近日,，《公共科學(xué)圖書館—綜合》發(fā)表了中國農(nóng)業(yè)科學(xué)院油料作物研究所博士后曾長立與合作導(dǎo)師伍曉明研究建立的能高通量檢測葉綠體和線粒體基因組遺傳變異的新方法。
據(jù)曾長立介紹,，葉綠體和線粒體基因組作為植物細(xì)胞質(zhì)基因組,，對光合作用、呼吸作用等重要生命過程具有重要意義,。
研究葉綠體和線粒體基因組變異不僅有助于揭示光合作用,、呼吸作用、育性轉(zhuǎn)變等重要生物學(xué)過程，還能提供有關(guān)植物起源進(jìn)化和生態(tài)分布規(guī)律等重要信息,。
然而,，現(xiàn)有技術(shù)和方法很難高通量分析葉綠體和線粒體基因組變異，成為阻礙植物細(xì)胞質(zhì)基因組研究深入發(fā)展的技術(shù)瓶頸,。
為了解決這個問題,，研究人員將基于CEL-I 酶切異源雙鏈核酸分子策略的定向誘導(dǎo)基因組突變技術(shù)運(yùn)用于檢測細(xì)胞質(zhì)基因組的單核苷酸多態(tài)性（SNPs）分析中，創(chuàng)建了適合高通量精準(zhǔn)檢測葉綠體和線粒體基因組遺傳變異的“ORG-EcoTILLING”技術(shù),。
該技術(shù)能高通量,、低成本、高效率地檢測葉綠體和線粒體基因組DNA多態(tài)性,，已成功應(yīng)用于十字花科植物7個族,、29個屬和45個種的187份植物材料的3 個葉綠體基因（accD、matK 與rbcL）和1個線粒體基因atp6的檢測中,。
新方法攻克了阻礙檢測大量個體葉綠體和線粒體基因組變異分析的技術(shù)瓶頸,，不僅能用于研究植物葉綠體和線粒體基因遺傳與功能變異，還能用于鑒定人類與動物線粒體基因DNA多態(tài)性,，鑒定與疾病相關(guān)的線粒體基因突變,，具有廣泛應(yīng)用前景。
2012年,，該課題組研究人員已獲得國家自然科學(xué)青年科學(xué)基金項目“甘藍(lán)型油菜葉綠體基因組特定區(qū)域高分辨率單倍型圖譜的構(gòu)建與分析”資助,，并將進(jìn)一步拓展應(yīng)用該方法，構(gòu)建油菜葉綠體基因組單倍型圖譜,。
目前,，這一能高通量檢測葉綠體和線粒體基因組遺傳變異的新方法的研究項目得到中國博士后科學(xué)基金項目和“973”計劃的資助。(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.
