| Gerbera hybrida is a perennial flower of the genus Gerbera Cass.in the Compositae family,which is one of the top five cut flowers in the global floricultural trade and is widely grown as pot flowers or garden plants in many countries.Over the last several decades,the Gerbera breeders has created numerous clonal cultivars and seed-propagated F1 hybrids.Breeding goals vary according to different breeding objectives of cut-flower,pot,and garden-type,yet bright flowers in multiple colors,flower types and coloration patterns,early and continuous flowering,high flower quality,high flower yield,tolerance to biotic and abiotic stresses,disease and pests resistance,cold resistance are important for all types of gerberas.Hybridization and inbreeding have been the primary approaches used in Gerbera breeding,which will continually play a decisive role in further improvement of Gerberas.However,most of the commercial varieties were created by the hybridization of Gerbera jamesonii Bolus ex Hook.f.and Gerbera viridifolia(DC.)Sch.Bip.,resulting the relatively narrow genetic basis,which brings some difficulties to non-directional conventional cross breeding.Therefore,broadened breeding methods and innovated technologies are particularly important to improve the efficiency of gerbera breeding.Haploids can be converted to doubled haploids,which can be used to replace inbreds as breeding parents for producing highly homogeneous F1 hybrid cultivars.Haploids and doubled haploids are also excellent materials for inheritance study and genomics research,such as genome sequencing,sequence assembly,and haplotype analysis.In this study,haploids were induced by in vitro culture from gerbera unfertilized ovules,and haploid transcriptome sequencing with target traits was used to study the molecular mechanism of gerbera cold resistance and the identification of recombinant suppressor genes.It will be provided some research and theoretical foundations for haploid breeding,cold-resistant breeding,increased recombination rates,and enriched genetic diversity in Gerbera hybrida.Firstly,the induction of gerbera haploids and double haploids were studied.In this study,45 gerbera genotypes were used as research materials.Unfertilized ovules from ray florets colored stage were stripped and cultured in vitro.Adventitious buds were induced 29 out of 45 genotypes,with an induction rate of 64.4%.Among them,12 genotypes had an induction rate of greater than 5% and 3 genotypes had an induction rate of greater than 10%.The induction rate of the ‘Baimawangzi’ was as high as 18.2%,which indicated that there were significant differences in the induction of adventitious buds used ovules of different genotypes.Four genotypes of ‘Baimawangzi’,‘Yangguanglu’,‘Hongjixin’ and ‘Duoli’,which have large differences in adventitious bud induction rate,were selected and further studied.The effects of different seasons and low temperature treatment at 4 ° C on adventitious bud induction were studied.The results showed that four genotypes can induce adventitious buds in winter and spring,but the best results were generated in spring,with an average induction rate of 10.4%,and the lowest average induction rate in autumn was 4.8%.There were great differences betweengenotypes.The induction rate of ‘Baimawangzi’ was 20% in summer,the shortest induction time was 39 days,and ‘Duoli’ was difficult to form adventitious buds,and the induction time was longer.In addition,the low temperature treatment at 4 ° C was conducive to the induction of adventitious buds.Compared with the non-treated control,the average induction rate after treatment was increased by 2-3%,and treatments had little effect on the induction time.Compared with the non-treated control,the induction times of ‘Baimawangzi’ and ‘Hongjixin’ were shortened by 1-5d.The chromosome ploidy of regenerated plants which induced by ovules in vitro from four gerbera genotypes‘Baimawangzi’,‘Hongjixin’,‘Yangguanglu’ and ‘Hongyundangtou’ were identified by flow cytometry.Among the 522 regenerated plants,288 plants were haploids with 25 chromosomes,accounting for 55.17%.218 plants were diploid with 50 chromosomes,accounting for 41.76%.16 plants were mixoploid,accounting for 3.07%,and the number of chromosomes were between 25-50.Compared with the corresponding hetero-diploid plants,haploid plants have significant differences in the growth rate of fresh weight,plant height,flower size,and plant development.However,there is no significant difference in the reproduction coefficient and ploidy correlation.The chromosome doubling of haploid plants by colchicine treatment showed that the doubling effect of colchicine at a concentration of 0.05% for 4 days was the best,and the doubling rate was 42%.From the perspective of the treatment method,the