Fine Mapping And Candidate Gene Analysis Of QKRN5.04,a Major QTL For Kernel Row Number In Maize

Kernel row number?KRN?is not only one of the components of maize yield,but also an important domesticated trait.Strengthening the genetic and developmental research of KRN is of great significance to maize breeding.Previously,our lab constructed F_2:3 families derived from reciprocally crossing Nong531?N531?with the four-rowed waxy corn?SLN?and identified a major QTL for KRN on bin5.04,designated qKRN5.04.Later,we continued to construct a series of advanced backcross populations and secondary separation populations with qKRN5.04 single fragment using N531 as the donor parent and marker-assisted selection.The genomic location of qKRN5.04 was certainly narrowed down to the interval of 136.3-140.0 Mb,explaining the effect value of 0.80-1.76 rows.In this study,we selected heterozygous individuals from BC₅F₄ using markers flanking qKRN5.04 and fine mapped qKRN5.04 by using a progeny test.Simultaneously,we combined the regional association analysis to find out the candidate gene.Sequence analysis,expression analysis and selection analysis of the candidate gene were also carried out.The main results were as follows:1.Using more InDel markers in the target section,six heterozygous recombinant groups were selected from BC₅F₅.We selected one recombinant family that contained more seeds within each genotype group and planted in Hainan,2016.By progeny test,qKRN5.04 was further delimited to a 739kb between markers A5S073 and A5S044.A new InDel marker N5M98 was added to determine the recombination breakpoints and important five genotype groups were planted in Beijing,2017.The same strategy was applied and we successfully narrowed qKRN5.04 to a 65-kb genomic region flanking N5M98-A5S044.2.A panel of 236 diverse inbred lines was used to carry out regional association analysis.Mixed linear model?MLM?tested at the region 136-142 Mb of chr5 and identified six SNPs?SNP370,SNP643,SNP651,SNP856,SNP859,SNP918?significantly associated with KRN.These SNPs are not only within a single gene GRMZM2G092154,but also located at the fine mapped region.Therefore,we consider GRMZM2G092154 a candidate gene for qKRN5.04 according to the combined results of progeny test and association mapping.3.GRMZM2G092154 has 1902 bp from ATG to TAG and there are five SNPs in the coding region between N531 and SLN.But these variations only result in one amino acid substitution.SIFT analysis was made,but the function of the protein was not been changed.The sequencing results of the regulatory region indicated that 46 SNPs and 5 InDels existed in the 1586 bp upstream from the ATG,containing one 308-bp InDel,named InDel308.Further analysis found that N531 and NIL-N531exhibited lower expression of GRMZM2G092154 at SPM-stage and SM-stage,but with higher KRN,while NIL-SLN showed higher expression of this gene and correspondingly lower KRN.Taken together,the expression of GRMZM2G092154 has a strong negative correlation with KRN.4.A total of 42 teosinte accessions and 145 diverse maize inbred lines were employed to identify InDel308 genotypes in the regulatory region of GRMZM2G092154 and calculate allele frequency.The results showed that allele frequencies of the 308 bp insertions and deletions are 0.26 and 0.74 in the teosinte entries,respectively and 0.45 and 0.55 in the maize inbred lines,respectively.It is obvious that the allele frequency of 308 bp insertion is higher in the modern maize.Therefore,we think that InDel308 in GRMZM2G092154 regulatory area might be under selection during domestication from teosinte to maize.