Molecular Mechanism Of Morphological Variation In The Tetraploid Chinese Cabbage

Chinese cabbage(Brassica rapa ssp.pekinensis,AA,2n=2x=20)is an important vegetable crop of the brassica,the most commonly eaten vegetable in northern China.In this study,the Chinese microspore DH(Double Haploid)line 'FT' was used as the test material for isolated microspore culture,and the naturally doubled diploid(AA,2n=2x=20)and tetraploid(AAAA,2n=4x=40)were selected in the regenerated plants.Their self-crossing progeny were uniform.Although both were naturally doubled from the same pure genome(A),many phenotypic differences were caused by different ploidy.In order to find out the molecular mechanism of polyploid morphological variation,the above diploid and tetraploid were used as test materials,and their growth and development characteristics,transcriptome and miRNA of leaves and petals were systematically analyzed.1.Phenotype differences between tetraploid and diploid of Chinese cabbageAfter continuous self-identification,the tested diploid and tetraploid lines were genetically stable.There was no significant difference between the morphology of diploid plants and the 'FT' of microspore cultured donor plants,but the tetraploid plants grew slow,the plants were short,the leaves became thicker,the flowering was delayed,the flower organs became larger,and the fertility decreased.2.Growth and development characteristics comparison between tetraploid and diploid of Chinese cabbageThrough seed germination and leaf growth and development observation experiments,it was proved that the seed germination and leaf growth rate of tetraploid Chinese cabbage were significantly slower than diploid;The observation of paraffin section showed that the thickening of tetraploid leaves was caused by the enlargement of mesophyll cells and the increase of cell layer;Through hormone content measurement,the content of brassinolide and abscisic acid were decreased in tetraploid leaves,the content of auxin and jasmonic acid were increased and the content of abscisic acid was decreased in tetraploid petals.by measuring the pigment content of petals,the content of lutein was significantly increased in tetraploid petals.3.Transcriptome and miRNA analysis of tetraploid and diploid leaves in Chinese cabbageUsing RNA-seq and sRNA-seq techniques,transcriptome and miRNA analysis were performed on tetraploid and diploid leaves of Chinese cabbage,and 13,225 differentially expressed genes and 56 differentially expressed miRNAs were screened.By association analysis of differentially expressed genes and differentially expressed miRNAs,35 differentially expressed miRNAs and 102 differentially expressed target genes were screened.Functional annotation of the target gene,combined with GO and KEGG Pathway enrichment analysis,speculated that 13 differentially expressed target genes regulated by 12 differentially expressed miRNAs were associated with leaf development,and their differential expression affected tetraploid dwarf and the leaf growth rate.We suggests two possible pathways for phenotypic variation induced by miRNA regulation of target genes.4.Transcriptome and miRNA analysis of tetraploid and diploid petals in Chinese cabbageUsing the RNA-seq and sRNA-seq techniques,the transcriptome and microRNA were analyzed separately for the diploid and tetraploid Chinese cabbage petals.A total of 14,412 differentially expressed genes and 34 differentially expressed microRNAs were screened.Among them,22 differentially expressed genes involved in the carotenoid biosynthesis pathway,affecting the synthesis of lutein,which is related to the darkening of tetraploid color.A combined analysis of the two sets of data showed that only 21 differentially expressed miRNAs and 82 of their target genes were differentially expressed.By performing gene function annotation on these target genes and combining GO and KEGG Pathway enrichment analysis,we finally speculated that 13 differentially expressed target genes regulated by 9 differentially expressed miRNAs were associated with flower development,which mainly related to flower shape,and flowering delay.