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Flowering time is a complex trait important for crop adaptation to local environments and an essential breeding target to face the challenge of global climate change. A major quantitative trait locus (QTL) for flowering time and number of nodes (ND), qVgt3.05 (Vgt3), was previously identified on chromosome 3, bin 3.05, in a maize introgression library (IL) derived from the cross B73 x Gaspé Flint (recipient and donor genotypes, respectively. Salvi et al. 2011). In order to clone Vgt3, B73 was crossed with its early isogenic line 39-1-2-33 which carries a 17-cM Gaspé Flint introgression on bin 3.05. Using this cross, Vgt3 showed an addictive effect of 1.4 nodes, explained 56.6% of the phenotypic variance and was mapped within 0.3 cM. For positional cloning, a total of 7,500 F2 plants were phenotyped and genotyped with SNPs and SSR markers flanking the QTL interval. One-hundred recombinants lines were derived and the QTL was further narrowed the target genomic region to a 380-kb interval. A MADS-box gene with no coding sequence variation between the two alleles was found in the physical interval. However, the MADS-box gene RNA expression profile and transgenics testing confirmed its effect on flowering time. We are currently searching for the Vgt3 causative regulatory region by studying chromosome structural variation between the B73 and Gaspé Flint alleles.

Occasional co-localization of CENH3-GFP foci during microsporogenesis. Representative images of pWOX2-CENH3-GFP fluorescence in uninuclear stage Arabidopsis thaliana microspores. Most microspores clearly exhibit five distinct centromeric GFP foci (A and B), reflecting the haploid chromosome number in diploid Arabidopsis thaliana (2x = 10). However, in some microspores, a lower number of centromeric GFP foci is observed due to the spatial co-localization or Z-axis based projection overlap of two or more GFP foci (C and D; see arrows). Scale bar, 5 μm. (DOC 151 kb)

Sequences of primers used in this study. (DOC 25Â kb)

DAPI-stained chromosome spreads of 3x Arabidopsis male meiocytes. DAPI-stained chromosome spreads of somatic (A) and male meiotic (B, C and D) nuclei of triploid Arabidopsis thaliana plants (3x = 15). The somatic nucleus exhibits 15 distinct chromosomes, reflecting the triploid somatic chromosome number. Male meiotic chromosome spreads reveal the formation of five trivalents at diakinesis (B) and metaphase I (C), that inherently lead to an unbalanced segregation of homologous chromosomes at anaphase I (D; 8–7 chromosome segregation). Scale bar, 10 μm. (DOC 206 kb)

Expression of pLAT52-CENH3-GFP in mature pollen grains. CENH3-GFP expression driven by the pollen-specific LAT52 promoter in mature Arabidopsis thaliana pollen grains exhibits a strong fluorescent signal in the cytoplasm and the vegetative nucleus. Scale bar, 20 μm. (DOC 294 kb)

CENH3-GFP does not complement cenh3 −/− seed lethality. Genotypic analysis of F2 progeny resulting from several F1 cenh3-1/CENH3 plants that harbor the pWOX2-CENH3-GFP transgene reveals an absence of homozygous cenh3 −/− plants, indicating that cenh3 −/− seed fertility is not restored by the incorporation of the pWOX2 transcribed CENH3-GFP fusion protein. (DOC 25 kb)

Centromeric localization of CENH3 GFP-tailswap in different tissues. Representative images of the expression and subcellular localization of CENH3 GFP-tailswap in different tissues of Arabidopsis thaliana; including a young developing petal (A), a mature petal (B), unicellular stage microspores (C) and a bicellular stage microspore (E). Corresponding bright field images of the developing microspores are also presented (D and F). Scale bars, 20 μm. (DOC 241 kb)

Cenh3-1/CENH3 plants harboring pWOX2-CENH3-GFP show seed abortion. Representative pictures of siliques of Arabidopsis thaliana wild type (A), cenh3-1/CENH3 (B), and cenh3-1/CENH3 plants hemizygous for the pWOX2-CENH3-GFP transgene (C). Heterozygous cenh3-1/CENH3 plants harboring pWOX2-CENH3-GFP show a similar frequency of seed abortion as seen in the control cenh3-1/CENH3 plants, supporting the notion that pWOX2-CENH3-GFP does not complement cenh3 −/− seed abortion. (DOC 425 kb)

Frequency distribution of pWOX2-CENH3-GFP signals in nuclei of late uninuclear and binuclear megaspores isolated from triploid Arabidopsis thaliana plants. (DOC 54Â kb)

Expression pattern of pWOX2-CENH3-GFP in early megasporogenesis. Representative images of pWOX2-CENH3-GFP fluorescence in early stage megaspores of Arabidopsis thaliana, displaying either the haploid number of five centromeric GFP foci in uninuclear megaspores (A, B and C) or ten GFP signals in binuclear megaspores (D; nuclei are spatially overlapping). Scale bar, 5 μm. (DOC 291 kb)

CENH3-GFP co-localizes with dense DAPI stained chromocenters. Expression of p35S-CENH3-GFP in DAPI stained guard and epidermal cells reveals that the CENH3-GFP signals co-localize with dense DAPI stained chromocenters, indicating that the CENH3-GFP fusion protein is targeted to the centromeric chromosome region. Scale bar, 5 μm. (DOC 311 kb)