Identifying crossover locations in an Arabidopsis thaliana Ler x pJ3-J3(G155R) Col/Ler backcross F1 population using genotyping by sequencing for male-specific crossover map

Genomic DNA from 96 Ler x pJ3-J3(G155R) Col/Ler backcross F1 population was extracted using CTAB and used to generate sequencing libraries as described (Rowan et al; Yelina et al), with the following modifications. DNA was extracted from 3 rosette leaves of 5 week old plants and 150 ng of DNA used as input for each library. DNA shearing was carried out for 20 minutes at 37°C with 0.4U of DNA Shearase (Zymo research). The barcoded adapters used for library construction are listed in Rowan et al. Each set of 96 libraries was sequenced on one lane of an Illumina NextSeq500 instrument (300-cycle Mid Output run). Sequencing data was analysed to identify crossovers as previously reported, using the TIGER pipeline.

采用CTAB法提取96份Ler × pJ3-J3(G155R) Col/Ler回交F1群体的基因组DNA（genomic DNA），并参照Rowan等人、Yelina等人已报道的方法构建测序文库，同时进行如下修改：该DNA提取自5周龄植株的3片莲座叶，每份文库的起始DNA投入量为150 ng；使用0.4U的DNA剪切酶（DNA Shearase，Zymo Research公司）在37℃条件下对DNA进行20分钟的剪切处理；文库构建所用的带条形码接头（barcoded adapters）详见Rowan等人的研究。每96份文库为一组，在一台Illumina NextSeq500测序仪的单个测序通道上完成测序，采用300循环中端输出运行模式（300-cycle Mid Output run）。参照已报道的方法，使用TIGER流程（TIGER pipeline）对测序数据进行分析，以识别交叉重组事件（crossovers）。