High-throughput SNP Development And Genome-wide Association Study In Maize Based On Next-generation Sequencing

Maize is one of the most important crops for food and fuel. Its quality and yield are always two of the main breeding goals. In this study, the major objects are identifying the genes affecting maize complex quantitative traits including kernel oil content, ear length and tassel main axis length by combing the high density single nucleotide polymorphisms(SNP) markers obtained by next-generation sequencing and SNP array technologies and existed phenotypic traits. The study will provide abundant candidate SNPs and genes for maize molecular breeding in the future. The major results are listed as follows:1. To build up the Illumina high throughput sequencing platform in our laboratory including whole genome sequencing, reduced representation sequencing( genotyping by sequencing, GBS) and RNA sequencing. The library construction, sequencing and SNP calling of 522 diverse maize inbred lines, 10 representative samples of recombinant inbred lines derived from Teosine and Mo17 and 186 accessions of Arundo donax have been completed.2. Using the built GBS platform, 670,412 SNP markers were identified from 522 diverse inbred lines. Combing the SNP identified by RNA-seq(3 M SNP of 368 lines), 50 K-Chip(513 lines) and 600 K-Chip(153 lines), an integrated map was merged based on comparison and imputation, 2.65 M unique SNP markers were obtained in 541 inbred lines. And 1.25 M(MAF ≥ 0.05) SNPs among the integrated map can be used for GWAS in further study.3. GWAS of maize kernel oil content using integrated map of 1.25 M SNP markers has verified the reported significant associated genes previously(19 /26), at genome-wide significance level(P < 9.83×10-7). Meanwhile, 18 new significant associated loci were identified, comparing to using the RNA-seq data of 0.56 M SNP markers. Among those loci, 7(1/3) loci located in nongenic region, and 14 loci were supported by expression Q TL(e Q TL). As an example of chromosome 4, 8 linked significant SNPs e QTL analysis how this locus may regulate three genes expressions(GRMZM2G066618, GRMZM2G162670, GRMZM2G125556). The homologous genes of three genes in Arabidopsis were reported previously to be involved in lipids metabolic pathway.4. Using the same 1.25 M SNPs, GWAS were also performed for ear length and tassel main axis length. 33 and 12 new significant associated SNPs were identified on chromosome 1 and 2 respectively, comparing to us ing the 0.56 M RNA-seq SNPs at genome-wide significance level(P < 9.83×10-7). Meanwhile, the new loci were also located on QTL regions mapped by YU87-1/BK and TEO/MO17 RIL populations for the same trait respectively. The identified candidate SNPs and genes provided useful information for further study.