Pollen-stigma Interactions Following Compatible And Incompatible Pollinations,Molecular Cloning And Expression Pattern Analysis Of Srk Genes In Corylus

Corylus,belonging to Betulaceae family,is one of the most important nut crops and woody oil plants,and has high economic and nutritional value.However,most of the plants of Corylus show the characteristic of self-incompatibility(SI),which have negative influence in breeding.Thus,reasonable pollination varieties are needed in field planting and culture.Recently,the germplasm enhancement of Corylus is mainly through cross breeding.Therefore,the study on the mechanism of self-incompatibility of this plant is of great significance to its high-yield cultivation and germplasm enhancements.Previous studies have shown that Corylus is one of the sporophytic self-incompatibility(SSI)plants,and the phenotype of pollen tube after incompatibility pollination is similar to that of Brassica.The molecular mechanism of SSI has been well-studied in Brassica(Brassicaceae),but little is known about the molecular mechanism of SSI in Corylus(Betulaceae).In this study,we discussed the Ping’ou hybrid hazelnut(Corylus heterophylla × Corylus avellana).we discussed the characteristics of pollen tube germination after compatible pollination and incompatible pollination,the time of pollen-stigma interaction as well from the aspect of cell biology.In addition,we also investigated the SSI mechanism of Corylus from the aspect of genetics.From the view of cell biology,we carried out the experiments of scanning electron microscopy and fluorescence microscopy.the results showed that there was no significant difference in stigma surface at 0,0.5,1 h after compatibility or self-incompatibility pollination.However,there were obvious differences in the morphology of pollen tubes at 4 h after pollination,and many abnormal pollen tubes,such as distorted pollen tubes and at the end of pollen tubes were knotted,could be observed on the stigma surface after self-incompatibility pollination,while pollen tubes penetrated papillae cells could be observed on the stigma surface after compatibility pollination.Plus,the studies showed that hydration took place 0.5 h or before0.5 h after pollination.From the perspective of genetics,in order to obtain two female determinants(SRK)of SSI in Corylus,we designed primers for SRK candidate genes which were selected by transcriptome and used the RACE cloning techniques,obtaining two SRK genes which were named Cha SRK1 and Cha SRK2 respectively.Bioinformatics approaches were used to analyze the c DNA sequences,protein sequences,and domains of the encoded proteins.The full-length Cha SRK1 c DNA was 2883 base pairs(bp)with a coding sequence(CDS)of 2,547 bp encoding the 848 amino acid Cha SRK1 protein.The full-length Cha SRK2 c DNA was 2,693 bp,with a CDS of2,433 bp encoding 811 amino acids.The Cha SRK1/2 proteins contained an S-domain(extracellular domain),a transmembrane domain,a Ser/Thr protein kinase active site(kinase domain),and DUF3660 and/or DUF3403 domains.Additionally,analyzing the expression patterns of Cha SRK1 and Cha SRK2 involved real-time quantitative PCR(q RT-PCR)and dualcolor fluorescence in situ hybridization Dual-color FISH.The expression patterns of Cha SRK1 and Cha SRK2 differed based on q RT-PCR analysis: Cha SRK2 was predominantly expressed in mature stigmatic styles,while Cha SRK1 was also expressed in other tissues.Using dual-color fluorescence in situ hybridization,Cha SRK1/2 expression was found to be localized in papillar cells and the expression of Cha SRK2 is relatively higher than that of Cha SRK1.Moreover,we cloned 18 partial SRK homologs from 4 Ping’ou hybrid hazelnut cultivar species,4 Corylus avellana species and 1 Corylus heterophylla species.The lengths of 18 partial SRK homologs ranged from 1347 bp to 1451 bp,and they contained the same structural domains as Cha SRK1 and Cha SRK2.Phylogenetic analysis revealed that all SRK homologs could be divided into two categories that were similar to the classification of SRKs in Brassica.However,SRK genes from Corylus and the SRK genes from Brassica are clustered in two different branches.Collectively,the expression pattern of Cha SRK gene and its classification is relatively similar to SRKs Brassica from genetic aspect and the characteristics of pollen tube after pollination has similarity.Therefore,we speculated that the SSI mechanism in Corylus might be more similar to the Brassica mechanism than to other SSI types.