MEMD Construction Procedure:

EMS Mutagenesis

B73 pollen was mutagenized with EMS and applied to the silks of B73 ears. The seeds harvested at 5-6 weeks post pollination were named M1. M1 seeds were planted and young leaves from each line were harvested for DNA sampling. The M1 were selfed to make M2 seeds, then families of twenty M2 siblings were planted and selfed to make the pooled seeds of M3.

Library Preparation

DNA samples isolated from M1 leaves were used to construct Illumina sequencing library. Exome enrichment of each sequencing library was performed using the Roche/NimbleGen Seq-EZ kit following NimbleGen's protocol. The exome captured libraries were resequenced by the Illumina HiSeq 2500.

Variation Calling and Annotation

SNP calling was done according to the Genome Analysis Toolkit (GATK, v2.1-9-gb90951c). The identified SNPs were categorized as variations in exonic, intronic, 5'UTR, 3'UTR and intergenic regions according to the maize genome annotation (release 5b) using SnpEff v3.6c.

Validation of EMS mutant

Generally we provide 20 M3-generation seeds for each mutation site of a single gene. These tweenty seeds may carry wild genotype (e.g. AA), homozygous (e.g. aa) or heterozygous mutation (e.g. Aa). A PCR-based method do need to be used to validate whether the mutation site was positive or negative. We call a mutation site positive if it is homozygous or heterozygous as shown in the following figure.

For convenience, we provide users the 1001-bp fasta sequence to design primers, which includes 500-bp upstream and 500-bp downstream of each SNP site from the B73 reference. The genome vesion that the 1001-bp from was kept as the same as that choosen when searching mutants.

We promise that we will provide another one allelic mutant for your interested gene if all these 20 lines were negative.