Nuclear/Cytoplasm Genotyping And Analysis Of Transcriptomics And Metabolomics In Remote Somatic Hybrids

Somatic hybridization techniques have great advantages in plant breeding. Fusing protoplast from intergeneric or interfamily species could overcome the barriers of traditional sexual hybridizations and make the resources of wild gene usable. It is also able to transfer not only nuclear genome but also cytoplasm genome to recipients simultaneous. The biparents' nuclear and cytoplasm genomes that coexisted and recombined in hybrids would created various aberrances. This could not be realized through traditional sexual hybridization. Recently great advancements have been made in remote somatic hybridizations, while the remote hybrids were hindered by the difficulty of plant regeneration. And there are few reports about the nuclear/cytoplasm genotyping, as well as transcriptomics and metabolomics in somatic hybrids between remote speciesIn the present study, nuclear/cytoplasm composition of two hybridization combinations Festuca arundinacea ( × ) Triticum aestivum and Bupleurum scoronerifolium ( × ) Gentiana strominea were studied, with protoplast and self fusion cultures as control. The aims were to understand the composition and interaction of nuclear/cytoplasm genome in hybridization of remote species; and how much somaclonal variation in protoplast culture contributes to the traits of hybrids. On the other hand, we study the transcritomics and metabolomics of the somatic hybrids cell lines of Chinese medicine herbs Bupleurum scoronerifolium/Gentiana strominea and the biparents so as to detect the variation of gene expression and metabolite pathway in hybrids. These researches will provide theoretical base for application of somatic hybridization in pasture breeding as well as biotechnology and metabolomics of Chinese medicine herbs.2. Phylogenetically remote somatic hybridization1) Nuclear/cytoplasm genome in hybrids of Festuca arundinacea with Triticum asetivum Thr protoplasts of common wheat (Triticum asetivum) irradiated with ultraviolet light (UV) at an intensity of 380uW/cm~2 for 30s, 1min, 1.5min, 2min and 3min respectively were fused by PEG method with protoplast of Festuca (Festuca arundinacea). In the same time, the protoplasts of Festuca and wheat were cultured as control. After 40 days of culture in dark, numerous clones produced in several fusion combinations and controls. In the early research of our lab, the culture of wheat protoplast had already been published, so we only focused on fescue in this report.Morphological observation showed that the phenotypes of most cell lines regenerated from combinations with dosage of 30s and 1min UV irradiation were Festuca-like, and grew vigorously. 25% cell lines could regenerate green plantlet. However the cell lines from UV dosage of 1.5min were non-regenerable and grew slowly; whiles protoplasts with higher UV dosage could not divide. The control produced series cell line with fescue phenotypes and grew vigorously. Isozymes profile indicated that somatic hybrids had bipareants' characteristic bands and new bands, while control cell lines had more similar profiles with fescue expected that a few cell lines had a few novel bands or lacked a few bands. Molecular marks showed that the hybrids had recombinant parents' genome, while the control had nearly the same genome with fescue. For cytological analysis, chromosomes of both hybrids and control were the same as fescue in appearance. Chromosome numbers of regeneration, non-regeneration hybrids cell lines and control lines distributed in a range of 26-45, 45-84 and 22-52. All lines had 1-3 chromosome fragments and 1-2 chromosomes with two/multi centromeres. GISH also indicated that either regeneration or non-regeneration cell lines had not intact wheat chromosomes but only a few its fragments. RFLP and SSR of the cytoplasm analysis showed that the somatic hybrids had a predominance of recipients' cytoplasm genome, and some recombination happened at some loci. The controls also had a few variations at some loci. In MSAP, the hybrids cell lines had series changes in a lot of DNA fragments. In diversity hybrid cell lines, different loci had distinct changes. But the contral cell lines from protoplasts had almost likely methylation-loci. 2). Somatic hybridization between Bupleurum scoronerifolium and Gentiana strominea.Protoplasts of Gentiana strominea irradiated with ultraviolet light(UV) at an intensity of 380uW/cm2 for 30s(Q), 1min(Q1'), 1.5min(Q1.5'), 2min(Q2') respectively were fused by PEG method with protoplast of Bupleurum scoronerifolium, and the self protoplasts of Bupleurum scoronerifolium were fused and cultured as control. After 70 days of culture in dark, numerous regenerated cell lines produced from combinations and Bupleurum. While Gentiana protoplasts could not divide when they were cultured alone in this culture condition.Respective 17 and 12 regenerated cell lines from combination Q and Q1' were verified as somatic hybrids on the basis of morphological observation and analysis of isozyme, cytology, 5SrDNA spacer sequences and RAPD. Both hybrid and Bupleurum cell lines could regenerate shoots and roots. Comparing with hybrids, the Bupleurum also had new bands expect donor characteristic bands in isozyme and RAPD profiles. Chromosome counting showed that hybrids' chromosome numbers were 9-14 and controls were 20-29 in self-fusion cell lines and 12 in protoplast cultures. RFLP analysis of cytoplasm genomes indicated that the hybrids had predominance of recipients' mt DNA around the atp6 and cox I loci, and the same ct DNA with recipients around rbcL locus.Based on above two experiments, complementary regeneration phenomenon was existed in remote somatic hybridizations both monocotyledonous and dicotyledonous. Regenerated cell lines from Bupleurum scoronerifolium cultures could recover regeneration ability not only by donors (+) recipients manner but also by themselves. It was found that the balance of genetic materials was other relative factors in this experiment. But some cell lines coming from protoplasm culture with relative balance genetic materials didn't regeneration. This implys that both complementation and genetic balance control the regeneration of cell lines. Cell lines from different UV dose (30s, 1min) combinations were similar in cytoplasm genomes constitution, so dosage effect was not present when UV doses were in the 30s-1min ranges. 2. Metabolomics and transcriptomics analysis of somatic hybrid between Bupleurum scoronerifolium and Gentiana stromineaThe main efficacious components of Gentiana strominea are gentiopicroside, swertiamarin and oleanolic acid.In this experiment gentiopicroside were not detected in any somatic hybrid, while some hybrids had swertiamarin but the contents were less than 0.03130mg/g dry weight. The content was much less than that in Gentiana strominea. Another efficacious component-oleanolic acid was existed in both parents. This content in most somatic hybrids was lower than in Bupleurum scoronerifolium. But there was a special cell line with the content of 1.729mg/g, much higher than biparents. Alcohol-soluble metabolites profiles of hybrids 3Q and 3Q1' showed that the components coming from Bupleurum scoronerifolium were 56.23% and 38.84%, while coming from donor were 24.39% and 36.30% and new members were 17.91% and 24.86%, respectively. Glucide contents of hybrids were much higher than that in biparents. This may imply that the basic metabolisms were strengthened in hybrids, and it may caused by interaction of metabolism pathway between donor and recipient.DDRT transferred transcriptomics showed that, in Comparation with recipient, 3Q and 3Q1' had 13.59% and 11.57% new expressed genes; 10.40% and 10.35% silenced genes and about 44.0% genes without information. Of them, 37.5% was remarked as hypothetical protein and others may have some relationships to basic metabolism, especially glucide metabolism.Beta-amyrin gene, which catalyzed the first step of oleanolic acid' biosynthesis were selected to discover how did donor's gene expressed in hybrids. The results showed that two different beta-amyrin genes expressed in Gentiana strominea and one in Bupleurum scoronerifolium, while the hybrids expressed the beta-amyrin from the donor.