| The leaves above the primary ear have an important influence on the plant morphology and yield of maize,and the proper number of leaves above the primary ear is an important goal for the improvement of the plant type of maize.In the early stage of the research group,a 9-leaf maize inbred line Y915 and a 5-leaf maize inbred line Zheng 58 were used to construct a segregating population.The candidate interval is reduced to between InDel-02 and Y250,and the physical distance is 1.53Mb.In this study,based on the early positioning results,the leafy inbred line Y915(9 leaves above the primary ear)and two normal inbred lines G86(5 leaves above the primary ear)and W373(6 leaves above the primary ear)were crossed to construct the F2 segregating population.We use the map position cloning method to finely locate QTL-qLN3-2,and construct a BAC library of the leafy inbred line Y915,which was used for sequence analysis of the finely-defined localization intervals to mine candidate genes.The main findings are as follows:1.Fine position of the main effect QTL-qLN3-2(1)Fine position with extreme recombinant plants.A total of 10038 F2 grains were harvested from the F2 population of Y915 X G86.The polymorphic markers InDel-O2 and InDel-58 were used to select 154 recombinant plants.Using 3 recombinant plants with 5 leaves above the primary ear and 10 recombinant plants with 10 or more leaves above the primary ear,we located the candidate interval between markers Y138 and L180-2,with a physical distance of about 97.6kb.A total of 12044 F2 grains were harvested from the F2 population of Y915 × W373.The polymorphic markers Y138 and Y205 were used to select 48 recombinant plants.Using 3 recombinant plants with 5 leaves on the ear and 5 recombinant plants with 10 leaves or more above the primary ear,we located the candidate interval between the markers Y152 and L30-2,with a physical distance of about 66.7kb.(2)Verification of offspring groups.In the Y915 × G86 population,six self-breeding progeny of recombinant plants were selected to be planted into plots,and the phenotypes of 3 plots were significantly different.Using polymorphic markers Y138,Y160,L180-2 and Y205 to determine the candidate interval between markers Y138 and L180-2.9 self-breeding progeny of 9 recombinant plants were selected from Y915 × W373 population to be planted into plots respectively,among which 7 plots had significant differences in phenotypes,using polymorphism marker Y141,Y152,Y160,L30-2 and Y169 positioned the candidate interval between the markers Y152 and L30-2.The experimental results validate the recombinant plants positioning results.2.BAC library constructionExtracting the whole genomic DNA of young female ears.After restriction enzyme digestion with HindⅢ,the pIndigoBAC536-S vector was used to introduce the digested fragments into E.coli DH10B to construct a BAC library of leafy maize Y915.The library has a total of 160 mixing pools,which are stored in the form of mixing pools,each containing at least 1200 clones,with an average insert of 143 kb,covering 11 times the corn genome.3.Mining candidate genesThe BAC library of the leafy inbred line Y915 was screened by gradient dilution method,and a positive monoclonal maize-177 covering the fine localization results was selected.The length of the positive monoclonal is about 142kb,and the length of the candidate interval is about 135kb.Compared with the B73 reference genome,it is found that there are two inserts with a size of 24.5kb and 52.8kb and a deleted segment with a size of 21.7kb in the candidate interval.Gene prediction of the candidate interval of Y915,a total of 20 candidate genes were predicted,real-time fluorescence quantitative PCR results showed that the expression levels of 7 genes in the candidate genes are different between the leafy inbred line Y915 and the normal inbred line Z58. |
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