Mapping Of BnaA.GL Gene In A Dominant Glossy Mutant And Cuticular Wax Analysis In Brassica Napus L

The aerial parts of land plants are covered with cuticular waxes. The wax was important and play role in limit non-stomatal water loss and gaseous exchange and protect plants from ultraviolet radiation and pathogen attack.Generally, the leaf and stem are covered with cuticular waxes, but the glossy mutants are shiny green without the waxes. There were extensively studies on the inheritance of glossy waxless character in Brassica, it demonstrated diversity among different species.A group of cuticular wax related genes were identified and cloned in Arabidopisis thaliana, but there were few researches on the cuticular wax in Brassica, especially there have been no reports of the molecular marker study on the glossy waxless character in Brassica napus. This study presented the first report on the characterization and genetic mapping of a novel dominant glossy mutant （BnaA.GL） in Brassica napus. The glossy mutant （GL mutant） exhibited abnormal cuticular wax deposition and increased water permeability. Biochemical analysis showed a decreased total cuticular waxes and alteration of wax composition. We did genetic analysis and mapped the BnaA.GL gene close to the end of chromosome A9. In search of clues underlying the molecular basis of the glossy phenotype, we conducted a microarray analysis and performed a comparison of the global transcript level between the wide type and the mutants. It was revealed that the wax biosynthetic genes were down-regulated in the glossy mutant. Data presented in the study on genetic mapping of the BnaA.GL gene and transcriptome alterations in the mutant pave the road for future efforts in cloning this gene and understanding the regulations of wax biosynthesis pathways in plants. The main points in this study as follow:1. The morphological observation about the glossy mutantScanning electron microscopy corroborated the reduction of wax on the leaf and stem surface in GL mutan, especially the leaf. Transmission electron microscopy revealed that the GL mutant epidermis had a cuticle membrane that was less osmiophilic, as indicated by the reduced electron density, but more than twice as thick compared with that of the wild type. Toluidine blue test showed that the GL mutant was stained more easily, in addition, the GL mutant rapidly lost chlorophyll content and showed a significantly higher water loss rate when compared with WT, which indicated the mutants were compromised in the strength of water permeability barrier. 2. Genetica analysis and mapping of the BnaA.GL geneThe F1（or RF1） plants of reciprocal crosses between WT and the GL mutant were all glossy, indicating that the glossy trait was dominant. The BC1progeny developed from the crosses between the F1plant and WT displayed a ratio of glossy to normal plants at a1:1ratio. Moreover, the glossy to normal phenotype ratio in the F2population was approximately3:1, indicating that this was a case in which one Mendelian locus controlled the glossy trait. Our initial mapping using the glossy leaf phenotype as a morphological marker （named Y） mapped the genetic lesion to linkage group A9from an F2population derived from WT x the glossy mutant. We then used an AFLP assay in combination with BSA and developed SSR markers, IP markers, in addition, an SNP Chip assay in combination with BSA was used. Finally, based on300normal individuals from a BC3F2population （1200）, we were able to map these markers to a region of0.67-1.33cM in the flanking region of the BnaA.GL gene. All markers linked to the gene were used to compare the micro-colinearity of the regions flanking the genes with B. rapa and Arabidopsis. By comparative mapping with B. rapa using http://brassicadb.org/brad/database, the distance from the closest marker and the last annotated gene on R9was calculated as only250kb, there were46genes in the region, the homologous region of BnaA.GL in Arabidopsis was between AT1G01190and AT1G02205. Interestingly, a homolog of Arabidopsis gene CER1（AT1G02205）, Bra032670was located in the mapped region of the BnaA. GL. CER1encodes aldehyde decarbonylase, the stem was glossy and the pollen was sterility in the mutant cerl-1. So we thought it was the candidate gene.3. Fatty acid composition of leaf and cuticular wax analysisGC-MS analysis of the leaf fatty acid composition indicated that the C16:0and C18:3were increased, but no C22:0was detected in GL mutant and DH line7, the total VLCFAs were decreased.Thin-layer chromatography （TLC） analysis revealed alkanes, ketones and secondary alcohols were not detected, but aldehydes were increased in the GL mutant. GC-MS analysis indicated that the primary alcohols, wax esters and fatty acids were decreased, and total waxes were decreased. 4cDNA microarray assayIn search of clues underlying the molecular basis of the glossy phenotype and providing more information for mapping. We then conducted a microarray analysis using RNA from leaf tissue of similar developmental stages and performed a comparison of the global transcript level between WT vs. the GL mutant and double haploid normal line bulk （DHNB） segregates vs. DH glossy line bulk （DHGB） segregates. These genes fell into several categories, including fatty acids biosynthesis, DNA-depend transcription, the responses to stress and to abiotic or biotic stimuli and photosynthesi, in addition, including genes related to protein biosynthesis, transcription, development and so on. A major functional category of the down-regulated genes emerged from the DHNB vs. DHGB microarray analysis included those encoding a subset of genes related to the fatty acid biosynthesis and wax biosynthesis pathway. It was revealed that wax biosynthetic genes were down-regulated, especially the decarbonylation pathway of wax biosynthesis was compromised and BnCER1was one of those most severely down-regulated in the glossy mutant. The different expression patterns of these genes in the GL mutants were verifiable by quantitative real-time PCR （qRT-PCR）. Based on those, we made a simplified cuticular wax biosynthetic pathway.5. Candidate gene analysisCER1was the aldehyde decarbonylase. The phenotype and biochemic character of Arabidopisis cerl-1mutant was similar with the GL mutant. In light of the fact that Bra032670at the end of the chromosome was the homologous to CER1（AT1G02205）, the genetic lesion responsible for the glossy phenotype in Arabidopsis.We thought it was the candidate gene.Sequence analysis indicated that there were4copies of CDS in WT, but we didn’t amplify any copies from the cDNA of GL mutant. Except the copies with introns, we amplified copies without introns. Promoter sequence was obtained by TAIL-PCR. However, there was no apparent sequence alteration between WT and the GL mutant except in the fifth intron where three SNPs were identified.The copy with SNPs differences obtainted from the GL mutant was transformed to the WT in Brassica napus, but we didn’t get the expected phenotype.The vectors of over expression BnCER1were construeted under the CaMV35S promoter and its own promoter in B. napus. Both of them were transformed in Arabidopisis cerl mutant （SALK₀08544C）, but no positive transformed plants were obtained.The vector of over expression BnCER1under the CaMV35S promoter was transformed in WT of Arabidopisis, but no significance differences were detected compared with WT. The vector of over expression BnCER1under its own promoter was transformed to WT in Arabidopisis, T1plants originating from independent TO kanamycin-resistant transformants exhibited glossy stem and reduced fertility. Scanning electron microscopy showed the reduction of wax on the stem surface in transformants, the pollen was abnormal and infertile. In transgenic plants, the expression level of CER1was lower than the WT, transgenes may be silenced together with the homologous endogenous genes in some cases. This phenomenon is called cosuppression.6. The difference of stress resistance between WT and GL muatantFour-weeks plants of WT and GL muatant were used for all stress treatments. NaCl stress was imposed by subirrigation with NaCl solution, for water deficit treatments, pots were deprived of water, ABA treatment consisted of spraying with ABA. Compared with the control plants, plants respond to all treatment by increasing the deposition of leaf cuticular waxes, meanwhile the chlorophyll-extraction rates and water loss rate were decreased, but the changing extent varied with different treatments. Results here also revealed that salt stress-induced accumulation of waxes involves accumulation of wax related gene CER1, but reduction of CER1under drought and ABA treatments.