Molecular isolation of the M gene suggests that a conserved-residue conversion induces the formation of bisexual flowers in cucumber plants

Abstract

Sex determination in plants involves a variety of mechanisms. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling gene M. M was identified as a previously characterized putative 1-aminocyclopropane-1-carboxylic acid synthase gene, while the m allele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.

Figure 1.—

M map-based cloning. (a) M genetic map. “X” indicates recombination events identified in population 5234 (the “right side” of the M locus) and population 1983 (the “left side” of the M locus), respectively. (b) Fine mapping M. New marker SNP8713, derived from the B87 end sequence, was mapped beyond the M locus with an additional recombination event. SSR70 was developed from the B70 inner sequence, which was mapped at one recombination event from M between S_ME8SA7 and SSR23487. SSR8501 and SSR8504, derived from the B85, had three and two recombination events, respectively, from the M locus at the other side beyond the SSR70 marker. The final contig was confirmed by an overlap test from STS877, STS867, and STS1399. BAC clones were not drawn to scale. (c) The final contig and the genomic organization for this region. A contig encompassing the M locus, which included three recombination events, was identified by the two markers, SSR8504 and SSR70. Broad arrows indicate the two predicted genes (p-CsPOD and CsACS2) and the predicted transcriptional orientations. Two new markers, S_CM2 and SNPCM2, were developed from the candidate gene CsACS2 and cosegregated with the M locus. The primer sequences are listed in Table S2.

Figure 2.—

Genomic structures of the M/m alleles. The monoecious plant line S52 had a unique 5-bp (CTATA) insertion/deletion in the second intron. Both of the unisexual parental lines, S52 and WI1983G, had the conserved “G.” The hermaphrodite lines, H34 and WI1983H, with an additional andromonoecious line, Lemon, shared the same framework with WI1983G, except for the nucleotide transition from G to T, which led to the missense mutation of Gly33Cys in the deduced amino acid sequence. Solid boxes represent the full-length mRNA sequence, and thin lines indicate introns. Dashed lines showed the conserved nucleotides in the dominant and recessive (marked with asterisk) alleles.

Figure 3.—

Alignment of the deduced amino acid sequence of ACS isozymes from cucumber, melon, and Arabidopsis thaliana. The conserved-residue conversions, G33C in C. sativus for the M/m gene and A57V in C. melo for the A/a gene, are shown above the alignment. Identical amino acids are against a solid background, and amino acid similarity levels >75% are against a shaded background.

Figure 4.—

Complementation of the E. coli integrative strain JAde 6 with various CsACS and AtACS7 cDNAs. Six plasmids were used to transform E. coli JAde 6 as described in File S1. The streaked sections in A and B correspond to the cDNAs in the pie chart. All the plates were grown at 37° overnight (on LB media) or for 3 days (on minimal media) and then photographed. (A) Growth of all six strains of JAde 6 on LB media for positive control. (B) Growth of all six strains of JAde 6 on minimal media.