Mining Of Key Genes Associated With Inter-morph Compatibility And Self-incompatibility In Primula Maximowiczii And P.forbesii

Self-incompatibility(SI)is a common mechanism that promotes outcrossing in angiosperms.Heterostyly has been reported in at least 28 families scattered throughout the angiosperm lineage and the most of heterostylous species are associated with SI.Dimorphic heteromorphic SI has been observed in 91%of Primula species,and they are often considered a model system for heterostyly.However,the molecular mechanism of heteromorphic SI remains unknown.Primula maximowiczii is perennial and native to northern China and P.forbesii is a biennial species and distributed mainly in Yunnan,China.In the current study,I used P.maximowiczii and P.forbesii as meterial to investigate the expression level of the genes after self-and cross-pollinations of pin and thrum morphs by RNA-seq.Our results provide the first transcriptomics analysis of the heteromorphic SI of Primula and offer new data for genetic and genomic studies on Primula SI in the future.The main results are the followings.1.P.maximowiczii and P.forbesii are the classic distylous plants with pin and thrum morphs.The lengths of stigmatic cell of the pin morphs of P.maximowiczii and P.forbesii were significantly longer than the thrum morphs.The diameter and length of P.maximowiczii style cell were not different between pin and thrum morphs.However,the length of style cell of P.forbesii pin morph was significantly longer than the thrum morph.2.1311 and 1048 DEGs were identified in the comparisons of pin and thrum morphs of P.maximowiczii by comparing incompatible pollination to compatible pollination(PS vs PT and TP vs TS),for a total of 2135 genes.A total of 89.51%of the 2135 DEGs were expressed with different patterns in the PS vs PT and TS vs TP comparisons.These results suggest that the majority of the DEGs involved in pin compatible and self-incompatible responses differ from those involved in thrum compatible and self-incompatible responses.The DEGs in the PS vs PT and TS vs TP comparisons were mostly enriched in carbohydrate metabolism,environmental adaptation and translation pathways,especially in plant-pathogen interaction.By comparing the pin combinations(PT and PS)to the thrum combinations(TP and TS)of P.maximowiczii,826 DEGs associtated with floral morphology were identified.Among them,554 genes were likely related with the pin morph and 268 genes wih high expression in the thrum morph.They were enriched in the process about the proteasome and ribosome pathways.3.In total,114,474 unigenes were identified from the transcriptomic data of P.forbesii.For functional annotation analysis,more than 42%(49,695)of total unigenes were annotated using various databases.There were 165 and 110 DEGs associated with the pin and thrum SI process of P.forbesii respectively,for a total of 242 genes.There were about 90%of the DEGs showed different expression patterns or were specific to the PS vs PT or TS vs TP comparisons.The results suggest that the majority of the DEGs involved in pin compatible and self-incompatible responses differ from those involved in thrum compatible and self-incompatible responses,same as the result of P.maximowiczii.The DEGs in the PS vs PT and TS vs TP comparisons were mostly enriched in carbohydrate metabolism and cell part pathways.The total of 28 DEGs were identified from the comparisons between pin and thrum combinations of P.forbesii.Among them,19 DEGs expressed higher in the pin morph than the thrum morph and 5 DEGs had higher expression in the thrum morph.They were enriched in the pathways associated with carbohydrate metabolism and cell part,incuding the auxin,GDSL and so on.4.According to the comparative transcriptome analysis of P.maximowiczii and P.forbesii,13,664 homologous gene families were identified from them and among them 7,154 pairs of putative orthologous contigs between them were identified.The genes specific to P.maximowiczii were enriched in the cell part pathways and the genes specific to P.forbesii were enriched in the mechanism process pathways.Estimations of non-synonymous/synonymous substitution rate ratios for these orthologs indicated that 282 of the pairs may be under positive selection(Ka/Ks>0.5).Five pairs of putative orthologous contigs expressed difirentially between the compatible and incompatible pollinations in pin morph were identified and 4 pairs of putative orthologous contigs associated with the thrum SI were identified.29 pairs of putative orthologous contigs could be associated with the distyly of P.maximowiczii and P.forbesii.5.Based on the transcriptomics data,17 genes were picked out and used as control genes for the analyses of P.maximowiczii and P.forbesii in the different type of plant tissues,including flower development,floral organs,plant organs,pistils after pollination and heat treatment.6.Using RT-RCR and the sequences from the transcriptomics data,the cDNA complete length of PmCML19 and PJCML19 were obtained.The PmCML19 and PJCML19 sequence are 435bp,encoding a protein of 144 amino acids.The plant expression vectors of PmCML19 and PfCML19 were constructed for the subcellular localization.The results indicated that PmCML19 and PfCML19 both produced fluorescence mainly in the nucleus and cell membrane.They had higher expression in the compatible pollinations than in the incompatible pollinations in pin and thrum morphs by qRT-PCR analysis.Using transcriptomics analyses,the current study investigated the expression patterns of genes involved in pin and thrum SI of P.maximowiczii and P.forbesii.Our results indicate that the SI systems of the pin and thrum morphs may be controlled by different molecular mechanisms.These results provide a lot of data about the SI process of Primula and promote further research into the molecular mechanisms of heteromorphic SI.