Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving “the travelling salesman problem”, and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.

Silene latifolia is a well-studied model system for plant XY sex determination. Three maleness factors are thought to function on the Y chromosome, gynoecium suppression factor (GSF), stamen-promoting factor (SPF), and male fertility factor (MFF), and their deletions result in hermaphrodites, anther defects, and pollen defects, respectively. Although a framework map of the Y chromosome exists, the sex determination genes have not been identified, and no markers close enough to potentially be used for BAC library screening are yet available. The analysis of Y deletion mutants by Y-chromosome-specific STS markers is an efficient way to isolate sex determination regions, but more Y-specific STS markers are needed to accelerate the exploration of sex determination factors. Herein, we report a marker design method that uses simple sequence repeats, which is especially effective on the Y chromosome of S. latifolia because it contains many simple sequence repeats. Six new Y-chromosome-specific STS markers were obtained, SmicSy1–6. These were used to detect relatively small Y deletion sites in heavy-ion beam irradiation-induced mutants. The mapping of male sex determination regions was narrowed down by using more markers and smaller-sized Y deletion mutants. One new marker, SmicSy6, is a proximal marker to SPF and, thus, a second index for SPF. The region including SPF is thought to be located between two SPF proximal markers. The flower phenotype correlates with the deletion size of SPF using SPF proximal markers. These findings represent new progress in isolating the sex determination factor, which has been studied for more than 50 years.

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The evolution of dioecy from hermaphroditism allows for avoidance of self-pollination, and its evolutionary background has been investigated both experimentally and theoretically since it was first proposed by Darwin. To reproduce this evolution, we screened hermaphroditic mutants of Silene latifolia using heavy-ion beam or γ-ray irradiation and characterized the phenotypes of their floral organs. Our scatterplots indicate severe deviations from the trade-off relationships between pollen and ovule numbers and between seed and germinated pollen numbers in hermaphroditic mutant S. latifolia. These deviations presumably led to promotion of dioecy from the ancestral state of S. latifolia. To infer the likely flower phenotypic characteristics of the ancestral plant of S. latifolia before evolving dioecy, the flowers of Silene viscosa, a naturally hermaphroditic plant related to S. latifolia were also characterized. S. viscosa exhibits both spatial separation of stamens from pistils within the flower (reverse herkogamy) and temporal separation of stamen and pistil maturation (dichogamy), raising the question of whether hermaphroditic mutant S. latifolia, which is thought to be the ancestral state, would possess these functions. We show that two hermaphroditic mutants of the dioecious plant S. latifolia exhibit signs of protogyny (reverse dichogamy) and approach herkogamy, as pistils were constantly longer than stamens. These findings illustrate the evolution of dioecy from hermaphroditism as a self-pollination avoidance mechanism and to balance the investments into male and female functions.

The dioecious plant Silene latifolia depends on nocturnal insects for pollination. To increase the chance of cross-pollination, pollen grains seem to be released and stigmas seem to be receptive simultaneously at night. We divided the floral development of S. latifolia into 1-20 stages, and determined the timetables of male and female function. The corolla of both male and female flowers opens at sunset (1900 hours) and closes at sunrise (0900 hours). To investigate the period of the reproductive phase of male and female function, we measured the germination rate on a pollen medium and the pollen germination rate on stigma during the period when stamens and stigmas were viable in the timetable. Male flowers had early- and late-maturing stamens that had the highest pollen viability, germination rate and pollen tube growth at midnight (0000 hours) at 1 day after flowering (DAF) and 0000 hours at 2 DAF. In contrast, female flowers maintained a germination rate of nearly 100 % from 1800 hours at 1 DAF to 1200 hours at 3 DAF. These results suggested that S. latifolia transferred the matured pollen grains from male flowers to female flowers only at night.