Gilchrist Erin J, Sidebottom Christine H D, Koh Chu Shin, Macinnes Tanya, Sharpe Andrew G, Haughn George W
Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada.
National Research Council Canada, Saskatoon, Saskatchewan, Canada.
PLoS One. 2013 Dec 20;8(12):e84303. doi: 10.1371/journal.pone.0084303. eCollection 2013.
We have generated a Brassica napus (canola) population of 3,158 EMS-mutagenised lines and used TILLING to demonstrate that the population has a high enough mutation density that it will be useful for identification of mutations in genes of interest in this important crop species. TILLING is a reverse genetics technique that has been successfully used in many plant and animal species. Classical TILLING involves the generation of a mutagenised population, followed by screening of DNA samples using a mismatch-specific endonuclease that cleaves only those PCR products that carry a mutation. Polyacrylamide gel detection is then used to visualise the mutations in any gene of interest. We have used this TILLING technique to identify 432 unique mutations in 26 different genes in B. napus (canola cv. DH12075). This reflects a mutation density ranging from 1/56 kb to 1/308 kb (depending on the locus) with an average of 1/109 kb. We have also successfully verified the utility of next generation sequencing technology as a powerful approach for the identification of rare mutations in a population of plants, even in polyploid species such as B. napus. Most of the mutants we have identified are publically available.
我们构建了一个由3158个经甲基磺酸乙酯（EMS）诱变的品系组成的甘蓝型油菜（油菜籽）群体，并利用定向诱导基因组局部突变（TILLING）技术证明该群体具有足够高的突变密度，可用于鉴定这种重要作物中感兴趣基因的突变。TILLING是一种反向遗传学技术，已在许多动植物物种中成功应用。传统的TILLING技术包括构建诱变群体，然后使用错配特异性核酸内切酶筛选DNA样本，该酶仅切割携带突变的PCR产物。随后使用聚丙烯酰胺凝胶检测来观察任何感兴趣基因中的突变。我们利用这种TILLING技术在甘蓝型油菜（油菜籽品种DH12075）的26个不同基因中鉴定出432个独特的突变。这反映出突变密度范围为1/56 kb至1/308 kb（取决于基因座），平均为1/109 kb。我们还成功验证了下一代测序技术作为鉴定植物群体中稀有突变的强大方法的实用性，即使在甘蓝型油菜这样的多倍体物种中也是如此。我们鉴定出的大多数突变体都是公开可用的。
