内各类群的划分均具有较高的支持率,这证明ITS区可以为解决苎麻属植物的系统发育问题提供较强的证据。
2.利用MP法和NJ法构建的系统发育树表明:外类群冷水花属的植物首先与苎麻属植物分开。而苎麻属14个种和8个变种的植物共可被分为四大类。其中,大叶苎麻组和序叶苎麻组植物聚成一类;帚序苎麻组植物和越南苎麻形成了第二个分支;苎麻组植物形成第三大组;而腋球苎麻单独成为一个分支,并与苎麻组植物具有较近的亲缘关系。它们的分支支持率都达到了50%以上。
3.苎麻ISSR分析的技术体系建立:25ul反应体系中,100ng模板DNA,0.4μmol/L的引物,1×PCR反应缓冲液,0.2mmol/LdNTP,1U的TaqDNA聚合酶,1.5mmol/LMgCl_2。反应程序为:94℃预变性5min;94℃变性45s,56℃退火45s,72℃延伸1min,35个循环;最后72℃保持7min。15个ISSR引物共扩增出258条带,其中多态性带247条,多态性比率(PPB)为95.82%。平均每个引物扩增出17.2条带。研究说明苎麻栽培品种间遗传差异较大。
4.依据ISSR标记谱带对供试苎麻栽培品种进行了UPGMA聚类分析,利用两条结合线可将56个苎麻品种划分成二个聚合等级。在遗传距离为0.635处的结合线可将56个苎麻品种分为5类。其中,湖南与湖北的51个苎麻品种聚为一类;贵州的2个苎麻品种聚为另一类;来自江西的2个苎麻品种和来自四川的1个品种各自成为一类。从分类结果可以看出,基本上相邻或相近地区品种的遗传距离较近,说明品种间的亲缘关系与地理位置密切相关。
Based on the difference in nrDNA ITS, we analyzed the phylogenetic relationships of Boehmeria; In the second part, by the usage of ISSR analyzing technique, we discussed the genetic diversities among 56 varieties of B.nivea. The thesis provide theoretical foundation for the evaluation, classification, selection of good rermplasm resources, utilization and protection. Major results were as follows:
1. The nrDNA ITS sequences of the 14 species and 8 varieties of Boehmeria and one outgroup were obtained by PCR and sequencing, which subsequently were used to analyze the phylogenetic relationships in the genus by homologous blast and clade anlaysis. In the data set of 14 species and 8 varieties of Boehmeria, the percentage of phylogenetically informative is 28.9% in ITS regions (including two spacers, without 5.8S subunit). According to the above statistics, there are relatively abundant informative sites in the ITS sequences of genus Boehmeria. According to the well bootstrap support among the most of clades, which proved that the ITS regions are valuable to resolve the phylogenetic relationships in Boehmeria.
2. According to the two ITS-based trees employing NJ and MP analyses: Pilea notata formed a single group, it has the most distant to the other species of Boehmeria. The 14 species and 8 varieties of Boehmeria formed four groups. The plants in Sect. Duretia and Sect. Phyllostachys formed the first monophyletic group; The plants in Sect. Zollingetrianae and B.tonkinensis formed the second monophyletic group; The plants in Sect. Tilocnide formed the third monophyletic group; B.malabarica formed a single monophyletic group, but it has the nearest distance to the plants of Sect. Tilocnide. Each clades has a well bootstrap support.
3. The establishment of an optimized PCR reaction system for ISSR analysis: PCR was performed in a 25ul reaction mixture with 100ng DNA, 0.4μmol/L primer, 0.2mmol/LdNTP, 1U Taq DNA polymerase, 1.5mmol/L Mg~(2+), 1×buffer. The reaction process went as following: the temperature profile used for PCR was 94℃for 5 minutes, followed by 35 cycles of 94℃for 45 seconds, 56℃for 45 seconds, 72℃for 1 second, and was terminated with a 7 minute DNA extension step at 72℃.
4. The primer sieving of B.nivea ISSR and amplification of PCR: in 56 experimental varieties, 258 DNA bands were amplified by 15 ISSR primers, in which 247 DNA bands were polymorphic, polymorphic ratio (PPB) was 95.82%. 17.2 bands were amplified by every primer. According to the widely distributing and adapting to various climate and soil condition of varieties of B.nivea, we can prove B.nivea heve strong adaption and genetic ability, potential of breeding amending. Based on the ISSR mark bands, the UPGMA cluster analysis was carried on, the results indicated that: the 56 varieties were divided into five groups by first combined line (D1=0.635). The fifty-one varieties in Hunan and Hubei province formed a group; the two varieties in Guizhou province formed the second group; the two varieties in Jiangxi and one variety in Sichuan formed three groups. The result was shown that there was relation of genetic relationship with geographical position.
引文
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[2]陈家瑞,Friis I.,Wilmot-Dear C.M..Flora of China[M].科学出版社,2003,164-174.
[3]Jiang Y.B.(蒋彦波),Jie Y.C.(揭雨成).Advances in research on the genetic relationships of Boehmeria in Chinap[J].Journal of Plant Genetic Resources(植物遗传资源学报),2005,6(1):114-118.
[4]Yang R.F.(杨瑞芳),Guo Q.Q.(郭清泉).Studies on the Karyotype and Giemsa C—Banding paaem of wild resources of Boehmeria[J].China's Fiber Crops(中国麻作),2000,22(2):6-11.
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