枇杷（Eriobotrya Lindl）核基因组DNA提取方法的改良及其SSR初步分析
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摘要
枇杷属蔷薇科(Rosaceae)枇杷属(Eriobotrya),原产我国,栽培品种为普通枇杷(E.japonica)。枇杷是一种亚热带常绿果树,秋萌冬花,春实夏熟,在百果中独具先天四时之气,被誉为独冠时新的佳果珍味。
枇杷幼叶表面的茸毛和内含的多糖和多酚等化合物常常影响基因组DNA提取的质与量,而用SSR分子标记构建枇杷的遗传图谱和基因定位时则需要对大量杂种后代个体进行DNA的提取。为此本实验探讨5种DNA提取方法,并改进了快速提取枇杷基因组DNA的方法,获得高质量基因组DNA。同时分别用以27对和36对适用苹果属扩增的SSR特异引物,对15种枇杷品种和两个杂交组合的90个后代单株进行了PCR扩增;分析了品种扩增片断多态性构建聚类关系树,统计了杂种后代等位性位点差异。结果表明:(1) 5种DNA提取方法中,改良CTAB法所提取的DNA具有良好的质量;(2) 27对引物15个品种中扩增出33个多态性片段,用NTYST2.0软件进行分析聚为六类;(3) 36对引物对两个杂交后代单株分别扩出35个和43个位点,可明确双亲均为纯合位点15个和29个,杂合位点分别为20个和14个,更多的位点有待进一步确定。这些结果为杂种后代群体进一步扩增,构建分子标记遗传图谱和性状分子标记奠定了基础。
Loquat (Eriobotrya japonica (Thunb) Lindl) is a subtropical evergreen fruit tree that blooms in fall and early winter. It belongs to the Maloideae subfamily of the Rosaceae beside apple or pear. The Maloideae, including loquat are functional diploids (2n =2x = 34) for which an allopolyploid origin has been suggested.Most major loquat cultivars derive from chance seedlings.
Restriction enzyme digestion and amplification of PCR to the genomic DNA extracted from loquat leaves will be often failed due to the contamination of polysaccharides and polyphenol compounds in the extracts and the tinsel of leaf surface. Furthermore when DNA molecular markers of SSR are used to construct the DNA molecular genetic maps or to map genes within a genome in hybridization progenies, it is essential to figure out a fast and efficient DNA extraction method for large numbers of sample individuals. In this experiment we compared and analysis five kinds of methods of extracting DNA, trying to improve the method of DNA extraction for high quality genomic DNA from loquat leaves. Then we adopted twenty-seven primers flank microsatellite sequences previously identified in Malus×domestica (Borkh.) were assayed in fifteen loquat cultivars and in progenies of two hybridization crosses.The amplification fragment polymorphism in the different cultivars was analyzed to construct the phylogenic tree and the numbers of allele sites were estimated in the hybridization progenies.
The results show that by the improved CTAB, we obtained high quality DNA needed the demands for PCR, comparing with the other four kinds of methods. Thirty-three polymorphic bands were amplified with the twenty-seven primers to the fifteen loquat cultivars. Unweighted Pair-group Method (UPGMA) analysis based on Nei’s genetic distance showed that the 15 genotypes were clustered into six groups. We use the thirty-six primers to amplify the progenies of the two different cross groups and we found thirty-five and forty-three amplification fragments were obtained respectively. Twenty-nine and fifteen alleles were definited, meanwhile twenty and twenty locus show polymorphic. These results will establish foundation for the further amplification of the hybridization progenies to construct genetic map of SSR molecular marker and to map the SSR marker with agronomic traits.
枇杷幼叶表面的茸毛和内含的多糖和多酚等化合物常常影响基因组DNA提取的质与量,而用SSR分子标记构建枇杷的遗传图谱和基因定位时则需要对大量杂种后代个体进行DNA的提取。为此本实验探讨5种DNA提取方法,并改进了快速提取枇杷基因组DNA的方法,获得高质量基因组DNA。同时分别用以27对和36对适用苹果属扩增的SSR特异引物,对15种枇杷品种和两个杂交组合的90个后代单株进行了PCR扩增;分析了品种扩增片断多态性构建聚类关系树,统计了杂种后代等位性位点差异。结果表明:(1) 5种DNA提取方法中,改良CTAB法所提取的DNA具有良好的质量;(2) 27对引物15个品种中扩增出33个多态性片段,用NTYST2.0软件进行分析聚为六类;(3) 36对引物对两个杂交后代单株分别扩出35个和43个位点,可明确双亲均为纯合位点15个和29个,杂合位点分别为20个和14个,更多的位点有待进一步确定。这些结果为杂种后代群体进一步扩增,构建分子标记遗传图谱和性状分子标记奠定了基础。
Loquat (Eriobotrya japonica (Thunb) Lindl) is a subtropical evergreen fruit tree that blooms in fall and early winter. It belongs to the Maloideae subfamily of the Rosaceae beside apple or pear. The Maloideae, including loquat are functional diploids (2n =2x = 34) for which an allopolyploid origin has been suggested.Most major loquat cultivars derive from chance seedlings.
Restriction enzyme digestion and amplification of PCR to the genomic DNA extracted from loquat leaves will be often failed due to the contamination of polysaccharides and polyphenol compounds in the extracts and the tinsel of leaf surface. Furthermore when DNA molecular markers of SSR are used to construct the DNA molecular genetic maps or to map genes within a genome in hybridization progenies, it is essential to figure out a fast and efficient DNA extraction method for large numbers of sample individuals. In this experiment we compared and analysis five kinds of methods of extracting DNA, trying to improve the method of DNA extraction for high quality genomic DNA from loquat leaves. Then we adopted twenty-seven primers flank microsatellite sequences previously identified in Malus×domestica (Borkh.) were assayed in fifteen loquat cultivars and in progenies of two hybridization crosses.The amplification fragment polymorphism in the different cultivars was analyzed to construct the phylogenic tree and the numbers of allele sites were estimated in the hybridization progenies.
The results show that by the improved CTAB, we obtained high quality DNA needed the demands for PCR, comparing with the other four kinds of methods. Thirty-three polymorphic bands were amplified with the twenty-seven primers to the fifteen loquat cultivars. Unweighted Pair-group Method (UPGMA) analysis based on Nei’s genetic distance showed that the 15 genotypes were clustered into six groups. We use the thirty-six primers to amplify the progenies of the two different cross groups and we found thirty-five and forty-three amplification fragments were obtained respectively. Twenty-nine and fifteen alleles were definited, meanwhile twenty and twenty locus show polymorphic. These results will establish foundation for the further amplification of the hybridization progenies to construct genetic map of SSR molecular marker and to map the SSR marker with agronomic traits.
引文
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