| Cucurbitaceae family has currently become the model plants in sex determination research, since it could have all seven sex forms in angiosperms. However, researches of inheritance and mechanism of sex determination in watermelon (Citrullus lanatus) was still relatively poor, compared with melon (Cucumis melo) and cucumber (Cucumis sativus). In this paper, we generated five pairs of crossing groups constructed by four watermelon germplasms with three sex forms, monoecious, andromonoecious and gynoecious, to investigate inheritance of sex forms. Based on inheritance results, two members of sex determination genes, the andromonoecious (a) gene CitACS4 and the gynoecious (gy) gene ClWIP1 were cloned using the same population as for inheritance study. Functions of CitACS4 and ClWIP1 have also been studied in this paper. Our researches were expected to explore the molecular mechanism of sex determination in watermelon, and to provide theoretical and technical basis of elite breeding production. The main results are as following:Five pairs of crossing groups were constructed using four watermelon germplasms, XHB (monoecious), XHBGM (gynoecious), SL3H (andromonoecious) and AKKZW (andromonoecious). These five crossing groups were forwarded to investigate the inheritance of sex forms in watermelon. Through the investigation of sex forms of segregating population plants in spring and summer, we found that andromonecy was controlled by a recessive allele a and gynoecy was controlled by another recessive allele gy. We also identified that trimonoecious was controlled by a new recessive allele tm, and a allele is epistatic to tm allele. Moreover we showed that environmental factors in different seasons have no effect on sex forms, but they do affect sex expression on individual plants.Suggested by genomic data in watermelon, a 1-aminocyclopropane-l-carboxylate synthase (ACS) gene,CitACS4, was cloned by homologous cloning. The sequence analysis showed that, C364W mutation plants are co-segregated with andromonoecious sex forms in re-sequencing germplasms. A dCAPs marker wma_dCAPs was designed at the C364W position and the marker wma_dCAPs was co-segergated with andromonoecy suggested by the segregating and germplasms. The functional study of CitACS4 showed its specific expression in the carpel primordia of female and hermaphroditic flowers.CifACS4 was also shown to be associated with the abortion of stamen primordia in early developing stages. The mutation on C364W position destroyed the structure of CitACS4 protein, and then leads to the reduced activity of CitACS4 enzyme which hampered the abortion of stamen primordia and result in hermaphroditic flowers’ developing.Through comparison of the genomic re-sequencing data between AKKZW and XHBGM, polymorphism markers to target the genomic regions that are associated with gy locus positioned on Chromosome 2 (Chr.2) and Chromosome 3 (Chr.3), were identified. Suggested by bioinformatics study and verified by PCR and FISH analysis, a reciprocal chromosome translocation on the gynoecious mutant XHBGM was identified. The translocation breakpoints are located at the genomic positions Chr.2:33348296 and Chr.3:4043234..4043236. The translocation breakpoint on Chr.2 is in the coding region of a C2H2 zinc finger transcriptional factor gene (gene ID:Cla008537), which is the ortholog gene of melon g gene CmWIP1,and thus is named as ClWIP1. Functional studies showed that ClWIP1 expressed specifically in carpel primordia and is related with the abortion of carpel primordia in the early developing stages. The chromosome translocation in XHBGM destroyed the transcription of ClWIP1, hampered the abortion of carpel primordia and led to gynoecy. |
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