Molecular Confirmation Of The Interspecific Hybrid Trait Of Cucumis×Hytivus Chen And Kirkbride And The Inheritance Analysis Of Organellar Genes In Interspecific Crossing

Cucumis X hytivus Chen and Kirkbride (2n=38) of genus Cucumis was created by Jin-feng Chen via interspecific crossing and embryo rescue, chromosome numbers were doubled through somaclonal variation. This new species was very useful in broadening the germplasm base of cultivated cucumber and transferring some valuable traits from the wild cucumis species to cultivated cucumber.In order to verify in molecular level that Cucumis X hytivus Chen and Kirkbride was originated from interspecific crossing between cultivated cucumber’Beijingjietou’and the wild Cucumis species and whether it still maintained hybridization trait after five generations breeding, in this study we selected cucumber cultivar’Beijingjietou’(C.sativus L.), the wild Cucumis species (C. hystrix Chakr.) and the S5progenies of C. hytivus Chen and Kirkbride.) as materials to investigate, the research results were as follow.1. The cloning and the analysis of6-phosphogluconate dehydrogenase gene (6PGDH) from cucumberDegenerated primers were designed according on conserved amino acids region of6-phosphogluconate dehydrogenase gene (6PGDH),a640bp in length cDNA fragment was amplified from Cucumis sativus’Beijingjietou’leaves through RT-PCR method, using fragment sequence as a querying probe, one highly homologous melon EST sequence AM715537.2was obtained from EST database. The full-length of cucumber6-phosphogluconate dehydrogenase gene(designated as Cs6PGDH) was obtained through RT-PCR method with primers designed basing on EST sequence and sequence assembling. Sequence analysis showed that Cs6PGDH (GenBank accession number was FJ610345) was1,829bp, which encompassed a1,488bp open reading frame (ORF) encoding495amino acid residues, no intron existed in ORF region and non-translation of5’and3’-terminal region were70bp,271bp, respectively. The results of Blast homologous analysis demonstrated that deduced amino acid sequence of Cs6PGDH had above74%identity with Arabidopsis thaliana, Glycine max, Oryza saliva, Zea mays, Spinacia oleracea et al. The expression of6PGDH was analyzed by semi-QRT-PCR indicating that the gene had expression in leaf, root and stem, the amount of expression was higher in heat stress than normal temperature, the result showed that expression of6PGDH was related to heat stress.2. Confirmation of the interspecific hybridization trait by nuclear gene6PGDHPrimers were designed based on the6PGDH gene of cucumber (GenBank accession number was FJ610345), fragments from allotetraploid, the wild Cucumis species and cucumber’Beijingjietou’were obtained in PCR reaction, respectively and sequences alignment were analyzed by DNAMAN software. Four polymorphic amino acid sites were detected among three species, the allotetraploid possessed three amino acids of polymor-phic sites from the wild Cucumis species, and one amino acid from cucumber ’Beijingjietou’; twenty-four polymorphic nucleotide sites were also detected in three species, the allotetraploid possessed eleven bases type of polymorphic nucleotide sites from cucumber’Beijingjietou’, ten bases type of polymorphic nucleotide sites from the wild Cucumis, two additive nucleotide sites possessed both different nucleotides sites from cultivar’Beijingjietou’and the wild Cucumis species simultaneously, while one base type of nucleotide sites differ from both species.The distribution of these polymorphic sites was congruence well with Mendel’s genetics law of inheritance. Therefore it was proven at molecular level that this synthetic allotetraploid species was the hybrid originated from the wild Cucumis species and cucumber’Beijingjietou’ interspecific crossing and still maintained hybridization trait after five generations breeding.To investigate the inheritance of organellar(mitochondrial and chloroplast) gene in Genus Cucumis, the Nad7, Nad1and cob gene fragments of mitochondrial DNA and the Matk-trnK and rbcL-accD gene region fragments of chloroplast DNA were amplified and sequenced from the S5progenies of the allotetraploid(Cucumis hytivus Chen and Kirkbride.), the wild Cucumis species(C. hystrix Chakr.) the maternal parents and cultivated cucumber(C. sativus L.’Beijingjietou’) the paternal parents, respectively. 3. The inheritance of mitochondrial(mt) genePolymorphic Loci in Nad7, Nad1and cob mt gene were detected among three species by DNA sequencing and comparison, the results showed that only one polymorphic locus existed in909bp length sequence of cob gene, the base type of polymorphic locus in allotetraploid was identical to cucumber’Beijingjietou’the paternal parents but different from the wild Cucumis species the maternal parents; seven polymorphic loci existed in943bp length intron sequence of NAD1gene, among them six base types of polymorphic loci in allotetraploid were identical to cucumber ’Beijingjietou’ but different from the wild Cucumis species, one base type of polymorphic locus was different from both parents; seventeen polymorphic loci were detected in880bp length intron sequence of NAD7gene, among them fourteen base types of allotetraploid were identical to cucumber ’Beijingjietou’ but different from the wild Cucumis species, three base types of polymorphic loci were different from both parents.This result revealed that mt genes was dominantly paternal transmission in hystrixxsativus interspecific crossing.4. The inheritance of chloroplast(cp) genePolymorphic Loci in Matk-trnK and rbcL-accD gene region of cp genomes were also detected among three species by DNA sequencing and comparison, the results showed that eighteen polymorphic loci existed in2,036bp length sequence of Matk-trnK region, among them the base types of sixteen loci in allotetraploid were identical to wild Cucumis species the maternal parents, only two loci’s base type in allotetraploid were identical to the cucumber’Beijingjietou’ the paternal parents; nineteen polymorphic loci existed in945bp length sequence of rbcL-accD region, among them the base types of eighteen Loci in allotetraploid were identical to wild Cucumis species only one base type of polymorphic locus was identical to the cucumber’Beijingjietou’.This study revealed that cp genes were dominantly maternal transmission in hystrixxsativus interspecific crossing.