Molecular evidence that the genes for dioecism and monoecism in Spinacia oleracea L. are located at different loci in a chromosomal region

Abstract

Spinach (Spinacia oleracea L.) is widely known to be dioecious. However, monoecious plants can also occur in this species. Sex expression in dioecious spinach plants is controlled by a single gene pair termed X and Y. Our previous study showed that a single, incompletely dominant gene, which controls the monoecious condition in spinach line 03-336, should be allelic or linked to X/Y. Here, we developed 19 AFLP markers closely linked to the monoecious gene. The AFLP markers were mapped to a 38.2-cM chromosomal region that included the monoecious gene, which is bracketed between flanking markers with a distance of 7.1 cM. The four AFLP markers developed in our studies were converted into sequence-characterized amplified region (SCAR) markers, which are linked to both the monoecious gene and Y and are common to both populations segregating for the genes. Linkage analysis using the SCAR markers suggested that the monoecious gene (M) and Y are located in different intervals, between different marker pairs. Analysis of populations segregating for both M and Y also directly demonstrates linkage of the genes at a distance of ~12 cM. The data presented in this study may be useful for breeding dioecious and highly male monoecious lines utilized as the pollen parents for hybrid seed production, as well as for studies of the evolutionary history of sexual systems in this species, and can provide a molecular basis for positional cloning of the sex-determining genes.

Figure 1

Crossing scheme to generate populations for mapping the monoecious gene (X^m or M) and the male-determining factor, Y. (a) 03–009 × 03–336 F₂ plants segregated for the monoecious gene. Index of femaleness (expressed as the percentage of female flowers per plant) for the parental, F₁ and F₂ plants grown during the autumn–winter season of 2008/2009 are given as mean with range in parentheses (Onodera et al., 2011). The symbol ‘X^m' or ‘M' is used to refer to the monoecious gene, depending on the assumption of the presence or absence of its allelism to the gene pair (X/Y) for dioecism. (b) 03–259♀ × 03–009♂ BC₁F₁ plants segregated for Y (Onodera et al., 2011). Genotypes at sex-linked SSR loci (SO4 and SO4b) located in a 5′ flanking region of the Nir gene (accession ID, X17031.1; Back et al., 1991) are also indicated. Alleles of SO4 and SO4b loci were named after numbers of the repeated-motif sequences (TTG and TCA, respectively).

Figure 2

Genetic linkage maps of the chromosomal region covering the sex-determining genes. The linkage maps were constructed from three different populations, 03–009 × 03–336 F₂, 03–259 × 03–009 BC₁F₁ and Test-cross population A (see Supplementary Figure 1). AFLP markers are indicated by ‘E' and ‘M' from the EcoRI/MseI selective primer pair (for example, E16M19) and SCAR markers are denoted by the prefix ‘SP'. In addition to the markers, SSR (SO4 and SO4b) on the 5′-flanking region of the Nir gene (Back et al., 1991) and male-specific DNAs (T11A and V20A; (Akamatsu et al., 1998) were also used for construction of the maps. Marker positions (cM) and names are written on the left and right sides of the map, respectively. Note that the linkage orders of SP_0008, Nir/SO4/SO4b, SP_0016, SP_0022 and SP_0007 are conserved across the populations.

Figure 3

Graphical genotypes and femaleness of F₃ families derived from two selections (F2-73 and F2-59) in 03–009 × 03–336 F₂. Black bars represent homozygous 03–336 segments, gray bars indicate heterozygous 03–009/03–336 regions, and white bars represent homozygous 03–009 segments. F₃ families are denoted by the prefix ‘F3'. Indexes of femaleness (expressed as the percentage of female flowers per plant) are indicated to the right of the bars. Marker positions (cM) and names are indicated above the bars.

Figure 4

Expected sex ratio in Test-cross population A and B. Assuming X/Y and M/m are located on an identical locus (no recombination occurs between X/Y and M/m; r=0), both test-cross populations can segregate at a ratio of one male to one monoecious. Alternatively, if X/Y and M/m are located on different loci (0<r<0.5), the crosses (Cross 2 and Cross 5) would give progenies segregating into three sex forms, that is, male, monoecious and female. See Supplementary Figure 1 for details of Cross 2 and Cross 5.