Genome-Wide Association Study And Comparative Genomics Analysis Of Quality Protein Maize

Quality protein maize is one of the most unexpected achievements in the field of plant breeding in the past.Its lysine and tryptophan content is higher than that of ordinary maize,thereby improving the quality of maize protein,and the nutritional value is comparable to that of milk,and has outstanding contributions to the development of the feed industry.But the mechanism of the endosperm modifer is complex.The main problem it faces is how to introduce multiple non-linked o2 modifiers that are present in tropical germplasm into a backbone inbred line with an invisible homozygous o2 background.Therefore,the development of new varieties is slow.Finding these non-linked o2 modifiers is the main task for accelerating QPM breeding.At present,the research on quality protein maize modifiers mainly focus on ?-zein and starch.However,the specific modification mechanism remains unclear.Existing low-resolution markers are difficult to map specific physical locations of QPM modifiers,although to a certain extent they can assist QPM breeding but are very limited.While maize is an incredibly diverse species,there are even as many as half as many differences between the two maize genomes.Therefore,maize needs multiple reference genomes to contain all its genome information.In order to analysis of o2 modified gene and its mechanism of restoring hard endosperm,the establishment of a quality protein maize reference genome are also essential.Therefore,the main content of this study is using the genotype data of 9,007,194 SNPs consisting of 464 common maize inbred lines and 38 QPM maize inbred lines to carry out genome-wide association analysis with 27-kDa ?-zein and amylose content,at the same time,RNAseq differential expression analysis of CM105?,CM105o2(o2 mutant)and CM105mo2(QPM)was performed.The purpose is to find more refined QPM modifiers intervals and loci,to further promote the research and development of QPM modifiers.At the same time,K0326y is used as material to construct high-resolution genome and full-length cDNA sequences by third generation sequencing technology and bionano optical map technology.It can provide sufficient sequence information for the study of QPM maize,thereby accelerating the mapping of o2 modified genes and promoting the process of QPM breeding in China.The main results of this study are:(1)Genotype data with 9,007,194 SNP sites consisting of 464 common maize inbred lines and 38 QPM maize inbred lines are used for genome-wide association analysis with 27-kDa ?-zein and amylose content.Among them,42 SNPs are found to be significantly associated with 27-kDa ?-zein expression levels,and within the candidate intervals are the gene GRMZM2G138727,which encodes a 27-kDa ?-zein,and the gene GRMZM2G138689,which encodes a 50-kDa ?-zein.The association analysis is performed with amylose content by removing 38 QPM materials,and Waxyl gene encoding GBSSI exists in the most significant SNP candidate interval and a number of genes involved in the amylose synthesis pathway are excavated.The association analysis is performed again with amylose content after adding 38 QPM maize inbred lines,find that 13 new SNPs are significantly associated with amylose content.The most significant SNP site has changed and the most important candidate gene becomes MFS-like.Two MADS-box transcription factors and ei13 genes may be involved in the formation of QPM vitreous endosperm.Fifteen genes unique in the RNAseq differential expression of 16-DAP CM105o2 and CM105mo2 endosperm coincide with GWAS results of 27-kDa ?-zein and amylose content,of which MADSil is upregulated in CM105mo2,which may regulate the expression of ?-zein and starch synthesis to promotes the formation of QPM maize.(2)Using Waxy maize materials in GWAS populations,three candidate markers within the Waxyl gene are developed by candidate gene association analysis techniques:30-33 bp InDe11773 and two SNPs markers.InDel primers can be directly distinguished by electrophoresis at 3%agarose gel.Using two SNPs to develop KASP primers WXT and WXT2.After verified,the WXT2 primer has the best typing effect,and can distinguish between Waxy maize and non-Waxy mazie.This high-throughput method will play an important role in the screening of Waxy maize materials.(3)De novo assembly of the quality protein maize K0326y genome using PacBio's third-generation sequencing technology and bionano optical map technology.The final assembled genome size is 2.14 Gb and 1221 contigs.The size is comparable to the latest B73 reference genome and the contig N50 is 6.99 Mb,which is 6 times higher than the latest version of the B73 reference genome contig N50(1.18 Mb).Our assembled K0326y is the most complete mazie genome,with a maximum contig length of 35 Mb.The contig data is further integrated with Bionano's optical map data obtaining a scaffold N50 of 28.5 Mb and the longest scaffold of 69.3 Mb,which is almost one third of the average chromosome length.