Fine Mapping Of The M Gene And Initial Research In Positive Cloning In Cucumber (Cucumis Sativus L.)

The sex type of plant is multi-gene regulated, which is the result of evolution. Variouspatterns of sexual expression in cucumber (Cucumis sativus L.) plants are advantageous forinvestigating the general mechanism of sex determination in higher plants. Studies show that,sex determination in cucumber (Cucumis sativus L.) plants is genetically controlled, largely,by F, M and A loci. The gene F promotes femaleness on the whole plant level while the geneM determines whether flowers are unisexual [M or bisexual (mm)]. Thus, the genotypeM_FF is gynoecious; the genotype M_Ff subgynoecious due to the incomplete dominance ofF; and the genotype mrnF is hermaphrodite. If F/f is homozygous recessive, the sexexpression is influenced by the gene A/a: MMffA genotypes are monoecious: MMffaagenotypes are androecious. Here we found out some molecular markers linked to M gene tofine map and clone it in future.In this study, the S52 and H34 Lines were used as the parents to develop an F₂ segregantpopulation, which composed of 167 individuals. Monoecious line S52 (ffMMAA) that wasfrom Dabie mountain of China was used as female parent, and hermaphroditic line H34(FFmmAA) that was from Europe was used as male parent. As to the segregation ofmonoecious character in the F₂ population, 46 individuals presented hermaphroditic and 121individuals presented monoecious. They were found to fit a segregantion ratio 3:1. Itconfirmed the monogenic inheritance of the monoecy/hermaphrodism in cucumber as waspreviously reported.A bulked segregant analysis (BSA) approach had allowed us to identify molecularmarkers lined to the M gene. We generated two bulks from the F₂ population, composed of 10plants, each one comprising monoecious plants and another with hermaphroditic plants. Atotal 736 SRAP primer combinations were used to screen DNA from the two parental lines(S52 and H34), and the result showed that 360 primer combinations of them (48.9%) werepolymorphic between two parents. And then DNA from the two bulks was screened withthese 360 SRAP primer combinations, the result showed that 8 SRAP primer combinations named: ME8SAT, MEIEM23, MEIEM24, MEIEM26, DCIEM9, ME67, ME42OD17,ME23SA4 had polymorphic bands between the two bulks. These 8 primer combinations werefurther analyzed in 167 F₂ individual plants, and the software Mapmaker3.0 was used toanalyze the genetic distance respectively. It was indicated that there were markers at the twosides of the M loci and the genetic distance between the 8 SRAP primers and M gene was 0,1.0, 5.4, 5.4, 8.3, 10.9, 21.6 and 25.5cM respectively.The polymorphic band amplified by the SRAP marker ME8SA7, MEIEM23,MEIEM24, ME1EM26 which linked closely were cloned and sequenced. Based on thesequence, pairs of SCAR primer were designed, which were all detected to get polymorphismbetween the parental lines and between the two bulks. Further analysis with 167 F₂ individualplants was indicated that these SCAR markers were mapped at the same loci with the randommarkers SRAP. To the co-segregated marker with the M loci, S ME8SA7, I developed anadditive enlarge population (F₂ population with 670 individuals and BC₁ population with 230individuals using the same parents, S52 and H34), and verify the recombinant events betweenthem. Unfortunately, I still had not found it. The PCR test with the marker S ME8SA7among 7 cucumber inbreds (of 5 were unisexual parents and 2 were bisexual parents) whichused widely in my lab was also indicated co-segregate. Above all, it illuminated that themarker S ME8SA7 linked closely to the M/m gene in the population from the two parentsS52 and H34, and it was conservative among different cultivated cucumbers. Further, I used itto screen the cucumber genomic BAC (bacterial artificial chromosome) library for thechromosome walking.