Abstract: 【Objective】 The CalS family plays an important role in regulating callose synthesis in plants. In this study, the members of the CalS family were identified and their bioinformatics and expression patterns were analyzed, which provided evidence for distant cross incompatibility of Paeonia lactiflora. 【Method】The pollen germination and tube growth in self- and cross-pollinated stigma were observed by fluorescence microscope. The callose content, endogenous ABA content and β-1, 3-glucanase activity in stigma were measured. Eight members of the CalS family were cloned and Expasy, MEME, TBtools, MEGA 7.0 and so on were used to predict the basic physicochemical properties and conserved motifs of proteins of the PlCalS family members and construct a phylogenetic evolutionary tree. The expression of eight PlCalS in stigma at 24 h of self-pollination, 24 h of cross-pollination and 36 h of cross-pollination were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Multiple sequence alignment of CalS5 was performed and a phylogenetic tree was constructed and the expression characteristics of PlCalS5 in response to ABA treatment at different concentrations were analyzed. 【Result】Fluorescence microscopic observation of pollen tube showed that callose plugging occurs in the stigma of hybrid, which restricted the pollen germination and tube growth. Callose content in cross-pollinated stigma was found to be higher in most periods than that in self-pollinated stigma. Then callose activity of β-1, 3-glucose and ABA content in stigma with different pollination affinity showed regular differences. The members of the PlCalS family of P. lactiflora were identified and named by integrity analysis of the structural domains and sequence matching, and 15 conserved motifs with stable distribution in the PlCalS family were encoded by every eight genes. Multi-species phylogenetic relationships showed that the CalS family could be divided into three branches, with the PlCalS family distributed in only two branches, of which PlCalS5 is more closely related to CalS5 in Paeonia suffruticosa, Arabidopsis thaliana and Solanum lycopersicum. Analysis of the bioinformatics showed that the eight family members encoded 1,745—1,951 amino acids, with a total number of 28,583—31,870 atoms and isoelectric points of 7.99—9.13. The analysis of the FPKM values in transcriptome showed that the PlCalS family members were highly expressed in the same period of hybridization and at 36 h of hybridization under the same treatment. qRT-PCR showed that the relative expression levels of 8 genes at 24 h of self-pollination were lower than those at 24 h and 36 h of cross-pollination. In addition, the PlCalS5 gene was found to be more sensitive to high ABA treatment. 【Conclusion】 There are eight gene members in the CalS family of P. lactiflora and play an important role in regulating the callose formation of P. lactiflora. The expression level of the PlCalS gene in cross-pollinated stigma was higher than that in self-pollinated stigma in most periods, which may be involved in callose abnormal deposition. Heterologous pollen stimulation to the stigma of P. lactiflora may enhance a certain ABA synthesis pathway. ABA induces callose deposition by positively regulating the expression of callose synthesis genes, thus inhibiting pollen germination and tube elongation, and thus affecting pollination compatibility.

Key words: Paeonia lactiflora, distant hybridization incompatibility, CalS, gene family, gene expression analyses