基于ssr的黑荆树DNA指纹图谱方法的建立

IF 1.5 4区 农林科学 Q2 FORESTRY
M. Bairu, W. Coetzer, Amelework Assefa
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引用次数: 1

摘要

背景:从植物叶片组织中提取DNA最常用的方法是十六烷基三甲基溴化铵,但一些物种,如Acacia mearnsii,含有高水平的次级代谢产物和多糖,会干扰这一过程。已经提出了各种修饰来有效去除这些生物分子,但这些方法可能很耗时。因此,本研究旨在优化十六烷基三甲基溴化铵提取高质量基因组DNA的方案,并开发一种使用跨物种可转移简单序列重复标记的指纹图谱工具,用于a.mearnsii的遗传多样性研究。方法:对5个基于CTAB的修饰进行了检测,并在四个不同群体中测试了49个为几种Acacia物种开发的跨物种微卫星标记。结果:在DNA纯化过程中,用高浓度NaCl去除多糖,用聚乙烯吡咯烷酮(PVPP)去除多酚类物质,得到了大量、高质量的蜂蜜曲霉叶片DNA。此外,在提取方案中省略了选择性沉淀和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。在测试的微卫星位点中,11个在所有四个多重面板中成功扩增出尖锐和高强度的条带,并且是多态性的。多态性水平在0.115到0.794之间,平均0.50,等位基因的平均数量在2到10之间,每个位点的总体平均值为6个等位基因。观察到的平均杂合度和预期杂合度分别为0.058至0.970和0.102至0.796。从A.mearnsii DNA中有效扩增的11个微卫星位点足以检测受试群体之间的遗传变异。结论:这些基于PCR的多等位基因共显性微卫星标记为a.mearnsii的遗传、育种和保护研究提供了强有力的工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of an SSR-based DNA fingerprinting method for black wattle (Acacia mearnsii De Wild)
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.
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来源期刊
CiteScore
2.20
自引率
13.30%
发文量
20
审稿时长
39 weeks
期刊介绍: The New Zealand Journal of Forestry Science is an international journal covering the breadth of forestry science. Planted forests are a particular focus but manuscripts on a wide range of forestry topics will also be considered. The journal''s scope covers forestry species, which are those capable of reaching at least five metres in height at maturity in the place they are located, but not grown or managed primarily for fruit or nut production.
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