Cloning And Characterization Of Unisexual Flower-controlling M/m Gene In Cucumber (Cucumis Sativus L.)

Sex determination in plants, which determines the pattern ofbreeding and cultivation, is a fundamental developmental process and hasimportant encomomic value. Due to its diversity in sex types and to theextensive physiological and genetics studies, cucumber (Cucumis sativusL.) is becoming a model plant for sex-determination research. In additionto environmental factors and plant hormones,3major genes—F/f, M/m,and A/a—regulate the sex types in the cucumber plant. In spite ofinvolving a variety of mechanisms in planta, a single gene locus, M/mgene, controlling unisexual expression is unique to the cucumber plants.Recently, the gynoecious-influence gene F/f has been cloned, and showedthat it encodes homologies of1-aminocyclopropane-1-carboxylic acidsynthase (ACS), which is related with phytohormone ethylene. In thisstudy, beginning with finding out some molecular markers linked to M/mgene, I report a map-based cloning and characterization of the unisexualflower-controlling gene M/m, and its function and regulation pattern isstudied.By combining the bulked segregant analysis (BSA) and the sequence-related amplified polymorphism (SRAP) technology,8markers were identified to link to the M/m locus. These8markers were further analyzed in167F2individual plants, and among them, the2closely linked SRAP markers flanking the M/m locus were the co-dominant marker ME1EM26and the dominant marker ME1EM23. Further, the co-dominant marker ME8SA7co-segregated with the M/m locus. With the chromosome walking method using the cucumber genomic bacterial artificial chromosome (BAC) library, a co-dominant SCAR marker S_ME1EM23from the ME1EM23sequence was developed successfully. Along with the other2co-dominant SCAR markers S_ME1EM26and S_ME8SA7(developed from ME1EM26and ME8SA7, respectively) in a larger segregating population (900individuals), the M/m locus was mapped between S_ME1EM26(5.4cM) and S_ME1EM23(0.7cM), and S_ME8SA7co-segregated with it.After testing in the final population (2700),I found2recombination events between the marker S ME8SA7and the M/m locus. When scanning the BAC library, a BAC clone named B87containing this marker was found. After end sequencing, a SNP marker from the M13end was developed and located beyond the M/m gene with one more ecombination event. Using the other end sequence, a further BAC clone named B70was identified. After whole BAC sequencing, the M13end of B70was located in the previous B87, and a new SSR70marker eliminated1recombination event. At the other end, a DNA segment wasused to identify the third BAC clone named B85. With the whole B85sequence, a SSR8504marker was found to have2recombination eventsat the other side of the M/m gene with SSR70marker.After sequences assembly, with the three recombination events, a52kb interval was found to encompass M/m. Sequence analysis of the M/minterval indicated the presence of two putative genes. Based onco-segregation analysis in the total population, polymorphism testsamong cucumber plants with different sex phenotypes, and geneexpression analysis among one set of near-isogenic lines, M wasidentified as a previously characterized putative1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene, CsACS2.In a prokaryotic expression analysis with Escherichia coli JAde6, the mallele that mutated at a conserved site of the synthase lost activity in theoriginal enzymatically active allele, while the ACC synthase activity ofCsACS1G, the well-characterized gynoecious controlling F gene, wasdemonstrated. Expression analysis of CsACS2revealed that endogenousethylene, which might derive from F or M itself, can activate theexpression of M gene.