福建省橄榄（Canavium album Raeusch）种质资源的ISSR分析
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摘要
橄榄(Canarium album Raeusch)属橄榄科(Burseraceae)橄榄属(Canarium L.),是我国南方特产水果之一,已有2000多年的栽培历史。橄榄原产于中国,主要分布于广东、福建、台湾、四川、浙江、广西和海南等省。目前对橄榄的研究主要集中在栽培、生理、药用成分分析等方面,在分子生物学方面的研究较少。
本研究以福建省60份橄榄遗传资源为材料,摸索出适合橄榄叶片基因组DNA的提取方法;建立并优化了橄榄ISSR反应体系;筛选出17条条带清晰,多态性好的ISSR引物;采用ISSR标记,结合聚类分析,进行了福建省橄榄遗传资源的亲缘关系与遗传多样性研究。为橄榄新品种的培育提供遗传背景,为橄榄种质资源的有效保护和合理利用提供科学依据。主要研究结果如下:
1.橄榄富含酚类化合物和多糖等物质,易导致DNA提取和纯化困难。针对此问题,本研究采用改良CTAB法提取橄榄基因组DNA,数量和纯度均可满足ISSR分析要求,探索出一套操作简便、快速的橄榄DNA提取和纯化技术。
2.建立了橄榄ISSR-PCR反应的技术体系。在20μL的反应体系中,采用40 ng的模板DNA,0.2 mmol/L dNTPs,0.25μmol/L ISSR引物,1 U Taq DNA聚合酶为橄榄1SSR-PCR扩增条件的最佳选择。按照此体系,在94℃预变性5 min,94℃变性30 s,最适退火温度退火60 s,72℃延伸90 s,40个循环,72℃延伸10 min。该体系扩增出的产物条带清晰易辨,效果良好,能够满足ISSR分析的要求。
3.从96条ISSR随机引物中筛选出17条,对60个橄榄品种进行了ISSR-PCR扩增。共得到151条带,其中多态性条带有102条,多态性百分率为67.6%。同时研究中发现含(TC)n、(CA)n序列的ISSR引物在对橄榄DNA扩增时多态性水平较高,说明橄榄因组中富含(AG)n/(GT)n序列。
4.对供试的橄榄品种进行聚类,分析结果表明:60份橄榄品种之间的遗传相似系数在0.819～0.991之间,揭示了不同橄榄品种之间的亲缘关系与遗传相似性。为以后有选择、有目的地引种、育种,缩短育种年限,提高育种效率奠定理论基础。
5.分析的到橄榄种群的遗传分化系数为GST=0.2529,说明橄榄的遗传分化主要存在于种群内,种群间的分化较小。根据遗传分化系数估算的基因流的值是Nm=1.4773,大于1,表明种群间存在一定的基因交流。
Olive(Canarium album Raeusch) belongs to Burseraceae Canarium L., which originated in China. It is one of the most well-known special fruit trees in south of China, which is distributed in Guangdong, Fujian, Taiwan, Sichuan, Zhejiang, Guangxi and Hainan . Olive has been cultivated in China more than 2000 years. Research on olive mainly focus on cultivation, physiology and medicine composition analysis, etc. But it is seldom that research in molecular biology.
Taking 60 Olive genetic resources in fujian as material, this research explored a extraction method for Olive leaf genomic DNA; The Olive ISSR reaction system is established and optimized; 17 primers have been selected which have clear and high polymorphism; Relationship and genetic diversity of fujian Olive genetic resources has been researched by ISSR combined with clustering analysis. The research provided scientific basis for effective protection and rational utilization of Olive germplasm resources. The main results were as follows:
1. Facing the fact that it is difficulty to extract DNA from Olive which contains phenolic compound and quite a lot of polysaccharose, the researcher used improved CTAB method to extract DNA of Olive, which meets ISSR requirements in quantity and purity. A set of simple and quick method for olive DNA extraction and purification technology has been found.
2. The technological system of ISSR-PCR reaction of Olive has been established. In the 20μL reaction system, the best condition of Olive 1SSR-PCR amplification is 40 ng DNA template, 0.2 mmol/L dNTPs, 0.25μmol/L primer, 1U Taq DNA polymerase, 2.0μL 10×Buffer solution. According to the system, the PCR amplification steps are as follow: 5 min predenaturation at 94℃,30 seconds denaturation at 94℃, 60 seconds annealing, 90 seconds extension at 72℃. The cycle repeats forty times and ends in 10 min extention at 72℃. The system can amplified clear and good effect products, which can meet ISSR analysis.
3. 17 ISSR primers were selected from 96 ISSR primers, and 60 Olive germplasm were researched by ISSR method. 151 amplification clips were detected,of which 102 were polymorphic clips, and polymorphic rate is 67.6% . In this research we found that primers containing(TC)n, (CA)n sequence resulted in higher levels of amplified polymorphism of Olive. This is due to the abundant content (AG)n/(GT) n sequence in Olive genome.
4. According to results of the clustering analysis to 60 Olive varieties by ISSR.:It proved that the genetic similarity index was between 0.819～0.991. From the result, genetic similarity and relationship among different olive breeds were revealed. It laid theoretical basis for breeding and species introduction on purpose, shortening the breeding period and improving the efficiency of breeding.
