Molecular Isolation of the M Gene Suggests That a Conserved-Residue Conversion Induces the Formation of Bisexual Flowers in Cucumber Plants

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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 themallele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.

SEX determination in angiosperms, including crop plants, evolves a variety of mechanisms that involve a number of different genetic and epigenetic factors (Tanurdzic and Banks 2004). Due to its diversity in sex types and to the extensive physiological and genetic studies conducted on it, cucumber (Cucumis sativus L.; 2n = 2x = 14) is becoming a model plant for sexdetermination research (Atsmon 1968; Tsao 1988; Perl-Treves 1999; Tanurdzic and Banks 2004). In cucumber plants, male and female flowers are generally produced separately in the same individual; however, certain lines also produce bisexual flowers. Preliminary genetic studies have indicated that three major genes are responsible for sex expression and segregation in the cucumber plantF/f, M/m, and A/a. The F gene may promote femaleness, while the m gene regulates the appearance of hermaphroditic flowers on the plant. Furthermore, in combination with the homozygous recessive f gene, the recessive a gene can intensify the androecious nature (Galun 1961; Robinson et al. 1976).

Sex expression in cucumber plants can also be modified by various environmental factors and plant hormones such as ethylene (Atsmon 1968; Takahashi et al. 1983; Takahashi and Jaffe 1984; Perl-Treves 1999; Yamasaki et al. 2005). A series of studies (Kamachi et al. 1997, 2000; Trebitsh et al. 1997; Yamasaki et al. 2003a;Mibus andTatlioglu2004;Knopf andTrebitsh 2006) have been conducted to investigate the F/f gene. These studies have shown that CsACS1G, which encodes a key enzyme of the ethylene-synthesis pathway, is the candidate gene for the F/f locus. However, the M/m gene has not been studied in as much detail as the F/f gene. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling M gene.

RESULTS

In the previous studies, the M/m locus was independently mapped into a genetic interval of 2.5 cM (Liu et al. 2008) and 6.1 cM (Li et al. 2008). In this study, we developed two larger segregating populations, which included 2830 F2 + BC1 (population 1983) and 2700 F2 (population 5234) individuals and constructed a highresolution collinear genetic map for the M/m locus (supporting information, Figure S1). After chromosome walking, a bacterial artificial chromosome (BAC) contig formed by two BAC clones (overlapped by an ~9.2-kb sequence) was found to be anchored to the genetic interval. The sequence of the entire contig covered an ~52-kb chromosome section, which had two complete candidate genes (Figure 1). One gene sequence showed limited similarity (68%) to a bacteriuminduced peroxidase precursor (GenBank accession no. AF155124) found in Gossypium hirsutum. However, the other gene, which was predicted to encode a 445-aminoacid protein, showed 100% sequence identity to the previously characterized CsACS2 gene, which encodes a putative 1-aminocyclopropane-1-carboxylate synthase in C. sativus (Kamachi et al. 1997).

The sequences of the entire genomic region of the putative peroxidase gene (p-CsPOD) and an ~2.0-kb 5' upstream region and 1.0-kb 3' downstream region were identical among the four parent lines. Therefore, we concluded that this putative peroxidase gene in cucumber was not the M gene.

We sequenced the entire genomic section of CsACS2 along with a 1.9-kb 5' upstream region and a 620-bp 3' downstream region from all four parental plants. These sequences revealed two types of polymorphism in the four lines used for mapping (Figure 2). First, a 5-bp insertion/deletion difference was found in the second intron between parent plants S52 and H34. …