Table 1

Molecular markers used for gene mapping"

Table 2

Primers used for Mn1 specific InDel markers and RT-PCR"

Fig. 1

Morphological analysis of wild type and mn1-m2 mutant A: mature ears of wild type (WT), heterozygous (+/mn1-m2), and homozygous mn1-m2, and arrows indicate the mutant kernels segregated from ears of the heterozygous plants; B: three-week old seedlings; C: mature kernels; D: kernel endosperm and embryo of 25 days after pollination (DAP); E: statistical analysis of hundred-kernel weight. Bar: 5 cm (A) and 1 cm (C-D)."

Fig. 2

Paraffin section observation of WT and mn1-m2 kernels at 20 DAP A: the endosperm observation of WT kernels at 20 DAP; B: the endosperm observation of mn1-m2 kernels at 20 DAP; C: the magnification of the area marked as red rectangle in (A); D: the magnification of the area marked as red rectangle in (B). Bar: 500 μm (A-B) and 100 μm (C-D)."

Fig. 3

Map-based cloning of mn1-m2 gene The polymorphic SSR and In-Del (IDP) markers were developed between mn1-m2 and B73, using the amplified F2 mapping population and, sequencing of putative genes in the target region, the mn1-m2 locus was finally mapped to Mn1, which encodes a cell wall invertase. An hAT transposon (166 bp) was inserted into the first exon at 18 bp from ATG. The gene marked in red was Mn1. Black boxes represent exons and lines represent introns. n: the number of individuals; Recombinants: the number of recombinants."

Fig. 4

Relative expression level of mn1 and identification of hAT transposon insertion in mn1-m2 mutant A: the relative expression level of Mn1 in 15-DAP kernels of WT and mn1-m2, FPGS: reference gene; B: the schematic diagram shows that primers P1 and P2 of the developed In-Del marker flank the hAT transposon; C: the In-Del marker was completely linked with the mn1-m2 mutant phenotype. The length of the PCR products was 495 bp for WT, and 661 bp for mn1-m2. M: DNA marker DL2000."

Fig. 5

Allelism test of mn1-m2 and mn1-89 A: gene structure of Mn1 and mutant sites of the two mutants; B: the ear phenotype of mutant mn1-m2, mn1-89 and mn1-m2×mn1-89; C: the kernels of WT for mn1-m2; D: the kernels of mn1-m2; E: the kernels of mn1-m2×mn1-89; F: the kernels of mn1-89; G: the kernels of W22. Bar: 5 cm (B) and 1 cm (C-G)."

Fig. 6

Relative expression levels of Mn1 gene in various maize tissues by RT-PCR A: RNA was extracted from young ear, bract, silk, tassel, and flag leaf for detecting Mn1 expression level; B: RNA was extracted from embryo and endosperm of 13, 15, 19, 23, and 29 DAP for detecting Mn1 expression level. FPGS: the reference gene."