**2. Materials and Methods**

### *2.1. Mutagenesis, Phenotypic Screening and Plant Growth Conditions*

ML10 is the maternal parent of the popular elite hybrid LVN10 in Vietnam [31]. ML10 was used to create the mutagenized population. The inbred was developed by selfing followed by sib-mating and was released in 1994. The pedigree is unclear, but historical records indicate that it was developed from a CP hybrid (Charoen Pokphand Group, C.P. Tower, 313 Silom Road, Bangrak, Bangkok 10500, Thailand). BL10 is the paternal parent of hybrid LVN10, also developed by selfing and maintained by sib-mating. B73 is the reference inbred strain and was obtained from Professor David Jackson, Cold Spring Harbor Laboratory. Maize was planted in two seasons in Ha Noi, Vietnam (map coordinate: 21◦06'23.8" N 105◦49'34.7" E): for the spring season, sowing was done in January and for the autumn season, sowing was done in August. These sowing times were optimized as shown in Table 1. The EMS pollen mutagenesis was carried out according to [5]. In brief, maize pollen was collected in the morning from tassel bags that had been put on the tassels overnight. To prepare the EMS stock solution, EMS (Sigma M0880-1G) was diluted to 1% (v/v) in mineral oil (Sigma 69794-500ML); the bottle was covered in aluminum foil, and mixed thoroughly by a magnetic stirrer overnight before the mutagenesis experiment. This stock solution was used on the next day for making the final EMS solutions in mineral oil. About 12−20 mL pollen was incubated with 70 mL of mineral oil containing EMS at the concentration stated in the results for 30 min. The pollen mixtures were then pipetted onto the silks. The pollinated ears were covered with shoot bags. The concentration of EMS of 0.1% (v/v) was used to generate a large population of over 6000 M1 seeds. The resulting M1 plants were then grown on the field and selfed to create M2 families. 40 seeds per M2 ear were sown and scored for visible phenotypes. Researchers can obtain seeds from this mutant population and/or arrange a screen for the specific phenotype in this population for research/breeding purpose via a Material Transfer Agreement by directly contacting the corresponding author S.L.V.