5. GST of the olive population is 0.2529 .The result showed that olive genetic differentiation mainly exists in the population . According to GST ,the gene flow was estimated Nm=1.4773 ,greater than 1 .It indicated that there exist certain gene exchange between species.
本研究以福建省60份橄榄遗传资源为材料,摸索出适合橄榄叶片基因组DNA的提取方法;建立并优化了橄榄ISSR反应体系;筛选出17条条带清晰,多态性好的ISSR引物;采用ISSR标记,结合聚类分析,进行了福建省橄榄遗传资源的亲缘关系与遗传多样性研究。为橄榄新品种的培育提供遗传背景,为橄榄种质资源的有效保护和合理利用提供科学依据。主要研究结果如下:
1.橄榄富含酚类化合物和多糖等物质,易导致DNA提取和纯化困难。针对此问题,本研究采用改良CTAB法提取橄榄基因组DNA,数量和纯度均可满足ISSR分析要求,探索出一套操作简便、快速的橄榄DNA提取和纯化技术。
2.建立了橄榄ISSR-PCR反应的技术体系。在20μL的反应体系中,采用40 ng的模板DNA,0.2 mmol/L dNTPs,0.25μmol/L ISSR引物,1 U Taq DNA聚合酶为橄榄1SSR-PCR扩增条件的最佳选择。按照此体系,在94℃预变性5 min,94℃变性30 s,最适退火温度退火60 s,72℃延伸90 s,40个循环,72℃延伸10 min。该体系扩增出的产物条带清晰易辨,效果良好,能够满足ISSR分析的要求。
3.从96条ISSR随机引物中筛选出17条,对60个橄榄品种进行了ISSR-PCR扩增。共得到151条带,其中多态性条带有102条,多态性百分率为67.6%。同时研究中发现含(TC)n、(CA)n序列的ISSR引物在对橄榄DNA扩增时多态性水平较高,说明橄榄因组中富含(AG)n/(GT)n序列。
4.对供试的橄榄品种进行聚类,分析结果表明:60份橄榄品种之间的遗传相似系数在0.819～0.991之间,揭示了不同橄榄品种之间的亲缘关系与遗传相似性。为以后有选择、有目的地引种、育种,缩短育种年限,提高育种效率奠定理论基础。
5.分析的到橄榄种群的遗传分化系数为GST=0.2529,说明橄榄的遗传分化主要存在于种群内,种群间的分化较小。根据遗传分化系数估算的基因流的值是Nm=1.4773,大于1,表明种群间存在一定的基因交流。
Olive(Canarium album Raeusch) belongs to Burseraceae Canarium L., which originated in China. It is one of the most well-known special fruit trees in south of China, which is distributed in Guangdong, Fujian, Taiwan, Sichuan, Zhejiang, Guangxi and Hainan . Olive has been cultivated in China more than 2000 years. Research on olive mainly focus on cultivation, physiology and medicine composition analysis, etc. But it is seldom that research in molecular biology.
Taking 60 Olive genetic resources in fujian as material, this research explored a extraction method for Olive leaf genomic DNA; The Olive ISSR reaction system is established and optimized; 17 primers have been selected which have clear and high polymorphism; Relationship and genetic diversity of fujian Olive genetic resources has been researched by ISSR combined with clustering analysis. The research provided scientific basis for effective protection and rational utilization of Olive germplasm resources. The main results were as follows:
1. Facing the fact that it is difficulty to extract DNA from Olive which contains phenolic compound and quite a lot of polysaccharose, the researcher used improved CTAB method to extract DNA of Olive, which meets ISSR requirements in quantity and purity. A set of simple and quick method for olive DNA extraction and purification technology has been found.
2. The technological system of ISSR-PCR reaction of Olive has been established. In the 20μL reaction system, the best condition of Olive 1SSR-PCR amplification is 40 ng DNA template, 0.2 mmol/L dNTPs, 0.25μmol/L primer, 1U Taq DNA polymerase, 2.0μL 10×Buffer solution. According to the system, the PCR amplification steps are as follow: 5 min predenaturation at 94℃,30 seconds denaturation at 94℃, 60 seconds annealing, 90 seconds extension at 72℃. The cycle repeats forty times and ends in 10 min extention at 72℃. The system can amplified clear and good effect products, which can meet ISSR analysis.
3. 17 ISSR primers were selected from 96 ISSR primers, and 60 Olive germplasm were researched by ISSR method. 151 amplification clips were detected,of which 102 were polymorphic clips, and polymorphic rate is 67.6% . In this research we found that primers containing(TC)n, (CA)n sequence resulted in higher levels of amplified polymorphism of Olive. This is due to the abundant content (AG)n/(GT) n sequence in Olive genome.
4. According to results of the clustering analysis to 60 Olive varieties by ISSR.:It proved that the genetic similarity index was between 0.819～0.991. From the result, genetic similarity and relationship among different olive breeds were revealed. It laid theoretical basis for breeding and species introduction on purpose, shortening the breeding period and improving the efficiency of breeding.
5. GST of the olive population is 0.2529 .The result showed that olive genetic differentiation mainly exists in the population . According to GST ,the gene flow was estimated Nm=1.4773 ,greater than 1 .It indicated that there exist certain gene exchange between species.
引文
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