Characterization and map-based cloning of miniature2-m1, a gene controlling kernel size in maize

Kernel development plays an important role in determining kernel size in maize. Here we present the cloning and characterization of a maize gene, nitrate transporter1.5 (NRT1.5), which controls small kernel phenotype by playing an important role in kernel development. A novel recessive small kernel mutant miniature2-m1 (mn2-m1) was isolated from self-pollinated progenies of breeding materials. The mutant spontaneously showed small kernel character arresting both embryo and endosperm development at an early stage after pollination. Utilizing 21 polymorphic SSR markers, the mn2-m1 locus was limited to a 209.9-kb interval using 9 176 recessive individuals of a BC1 segregating population from mn2-m1/B73. Only one annotated gene was located in this 209.9 kb region, Zm00001d019294, which was predicted to encode nitrate transporter1.5 (NRT1.5). Allelism tests confirmed that mn2-m1 was allelic to miniature2-m2 (mn2-m2) and miniature2-710B (mn2-710B). The mn2-m1 and mn2-m2 alleles both had nucleotide deletions in the coding region resulting in premature termination, and the mn2-710B allele had some missence mutations. Subcellular localization showed that Miniature 2 (MN2) is localized in the plasma membrane. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of MN2 and some genes involved in the basal endosperm transfer layer (BETL) and embryo surrounding region (ESR) development were affected in mn2-m1 seeds. These results suggested that MN2 plays an important role in maize seed development.