Establishment Of Polymorphism Marker Development Method And Its Application In The Map-Based Gene Cloning

Maize（Zea mays）kernel size is a major determinant of final yield and a major target trait for domestication and improvement.Therefore,it is important to investigate the key genes affecting maize kernel size to resolve the molecular mechanism of maize kernel development.Map-based cloning using high-density molecular markers to locate and progressively narrow down candidate intervals is an important tool for cloning target genes.However,for genotypes without reference genome,the design of molecular markers is still difficult and the success rate is low.Meanwhile,due to the rapid development of sequencing technology,a large number of high-quality reference genomes of maize have been published,bringing new opportunities for molecular marker design.Therefore,in this study,we developed a method for designing InDel molecular markers for reference-free genomes by using B73 as the main reference genome and comparing multiple reference genomes with it,and applied it to the cloning of two kernel development-related genes.The specific results were as follows:1.Based on the published genomes of B73,Mo17,W22,PH207 and Oh7B,covariance analysis was performed to identify the InDel present on these genomes within the candidate intervals.By verifying the published data,we found that some InDel sites only exist on one genome,so increasing the number of comparative genomes can obtain richer InDel sites.2.Our group identified two kernel size mutants that matched the 3:1 segregation ratio,and named them SY189 and L191.The mutants were crossed with B73 to construct an F₂ locus population,and BSR-Seq analysis was performed on the two mutants to obtain the initial localization intervals.Using this method to develop markers in each of the two candidate regions to identify polymorphisms,InDel were found to be present on one,two,three,and four genomes with success rates of 0.00%,23.08%,69.23%,and 82.50%for the designed primers,respectively.These results suggest that insertion/deletion sites that are present on multiple known maize genomes do tend to be more polymorphic on an unknown genome,and this possibility increases with the number of genomes.3.Using the InDel molecular marker development method established in this project,a total of 11 pairs of InDel molecular markers were developed within the SY189locus interval,and the interval was narrowed to 1.6 Mb.The Zm00001d039808 gene was identified as a candidate gene.We cloned the gene in the wild type and mutant separately,and the sequencing results showed that a 2,858 bp fragment was inserted764 bp downstream of the first exon start codon,which was identified as an LTR-type transposon,causing the reading frame to be altered and the translation of the coding region to be terminated prematurely,thus affecting the translation and function of the protein.The gene encodes a P-type PPR protein containing 19 P motifs that is expressed at all stages of maize kernel development.Mutations in this gene affect maize kernel development and ultimately lead to a maize kernel defective phenotype.4.Using the InDel molecular marker development method established in this project,a total of 15 pairs of InDel molecular markers were developed in the L191interval,narrowing the interval to 391 Kb.We designed primers for PCR amplification and sequencing of these potentially variable regions,but no corresponding variants were detected at the DNA level.We speculate that the variants may occur in the intergenic region or intron,and we will continue to identify candidate genes by further narrowing the localization interval through fine localization.