Research On Isolating Mutator Transposon Insertion Flanking Sequences And Preliminary Gene Mapping On Albino Mutants In Maize

Maize not only is the important food and cash crops, but also is the model plant inthe theoretical researches of genetics and crop breeding. The availability of the B73reference genome sequence brings about opportunities and challenges for researches andapplications of maize genome. Mutator, a huge family among maize genome,characterized by a high mutagenic activity and copy number, occupies an importantplace in the mutants creating.Elite Maize Inbred Lines Zong31（Z31） and B73with the MuDR-active lineW22::Mu build mutagenesis populations. The MuDR insertion mutants were obtained bygenetic analysis, selfing and backcross with Z31or B73. The MuDR insertion mutantswere used to construct the MuTAIL-PCR system and its effectiveness evaluation ofseparation flanking sequences. Two mutants of them were sequenced by Mu-illumina toisolate the MuDR flanking sequences, and primers were designed to verify theauthenticity of the MuDR insertions basing on B73genome sequence. The results are asfollows:1. The MuDR insertion material were amplified by PCR using four pairs of MuDRspecific primers. This revealed that the plant materials used in our study containedMuDR;2. The two specifical MuTAIL-PCR primers, TIR9-1and TIR9-2, were designedaccording to the MuDR-TIR sequence, located in the3’-TIR at positions150–181bp,and167–190bp, and used to constructed MuTAIL-PCR detected system;3. Using the constructed MuTAIL-PCR system,150flanking sequences containedMuDR were obtained,79of them were Unique MuDR sequences, accounting for52.67%;5MuDR single-copy inserted sites were authenticity verified and detected ofgenotype in population;4. To optimize the constructed MuTAIL-PCR system, the appropriate MuTAIL-PCRamplification products were obtained. The optimal conditions were follows: first,adding glycerol and dimethylsulfoxide （DMSO） in MuTAIL-PCR reaction system;second, optimizing the annealing temperature of primary step to72℃;5. The two MuDR inserted mutants were obtained148and152MuDR inserted flankingsequences using Mu-illumina method, but none of authenticity sites were verified byPCR using primers designed based on the B73sequence. Leaf color closely related to the chloroplast structure and components inmaizedetermines the photosynthetic outputs of plants. Thus, genetic analysis for genescontrolling leaf color would facilitate the improvement of photosynthetic efficiency andyield in maize.In this study, albino mutants of maize were used to evaluate the mutability and thengenetic assessment and analysis were conducted. Coarse QTL mapping for albinism wasperformed based on molecular marker technology and BSA method. The results are asfollows:1. The BC₃F₂segregation population derived from albino mutants was plant both underfiled and greenhouse conditions with the segregation ratio of3:1occurred, indicatingthat albino mutability were controlled by recessive nuclear genes;2. Comparing chlorophyll a, chlorophyll b and carotenoid content in albino lines with incontemporaneous normal lines of the BC₃F₂population, we found that chlorophyll a,chlorophyll b, carotenoid and total chlorophyll content in albino lines were1.78,0.79,0.57and2.57, and were9.80%,16.60%,19.79%and11.25%of contemporaneousnormal lines;3. Analysis of the parameters for chloroplast fluorescence showed that the value of PSⅡin mutant lines decreased96.21%～98.32%than normal lines;4. Investigation on mesophyll cell in mutants showed that mesophyll cells of normallines were full of chlorophyll, while litter was found in albino mutants. Meanwhile,almost none of complete structures of chloroplast, the existed chloroplast shapeellipse, the bilayers was incomplete, the volume was relatively small, and the internalthylakoid lamellar disorganized, and almost no grana;5. With732SSR primer pairs uniformly scattered on all10maize chromosomes toscreen parents, we obtained140polymorphic markers; with the140polymorphicmarkers between parents to screen green and albino pools in BC₃F₂population, weobtained8polymorphic markers; with30albino plant to test, parents and green poolof in control, we obtained umc2281and umc2075, closely linked to the albino mutantgene.