The Development Of The Foreground And Background Integrated Genotyping (FBI) Technology And Its Application In Rice Breeding

Molecular Marker-Assisted Selection(MAS)is an important strategy for breeders to improve the efficiency of breeding screening,increase the accuracy of target gene screening,and obtain high-quality varieties conveniently and quickly during the research process.With the rapid development of molecular detection technology,in the era of “big data”,high-throughput single nucleotide polymorphism(SNP)markers have been widely used in breeding research due to their characteristics of great quantity,high-throughput,and a large scale.Our laboratory earlier invented a new high-throughput SNP molecular marker method through improving transposon display technology.The method was using a transposon primer and a specific primer for the foreground gene to generate a second-generation sequencing library through one-step transposase reaction and two rounds of PCR amplification,and the sequencing data can be analyzed to know the target genotype and genetic background of the tested sample.Based on this,the present study improved and optimized the experimental procedure,using primer-template perfect annealing(PTPA)to amplify the target fragment as the foreground markers while using the primer-template mismatches annealing(PTMA)to amplify hundreds of thousands of other fragments as background markers for the whole genome,and finally developed FBI(Foreground and Background Integrated genotyping)technology that can simultaneously detect multiple target foreground loci and the whole genetic background.This technology is not restricted by species,and which can provide more convenience for the research work of breeders.Generally,the following results were obtained in research:1.Complete the optimization of the experimental method for screening one foreground gene or multiple foreground genes,so that the PTMA and PTPA amplification effects of the primers are stronger,and the final optimal system of the FBI method is established through Q-PCR detection.2.The versatility research of different primers of FBI technology in different crop varieties has been done,which proved that the method is not restricted by species,and any one primer can get the genetic background marking for different crops.3.Using high-fidelity enzyme and non-high-fidelity enzyme comparison experiments,it is determined that the matching mode of the PTMA for the specific primer in the FBI technology,and the mode is the5-7 bases in the middle of the primer matching perfectly with a sliding window moving combined amplification method.4.In the application research of FBI-seq,we have tested the whole background and 5 foreground genes of blast resistance gene Pi2,aroma gene BADH2,fertility-restoration genes Rf3 and Rf4,and quality gene Waxy in 46 rice lines.These results show that the method of FBI-seq only needs a common primer that does not require special design and optimization to realize the simultaneous detection of the foreground marker and hundreds of thousands of background markers,and can easily switch to different foreground genes and different species,especially those with less genomic information.In addition,this method can simultaneously detect up to 6target foreground genes.Compared with other current whole-genome marker detection methods,the FBI technology based on the combination of LM-PCR and NGS sequencing proposed in this study has simple experimental procedures and is a kind of simple,fast and trendy new-type breeding genome-wide molecular marker method,and has wide application prospects in the genotyping for breeding research.