effect of adding colchicine to the medium was better than that of whole plant immersion and stem tip immersion.Compared with haploid plants,double haploid plants had larger stomatal guard cells and more chloroplasts.The plants restored fertility,but their fertility was weaker than that of hetero-diploid.This study provides a method and research basis for gerbera haploid induction,and provides ideal materials for its ploidy breeding,genetic and genomics research.Secondly,the transcriptome analysis of gerbera haploids for cold resistance was analyzed.Gerbera hybrida ‘Hongjixing’ with cold resistance and ‘Qiuri’ with non-cold resistance haploid plants were selected.Through high-throughput transcriptome sequencing,we compared the transcripts of these two haploids under low temperature treatment(-2 °C)and obtained 9.50 Gb and 9.59 Gb transcriptome data,respectively,with99,160 assembled Unigenes and annotated 58127 Unigenes,respectively.After analyzing the expression levels of Unigenes in both ‘Hongjixing’ and ‘Qiuri’,19100 expression upregulated genes and 13931 expression-down genes were screened.The 20 most differentially expressed genes of the top 30 highest difference in up-regulated expression genes were further studied,which were found to be mainly involved in carbon metabolism,nucleotide metabolism,secondary metabolism,transcription regulation,and defense related response.The molecular regulatory pathways of these genes are basically related to a variety of abiotic stress responses.Thus,we speculated that the cold resistance of Gerbera hybrida ‘Hongjixing’ was related to these genes.To verify the correlation of differentially expressed genes in the regulation of cold resistance,we analyzed the expression levels of these 20 genes between ‘Hongjixing’ and ‘Qiuri’.The results of q RTPCR showed that 12 differentially expressed genes displayed significant expression differences between ‘Hongjixing’ and ‘Qiuri’,which was consistent with the expression trend of transcriptome sequence.Among them,Unigene49221,Unigene49372,Unigene50734,Unigene44870,Unigene45349,Unigene45911,Unigene46132,Unigene48047,Unigene48068,Unigene48783 and Unigene49105 in ‘Hongjixing’ have significantly up-regulated expression compared to ‘Qiuri’.It demonstrated that these genes are closely related to the cold resistance of Gerbera hybrida,and the cold resistance is mainly related to nucleotide metabolism,transcriptional regulation,secondary metabolite synthesis and stress response.Thirdly,the cloning and expression of the gerbera recombinant suppressor gene TOP3α was conducted.According to the haploid transcriptome data of Gerbera hybrida‘Hongjixing’ and ‘Qiuri’,20 reference candidate genes were further screened and identified.Through the expression analysis and stability expression evaluation by realtime PCR in flower buds among the 20 reference candidate genes,PGK2 was identified as the most stable expressed gene,and thus most suitable as the reference gene for gene expression study during meiosis in Gerbera hybrida.What’s more,six recombination suppressors(FANCM,TOP3α,RECQ4,FIGL1,RMI1 and FLIP)were identified in Gerbera hybrida using the transcriptome data.The RACE technique was subsequently used to clone a 2808 bp full-length c DNA sequence of TOP3α,which encodes 935 amino acids with three conserved domains,namely topoisomerase-primase(TOPRIM),DNA topoisomerase(TOP1Ac)and zinc finger(zf-GR).Amino acid sequences analysis of TOP3α from different species showed that TOP3α of Gerbera hybrida harbored the same conserved sequence regions with highly similar to other plants,which was closely related to homologs in Cynara cardunculus L.and Helianthus annuus L.,with 90% and 89%amino acid sequence similarity,respectively.This demonstrated that the meiotic recombination suppressor TOP3α was highly conserved during the evolution of plants,and TOP3α may be associated with the regulation(suppression)of meiotic recombination in the species of Gerbera hybrida.The transcript levels of TOP3α were normalized to that of the reference gene PGK2 during meiosis.The results showed that TOP3α was strongly expressed in flower buds.In terms of its expression over the course of flower development,TOP3α expression decreased sharply after the flower bud stage and remained low.This finding further suggests that TOP3α is associated with the regulation of meiotic recombination in Gerbera hybrida,which was consistent with the reported function of TOP3α in Arabidopsis thaliana that limiting meiotic recombination.The identification and expression analysis of TOP3α provides a new method and a new way for gerbera breeding.It also laid the research and theoretical foundation for increasing its recombination rate,enriching genetic diversity,and improving breeding efficiency. |
