Full Length Transcriptome Analysis And Identification Of Self-incompatibility Related Genes At Different Developmental Stages Of Stigma In Ornamental Kale

Ornamental kale(Brassica oleracea var.acephala)is mainly ornamental plant in northern China.Plant self-incompatibility(SI)refers to the process in which the pistil recognizes the self and cross pollen,and ultimately inhibits the growth of self-pollen and prevents fertilization through intracellular signal transduction and cascade reaction.The main site of self-incompatibility reaction in Brassica is papillar cells of stigma,which inhibit the growth of self-pollen through intracellular signal cascade.In this study,stigma development was divided into three stages according to the length of bud:SS stage(bud size 0-4 mm),SM stage(bud size 6-8 mm)and SL stage(bud size>10 mm).The stigma of unopened buds at three developmental stages was used as the research material,and the full-length transcriptome was sequenced.Through bioinformatics methods,we explored alternative splicing(AS),alternative polyadenylation(APA),predicted new genes and long non coding RNA(LncRNA)during stigma development.The key genes in stigma development were analyzed by quantitative analysis of differential transcripts.The key genes of Self-incompatibility pathway were analyzed by full-length transcriptome sequencing,and the expression levels of these key genes were verified by qRT-PCR.The main results are as follows:(1)The flower buds of self-incompatibility lines S13bS13b of ornamental kale at SS,SM and SL stages were observed the morphology of their stigmas by body microscope and scanning electron microscope.The results showed that the mastoid cells of SS stigma at early development stage were shriveled and round;the mastoid cells of SM stigma at middle development stage were oblate and overlapped with each other;at the mature stage,the stigma of SL was pointed and round,the papillar cells were full and nearly round,and the surface of cells in the center was smooth and arranged closely.(2)The stigma of ornamental kale at SS,SM and SL stages were pollinated by compatible pollination(cross pollination)and incompatible pollination(self-pollination).The number of pollen grains and pollen tubes of SM self pollination was different from that of SS self pollination,and the number of pollen grains and pollen tubes of SM self pollination was significantly different from that of SL self pollination;the number of pollen grains and pollen tubes of SS cross pollination was significantly different from that of SM cross pollination,and the number of pollen grains and pollen tubes of SS cross pollination was significantly different from that of SL cross pollination.(3)The length of seed pod and the number of seeds were observed following pollination after 30 days.There was no significant difference between the pod length and seed number of self-pollinated seeds and that of self pollination in SS and SL.The length and number of seeds of self-pollinated seeds were significantly different from that of sl.the pod length and seed number of SS cross pollinated seeds were not significantly different from that of SL and SM.The length and number of seeds of SS cross pollinated seeds were not significantly different from that of SL.(4)The full-length transcriptome of stigma in three developmental stages of ornamental kale was analyzed by PacBio single molecule real-time sequencing and Illumina RNA sequencing.A total of 22 389 SSRs,21 816 complete ORF sequences and 4 591 lncrnas were predicted,and 31 269 new transcripts were annotated.A total of 41 786 349 and 5 158 differential genes(DEGs)were identified in stigma of ornamental kale.at different developmental stages of SS,SM and SL.WGCNA method was used to construct the predicted co expression network of 8 619 transcription factors(TFs),and 12 highly related TFs modules were identified in the three stages of stigma development of kale.(5)Based on the quantitative analysis of transcriptome,the differentially expressed genes were divided into three parts:compatible genes,incompatible genes and developmental genes.KEGG metabolic pathway annotation and enrichment analysis were performed for genes belonging to "incompatible" part.By KEGG annotation analysis,starch and sucrose metabolism(including 50 DEGs),plant hormone signal transduction(including 47 DEGs),plant pathogen interaction(including 44 DEGs)and amino acid biosynthesis(including 45 DEGs)were more DEGs.By KEGG enrichment analysis,flavonoid biosynthesis,fatty acid biosynthesis,plant pathogen interaction and fatty acid metabolism were significantly enriched.qRT-PCR showed that the expression of BoCHS2 and BoCHS3 was up-regulated in SS and SM stages of stigma development;The expression of BoCHSl was down regulated;The expression of BoF3H1 was almost unchanged;During the mature period of stigma development,the expression of genes related to flavonoid synthesis pathway was down regulated.This result is consistent with the result of transcriptome quantitative analysis.