Cytological Basis Of Self-Incompatibility And Cloning Of Potential S Genes In Chrysanthemum

Self-incompatibility(SI)is a kind of reproductive isolation that facilitates cross-pollination during long-term evolution of plants.It is an important mechanism to prevent species degradation and maintain genetic variation.Therefore,the study of SI is always a hotspot in the field of reproductive biology.However,current research on SI mainly focuses on plants such as Brassicaceae,Solanaceae,Rosaceae,Papaveraceae and so on,less on Asteraceae.Chrysanthemum morifolium is a representative species of Asteraceae with high ornamental value and important economic value,and it has the phenomenon of SI.It is of great theoretical and practical significance to carry out SI research with chrysanthemum as experimental material.In this study,spray cut chrysanthemum was used as research material,and the mechanism of SI was studied from the aspects of cell biology and molecular biology.The study results not only enrich the theoretical knowledge of plant self-incompatibility research,but also lay the foundation for improving breeding efficiency of chrysanthemum.The main contents and conclusions are as follows:1.To understand the distribution of SI characteristics among chrysanthemum cultivars,we investigated pistil receptivity,seed set,and compatible index of 24 chrysanthemum cultivars.It was found that seed set of 15 chrysanthemum cultivars were 0(such as ’Q 10-22-2’),seed set of 5 cultivars were lower than 0.50%,seed set of 3 cultivars were 0.50%to 1.24%,and seed set of 1 cultivar(’Q10-33-1’)was 56.50%.The results indicated that in the 24 chrysanthemum cultivars,most cultivars were self-incompatible,while a few cultivars were self-compatible.2.Among the above 24 chrysanthemum cultivars,seed set of ’Q10-33-1’ was as high as 56.50%,but the seed set of its progenies were varied,which ranged from 0 to 37.23%.To understand the cellular reasons for differences in seed set,four progenies of ’Q10-33-1’,‘Q10-33-1①,‘Q10-33-1③’,‘Q10-33-1④’and‘Q10-33-1⑩’(seed set were 37.23%,26.77%,7.97%and 0,respectively),were used as experimental materials.We systematically investigated their pollen viability,pistil receptivity and embryo development.It was found that seed set of ’Q10-33-1①’,’Q10-33-1③’and‘Q10-33-1④’were positively related to their pollen viability.‘Q10-33-1⑩’was self-incompatible,because the pollen tube stopped growing and failed to penetrate the stigma’s surface.That is,the differences in seed set among four progenies were mainly attributable to the differences in pollen viability and pistil receptivity.3.To understand the reasons for chrysanthemum SI,a self-incompatible cultivar,’Q10－22-2’,was picked out from 24 chrysanthemum cultivars as experimental material.The number of pollen grains germinating on per stigma of ’Q10-22-2’ was only 3.7,and double fertilization did not happen in its embryo sac.The stigmas and anthers of ’Q10-22-2’ were sampled at different developmental stages for transcriptome sequencing.After data analysis,15 potential stigma S genes and 6 potential pollen S genes were found out.In potential stigma S genes,13 genes were in the same family as the stigma S genes in Brassicaceae plants(belonging to sporophytic self-incompatibility,SSI),and 2 genes were in the same family as the stigma S genes in Solanaceae plants(belonging to gametophytic self-incompatibility,GSI).Meanwhile,in potential pollen S genes,5 genes were in the same family as the pollen S genes in Brassicaceae plants,and 1 gene was in the same family as the pollen S genes in Solanaceae plants.The results indicated that the genes in the same families as the S genes of SSI and GSI system both existed in chrysanthemum.4.According to the transcriptomic data,two genes belonging to the families of the S genes in Brassicaceae plants were cloned from ’Q10-22-2’.The stigma S gene was named as CmSRKl,and the pollen S genes was named as CmPCP1.Quantitative real-time PCR(qRT-PCR)of different tissues revealed that CmSRK1 was specifically expressed in mature stigmas,while CmPCP1 was specifically expressed in anthers 3 d before maturation.Subcellular localization showed that both CmSRK1 and CmPCPl were localized in the nucleus and membrane.Transcriptional activation activity analysis indicated that none of the two proteins had transcriptional activation activity.Yeast two hybridization showed that there was no interaction between CmSRKl and CmPCP1.CmSRK1 was constructed on the expression vector containing stigma-specific promoter,and CmPCP1 was constructed on the expression vector containing anther-specific promoter,then they were transformed into Arabidopsis thaliana.Artificial hybridization was performed with transgenic lines containing CmSRKl as the female parents,and transgenic lines containing CmPCP1 as the male parents.The hybridization results showed that seed set of two transgenic lines were 19.62%and 11.64%,respectively,while cross-pollinated seed set of Col-0 was 84.43%.It was speculated that CmSRKl and CmPCP1 were stigma and pollen S genes of chrysanthemum,respectively,and SI of chrysanthemum belonged to SSI.5.According to the transcriptomic data,three genes belonging to the families of S genes in Solanaceae plants were cloned from ’Q10-22-2’.The stigma S genes were named as CmRNSl and CmRNS2,respectively,and the pollen S gene was named as CmFB1.qRT-PCR of different tissues revealed that CmRNSl and CmRNS2 were not specifically expressed in the stigmas.Meanwhile,CmFB1 was not specifically expressed in the anthers.Subcellular localization showed that CmRNS1,CmRNS2 and CmFB1 were all localized in the nucleus and membrane.Transcriptional activation activity analysis indicated that none of the three proteins had transcriptional activation activity.Yeast two hybridization showed that both CmRNS1 and CmRNS2 did not interact with CmFB1.The results implicated that all the three genes were not S genes in chrysanthemum,and the GSI system similar to Solanaceae was not likely to function in chrysanthemum SI.