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Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants and has been applied in the commercial production of hybrid seeds. Two CMS lines A1 and A2 of maize were obtained previously by a transgenic experiment. In this study, we conducted cytological observation of developmental microspores with CMS line A1, A2 and their maintainer line (18 red) using paraffin section technology. We also analyzed DNA methylation levels at different developmental stages using high performance liquid chromatography (HPLC). Our results showed that the pollen abortion of A1 and A2 mainly happened from the tetrad stage to the middle of mononuclear stage. Another abortive phenomenon found in CMS line A2 occurred at the pollen mother cell stage. The DNA methylation level of leaf increased rapidly from the seedling stage to the shooting stage in 18 red, while it remained constant in A1 and A2. For the tassel, the DNA methylation levels in 18 red increased gradually during the anther development, while a peak of DNA methylation level occurred in A1 and A2 at the tetrad stage, corresponding to the abortion period of microspore. This result suggested that the level of DNA methylation in the tassels is associated with the pollen abortion characteristics in CMS lines. In summary, our results implied a connection between pollen abortion and epigenetic regulation in maize CMS.

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism (AFLP) to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP (methylation sensitive amplification polymorphism) ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5' -CCGG-3' sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.

Key message: Retrotransposon insertion in Brachytic2 generated a new incomplete recessive dwarf allele after spaceflight can moderately reduce plant height in heterozygous and potentially improve maize yield. Plant height and ear height are two important agronomic traits in maize breeding. In this study, two dwarf mutants short internode length1 (sil1) and short internode length2 (sil2) were obtained from two of 398 spaceflighted seeds of inbred line 18-599. The decrease in longitudinal cell number and cell length led to the shortened internodes of sil1 and sil2. A Ty1-copia LTR-retrotransposon, termed ZmRE-1, inserted in the fifth exon of Brachytic2 (Br2) was identified in sil1 and sil2 at exactly the same site, which indicated the transposition of ZmRE-1 probably correlated with the spaceflight. This new dwarf mutant allele was named as br2-sil in this study. The insertion of ZmRE-1 not only led to the loss of normal transcript of Br2 allele, but also reduced the transcript expression of br2-sil allele. Chop-qPCR displayed that the promoter region DNA methylation level of br2-sil allele in sil1 was higher than that of Br2 allele in WT-sil1. We speculated that the increased methylation level might downregulate the br2-sil expression. There was no difference in the seed-setting rate between sil1 and WT-sil1. Meanwhile, br2-sil could reduce plant and ear height effectively in Br2/br2-sil genotype without negative effects on grain yield. Therefore, the application of br2-sil in breeding has the potential to improve the grain yield per unit area through increasing the planting density. [ABSTRACT FROM AUTHOR]