Mapping And Cloning The Genes In Chlorophyll-Deficient And Glossy Mutants In Brassica Napus L

Leaf mutants of crops are abundant and diverse such as the mutation of leaf coloration, leaf morphology and so on. Leaf mutants may be good materials for the researchs about photosynthesis, respiration, energy use, plant development, gene function, pigment metabolism, wax metabolism and so on.The mutation of Leaf is very obvious trait mutation and the mutation frequency is high. They may also be used as a morphological marker for crossbreeding. In our study, we obtained a chlorophyll-deficient mutant and a gossly mutant of Brassica napus. The researchs about preliminary phenotypic analysis, genetic analysis and mapping of the chlorophyll-deficient mutant (BnaC.ygl) and the gossly mutant (Nilla glossy) had been done. And the BnaC.ygl gene was isolated by a map-based cloning approach with the candidate gene approach. The major points were described as follows:1The mapping and coloning of Bna C.YGL gene in a chlorophyll-deficient mutant of Brassica napus1) There was no changes in the pigment composition of BnaC.ygl mutant.The Chl a and Ch1b levels in BnaC.ygl mutant and the wild type were significant differences at seeding stage, but not at flowering stage.2) In BnaC.ygl mutant, the chloroplasts displayed less dense granal stacks and fewer granal membranes compared with those of the wild type. However, in twenty-week-old plants, Chloroplasts in the BnaC.ygl mutant and wild-type had well-ordered granal stacks and normal granal membranes. There was no apparent difference in the structures of chloroplasts in the BnaC.ygl mutant and wild-type.3) Mapping of the gene was subsequently conducted in two populations with yellow-green leaves (population â…  BCs and â…  BC4, which comprised3472and5288individuals respectively).And the IP, SSR makers were developed from the sequences of B. oleracea. Then the BnaC. YGL gene was narrowed into a0.35cM region in the linkage group N17of Brassica napus. And in the two populations,18makers cosegregated with BnaC.YGL. The results suggested the recombination between cosegregated markers and BnaC.YGL was severely suppressed. BLAST analysis revealed that the sequences of the makers displayed highly conserved homo logy with C07of B. oleracea. The analysis of the information342genes from B. oleracea homologous region indicated that an ORF(Bol027145) in the vicinity ofSC25maker had homologous to HOI which can catalytic decomposition of heme in the heme/bilin branch. Multiple mutant of HOI in Arabidopsis exhibited yellow-green leaves. Thus we hypothesized that this gene in B.napus, the ortholog of atHO1may be a candidate gene of BnaC. YGL. Comparative sequencing results showed that homologous copy (BnaC.HO1) of HO1on the C genome was losted in the mutant. Two resulting complementation constructs (gDNAå’ŒcDNA) were introduced into the BnaC.ygl mutant by Agrobacterium tumefaciens-mediated transformation. The genetic complementation suggested BnaC.HO1corresponds to the mutant gene in the BnaC.ygl mutant.4) Comparison of the deduced amino acid sequences between BnaC.HO1and other Brassica HO1s further revealed several highly conserved regions as signature sequence and conserved amino acid residues. These results suggested that BnaC.HO1is highly homologous to BoHO1. Moreover, BnaC.HO1shared more than90%,80%,80%amino acid identities with BnaA.HO1, AtHO1, BrHO1.5) Semi-quantitative RT-PCR analysis indicated that BnaC.YGL displayed higher expression level in young leaves and pods and lower in germinating seeds and roots. The subcelluar localization assay showed the BnaC. YGL protein was localized in the chloroplast of the protoplast.6) The analysis of16cosegregated makers covering C07at least1.2Mb in B. oleracea showed that16cosegregated makers were detected polymorphism in the BnaC.ygl mutant and wild-type. And comparative sequencing results indicated that the C7homologous copy of three genes were losted. These suggested that there may be a large fragment deletion on N17in the BnaC.ygl mutant.2Genetic characterization and fine mapping of a gossly mutant (Nilla glossy)in Brassica napus1) The wax of stems, leaves, buds and pods in the glossy mutant was defective in the entire growth period. Scanning electron microscopy indicated that the wax on the leaf and stem surface in Nilla glossy mutan was significantly reduced.2) The phenotype of the filial generations obtained by the crosses of the BnaC.ygl mutant and the normal parents (No2127-17and Bing409) was thoroughly investigated. The reciprocal F1plants exhibited glossy leaves as No2127-17and Bing409, which indicated that the phenotype of the glossy mutant was controlled by nuclear genes. The F2populations from both crosses showed an expected Mendelian inheritance ratio of15:1. And the ratio of normal plants to glossy plants in the BC1progenies was approximately3:1. These data indicated that the phenotype of the Nilla glossy mutant was controlled by two recessive genes.3) Five possible polymorphic markers from F2population were used to detect the polymorphisms in16lines with1:1separation in BC2generation of G1combination.The results showed that CB10443had polymorphisms in7lines, which indicated that CB10443was linked to one of the two genes and the gene linked with CB10443was named as "BnaC.NGLl".4) The rough mapping mapped BnaC.Ngll to linkage group N11. Eight AFLP makers were developed by AFLP technique combined with BAS. The sequences of the CB10443and AFLP markers were submitted to the public database for identification of putative orthologues, which showed sequence homology to the CO I of B. oleracea. The SSR makers were designed from the sequence of C01in B. oleracea and analyzed in population G1BC2with1191plants. Finally, BnaC.YGL locus was mapped to the interval between the markers gls41and gls79, corresponding to a genetic distance of0.16cM. Compared with C01in B. oleracea, the interval between the markers gls41and gls79was about230kb, including51annotated genes.