Fine-Mapping And Transcription Expression Profiling For Sex Determination In Cucumber

Sex differentiation in plants is a fundamental developmental process of economic importance since the sexual phenotypes of crops determine the processes of breeding and cultivation. The diverse sex types of cucumber allow this organism to serve as a model system for studying sex expression in flowering plants. The cucumber has three types of flowers:male, female, and bisexual. Morphologically, all cucumber floral buds are initially hermaphroditic with both male and female reproductive organs. Pistil development is then arrested in floral buds that develop into male flowers, whereas stamen development is arrested in floral buds that develop into female flowers. Bisexual flowers form from the buds in which neither pistil nor stamen development is arrested. Three primary genes that influence sex expression in cucumber have been described including F, M and A. In addition, sex expression is also influenced by environmental conditions and plant hormones. Long days, high temperatures, and gibberellic acid promote the formation of male flowers, whereas short days, low temperatures, auxins, and ethylene enhance the development of female flowers. The model of sex determination in cucumber is presumed that ethylene serves as both a promoter of the female sex and an inhibitor of the male sex. The model predicts that the F gene encodes a molecule that influences the range and gradient of ethylene production along the shoot, thereby acting to promote femaleness, whereas the M gene encodes a molecule that detects this ethylene signal and inhibits stamen development when ethylene levels reach a threshold. Recent studies have provided molecular evidence in favor of the ethylene model of sex determination in cucumber: the F gene encodes an ACC synthase, is cloned. The result is accord with above ethylene model of sex determination. In addition, the product of the M locus mediates directly the inhibition of stamen development by ethylene. For further testify the authenticity of the ethylene model, the M gene must be cloned and fine-mapping is the basic of map-based cloning. In this study, nearly isogenic cucumber lines WI1983G （gynoecious; MMFF） and WI1983H （hermaphrodite; mmFF）, F2 and BC1 individual plants were used to clone Mgene and analysis transcription expression profiling of female and bisexal flower. This study provides theoretic evidences for further understanding the ethylene fuction for sex determination in cucumber, revealing the evolutional history of sex expression in Cucurbitaceae and controling sex expression of other crops for speedup the utilization of heterosis by the way of gene engineering. The main conclusions are as follows:1. The mother plant WI1983G bears only pistillate flowers, whereas the mother plant WI1983H bears bisexual flowers. All F1 progeny that were derived from the cross between WI1983G and WI1983H lines bore pistillate flowers. This finding confirmed the complete dominance of the maternal sex type over the paternal sex type. In the 638 F2 individuals, 473 had only pistillate flowers and were scored as gynoecious （M_FF）. In addition,165 F2 individuals had only bisexual flowers and were, thus, scored as hermaphroditic （mmFF）. These results fit the Mendelian 3:1 ratio （x2= 0.38; p> 0.5）; In the 751 BC1 individuals, 373 had only pistillate flowers.378 individuals had only bisexual flowers. These results also fit the Mendelian 1:1 ratio（x2=0.03; p>0.95）, indicating the single M locus controlled the segregation of the sex expression in the F2 and BC1 progeny. The result is consonant with the conclusion of others’study.2. we obtain four AFLP markers were linked to the M locus using high-throughput AFLP technology. The local map spanned a genetic interval of 5.0 cM, which was defined by the AFLP markers PGGMCCC₄50/453 on one side and PGTMCTA₁85 on the other side. The M locus co-segregated with the CsEIL1 marker （no recombinants in 96 F2 individuals）, which developped according to the pivotal gene EIL1 in ethylene singal, and was flanked by the AFLP markers EACAMCAT₂02/203 and EATGMCAA₃80 that defined a 2.5 cM interval. The linkage map provides a solid basis for high-resolution mapping and ultimately for molecular isolation of the M gene.3. With flanking markers （PGGMCCC₄50/453 and PGTMCTA₁85） of the M gene, recombinant plants in M interval were identified from 1984 F2 and 751 BC1 segregating population and we obtain 6 AFLP,8 SSR,2 SCAR,1 CAPS markers by the way of screening 896 AFLP primer combinants,2000 SSR primers, relating to superscaff sequence information. A high-resolution genetic map of the M gene was constructed. The M gene was delimited into a genetic interval of 0.2 cM between SSR23487 （0.1 cM） and S_ME8SA7 （0.1 cM）. A SNP marker SN1, co-segregating with the M gene based on the linkage analysis （no recombinants in 2,080 F2 and 751 BC1 individuals）, was obtained by the way of chromosome walking.4.199,032 ESTs in female flower （WI1983G） and 176,139 ESTs in bisexal flower （WI1983H） were obtained from sequencing of cDNA. Sequence assembly by Phred/Phrad/Consed software revealed that 23,627 contigs were acquired except for 32521 singlet sequences in female flower （WI1983G） and 33494 singlet sequences in bisexal flower （WI1983H）.1,256 Unigenes, which have distinctly different expression （p<0.05） between female and bisexal flower, were ultimately found out using blast （Nr, non-redundant database） and chi test in the IDEG.6 software. These genes involved in several aspects such as substance transport, energy metabolism, signaling, stress response et al. In addition, there are mostly ESTs with unknown functions （No hits）.1,256 Unigenes, which have distinctly different expression （p<0.05） between female and bisexal flower, were done with blast, EST annotation and GO classification. By homology search and gene ontology analyses,435 identified ESTs, which were annotated, were mainly categorized as belonging to:Cellular Component （108,36% involving with nucleus, membrane, mitochondrion et al.）, Molecular Function （155,51% including seven kinds of molecular activity）, and Biology Process （39,13% involving with signaling pathway, biosynthesis, metabolization et al.）.The fine-mapping of M gene, which privides a basis for map-based cloning M gene, was constructed based on the nearly isogenic lines and AFLP, SSR molecular marker et al.By sequence of about 380,000 ESTs in female flower （WI1983G） and bisexal flower （WI1983H） and study of related transcription expression profiling for sex determination in cucumber, the biological processes in which these related genes for sex determination involved in came to be primary understood. In addition, partial unknown-fuction annotation ESTs were obtained in our study, in which provides more candidate genic resources for our further studies the regulative mechanism of sex expression in cucumber.