Mapping Of The Epicuticular Wax Deficiency Mutant Gene Brcer1 In Chinese Cabbage (Brassica Campestris L. Ssp. Pekinensis)

The epicuticular wax of Chinese cabbage（Brassica campestris L.ssp.Pekenensis）is a kind of gray substance covering the surface of Chinese cabbage.It is mainly composed of long-chain fatty acids（C>18）and aldehydes,alcohols,alkanes,ketones,lipids derived from long-chain fatty acids.It is an important barrier to resist the harmful factors of the external environment Chinese cabbage leaves as product organs,lack of epicuticular wax,the surface of leaves is smooth and bright green,looks more tender,loved by people,and often has high commercial value.In this study,EMS was used to mutate the germinating seeds of double haploid Chinese cabbage’FT’,and a waxy epicuticular deletion mutant cer1 was obtained.Based on the analysis and identification of wax morphology,composition and genetic characteristics of mutants,mutmap and KASP genotyping techniques were used to locate and verify the mutant gene,and the expression patterns and cloning and sequencing of candidate gene were analyzed.The main results are as follows:1.Cuticular wax composition and morphological structure of mutant cer1The results showed that the surface of stem,leaf and seed pod of the mutant was bright green and glossy,especially at bolting stage,while the surface of the wild type had a layer of gray wax.In addition,the mutant and wild-type plants have similar characteristics.2.Morphological structure of epicuticular wax of cer1,a wax deficient mutant of Chinese cabbageGas chromatography-mass spectrometry（GC-MS）was used to analyze the composition of epicuticular waxes.The results showed that the total content of epicuticular waxes in the mutants decreased significantly,and the content of alkanes,ketones,alcohols and lipids decreased significantly,while the content of aldehydes increased.Scanning electron microscope（SEM）was used to observe the epicuticular wax of wild type and mutant plants.It was found that the epicuticular wax of wild type plants was thicker,showing rod-shaped and flaky morphology,while the surface of mutant plants was very smooth,almost without wax.3.Genetic characteristics of epicuticular wax deficiency mutant in Chinese cabbageWild-type‘FT’crossed with mutant cer1 to obtain F₁generation,F₁generation was backcrossed with‘FT’and mutant cer1 to obtain BC₁P₁and BC₁P₂,and F₁generation selfing to obtain F₂generation.The results of the above generation materials showed that the wax deficiency mutant cer1 was controlled by a pair of recessive nuclear genes.4.Mapping of the epicuticular wax deficiency mutation geneDNA was extracted from 50 F₂plants with epicuticular wax powder deletion phenotype,and the mutant DNA pool（W-pool）was constructed.The candidate genes were mapped on A09 chromosome by mutmap analysis.The candidate gene was Bra A09g066480.3c,and its Arabidopsis homologous gene was CER1,which encoded aldehyde decarboxylase and participated in the formation of wax powder,so it is named Brcer1.5.Structural variation and expression characteristics of candidate geneThe full length of the candidate gene was cloned and sequenced.The results showed that there was a substitution of C→T in exon 4 of Bra A09g066480.3c resulting in the 255th amino acid conformation changed from hydroxyl to benzene ring.The amino acid sequence changes from serine to phenylalanine（S to F）.The q RT-PCR results showed that the expression of Bra A09g066480.3C in the same plant organs of wild-type‘FT’and mutant cer1 was not significantly different.Bra A09g066480.3C has the highest expression in flowers and almost no expression in roots.