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首页> 外文期刊>New Zealand Journal of Forestry Science >Development of an SSR-based DNA fingerprinting method for black wattle (Acacia mearnsii De Wild)
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Development of an SSR-based DNA fingerprinting method for black wattle (Acacia mearnsii De Wild)

机译：基于SSR的DNA指纹识别方法对黑色荆棘（Acacia Mearnsii de Wild）

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Background: The most commonly used method for extracting DNA from plant leaf tissue involves cetyl trimethylammonium bromide but some species, such as Acacia mearnsii, contain high levels of secondary metabolites and polysaccharides that interfere with this process. Various modifications have been proposed for effective removal of these biomolecules but these methods can be time consuming. Therefore, this study was initiated to optimise the cetyl-trimethylammonium bromide protocol for the extraction of high-quality genomic DNA and to develop a fingerprinting tool using cross species transferable simple sequence repeat markers for genetic diversity studies in A. mearnsii. Methods: Five CTAB-based modification were examined and 49 cross-species microsatellite markers, developed for several Acacia species, were tested in four multiplex panels of A. mearnsii populations. Results: The modified protocol yields high quantity and quality DNA from A. mearnsii leaves using high concentration of NaCl to remove polysaccharides and polyvinylpolypyrrolidone (PVPP) to eliminate polyphenols during DNA purification. In addition, omitting the selective precipitation and NaCl gradient steps in the extraction protocol, enabled us to extract DNA 10–20 min faster than the normal protocol. Of the tested microsatellite loci, 11 were successful in amplifying sharp and high-intensity bands in all the four multiplex panels and were polymorphic. The level of polymorphism ranged from 0.115 to 0.794, with a mean 0.50 and mean number of alleles varied from 2 to 10, with overall mean of 6 alleles per locus. The mean observed and expected heterozygosity ranged from 0.058 to 0.970 and 0.102 to 0.796, respectively. The 11 microsatellite loci that were effectively amplified from A. mearnsii DNA were adequate in detecting genetic variation among the tested populations. Conclusions: These PCR-based, multi-allelic, co-dominant microsatellite markers provide a powerful tool for genetic, breeding and conservation studies in A. mearnsii.
机译：背景：从植物叶组织中提取DNA的最常用方法涉及十六烷基三甲基溴化物，但有些物种（例如金合欢）含有高水平的次级代谢物和多糖，其干扰该过程。已经提出了各种修改以有效去除这些生物分子，但这些方法可能是耗时的。因此，启动该研究以优化用于提取高质量基因组DNA的三甲基三甲基溴化铵方案，并使用交叉物种可转移简单序列重复标志物在A.Mearnsii中进行遗传多样性研究。方法：检查五种基于CTAB的修饰，在A.Mearnsii种群的四个多路复用板上测试了49种跨物种微卫星标志物，用于若干合并物种。结果：改性方案使用高浓度的NaCl从A.Mearnsii叶中产生高量和质量DNA，以除去多糖和聚乙烯醇吡咯烷酮（PVPP），以在DNA净化期间消除多酚。此外，省略提取方案中的选择性降水和NaCl梯度步骤，使我们能够比正常方案更快地提取DNA 10-20分钟。在测试的微卫星基因座中，11在所有四个多重板中的放大夏普和高强度的频带中成功，并且是多态的。多态性水平范围为0.115至0.794，平均值0.50，平均等位基因数量从2〜10变化，总均值为每位轨迹6等位基因。平均观察和预期的杂合子分别为0.058至0.970和0.102至0.796。从A.Mearnsii DNA有效扩增的11个微卫星基因座足以检测测试群体中的遗传变异。结论：这些基于PCR的多个等位基因共同的微卫星标记为A. Mearnsii的遗传，繁殖和保护研究提供了强大的工具。

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