近日,《公共科学图书馆—综合》发表了中国农业科学院油料作物研究所博士后曾长立与合作导师伍晓明研究建立的能高通量检测叶绿体和线粒体基因组遗传变异的新方法。
据曾长立介绍,叶绿体和线粒体基因组作为植物细胞质基因组,对光合作用、呼吸作用等重要生命过程具有重要意义。
研究叶绿体和线粒体基因组变异不仅有助于揭示光合作用、呼吸作用、育性转变等重要生物学过程,还能提供有关植物起源进化和生态分布规律等重要信息。
然而,现有技术和方法很难高通量分析叶绿体和线粒体基因组变异,成为阻碍植物细胞质基因组研究深入发展的技术瓶颈。
为了解决这个问题,研究人员将基于CEL-I 酶切异源双链核酸分子策略的定向诱导基因组突变技术运用于检测细胞质基因组的单核苷酸多态性(SNPs)分析中,创建了适合高通量精准检测叶绿体和线粒体基因组遗传变异的“ORG-EcoTILLING”技术。
该技术能高通量、低成本、高效率地检测叶绿体和线粒体基因组DNA多态性,已成功应用于十字花科植物7个族、29个属和45个种的187份植物材料的3 个叶绿体基因(accD、matK 与rbcL)和1个线粒体基因atp6的检测中。
新方法攻克了阻碍检测大量个体叶绿体和线粒体基因组变异分析的技术瓶颈,不仅能用于研究植物叶绿体和线粒体基因遗传与功能变异,还能用于鉴定人类与动物线粒体基因DNA多态性,鉴定与疾病相关的线粒体基因突变,具有广泛应用前景。
2012年,该课题组研究人员已获得国家自然科学青年科学基金项目“甘蓝型油菜叶绿体基因组特定区域高分辨率单倍型图谱的构建与分析”资助,并将进一步拓展应用该方法,构建油菜叶绿体基因组单倍型图谱。
目前,这一能高通量检测叶绿体和线粒体基因组遗传变异的新方法的研究项目得到中国博士后科学基金项目和“973”计划的资助。(生物谷Bioon.com)
doi:10.1371/journal.pone.0047284
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PMID:
High-Throughput Discovery of Chloroplast and Mitochondrial DNA Polymorphisms in Brassicaceae Species by ORG-EcoTILLING
Zeng CL, Wang GY, Wang JB, Yan GX, Chen BY, Xu K, Li J, Gao GZ, Wu XM, Zhao B, Liu L.
BACKGROUND: Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used. METHODOLOGY/PRINCIPAL FINDINGS: To address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results. CONCLUSIONS/SIGNIFICANCE: These results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.