A Mutant Brassica napus (Canola) Population for the Identification of New Genetic Diversity via TILLING and Next Generation Sequencing

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.

本研究构建了包含3158个乙基甲磺酸（Ethyl Methanesulfonate，EMS）诱变株系的甘蓝型油菜（Brassica napus，俗称canola双低油菜）群体，并通过TILLING技术（Targeting Induced Local Lesions In Genomes，靶向诱导基因组局部损伤技术）验证该群体具备足够高的突变密度，可用于识别这一重要作物中目标基因的突变。TILLING技术是一种反向遗传学技术，已在多种动植物物种中成功应用。经典TILLING技术流程首先构建诱变群体，随后利用错配特异性核酸酶对DNA样本进行筛选，该酶仅能切割携带突变的PCR产物；后续再通过聚丙烯酰胺凝胶电泳检测，可视化任意目标基因中的突变位点。本研究利用该TILLING技术，在甘蓝型油菜品种DH12075（canola cv. DH12075）的26个不同基因中，共鉴定出432个独特突变位点。该群体的突变密度范围为每56 kb至每308 kb一个突变（因基因座而异），平均突变密度为每109 kb一个突变。此外，本研究还成功验证了下一代测序（Next Generation Sequencing，NGS）技术作为高效手段，可用于鉴定植物群体中的稀有突变，即便对于甘蓝型油菜这类多倍体物种亦适用。本研究鉴定出的多数突变体均已公开可获取。