Computational protocol: KRN4 Controls Quantitative Variation in Maize Kernel Row Number

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Protocol publication

[…] A subset of an association mapping panel with 368 diverse inbred lines was genotyped with 500K SNP markers []. KRN of these 368 lines was evaluated in five environments and reported in previous study, including Ya'an (30°N, 103°E), Sanya (18°N, 109°E), and Kunming (25°N, 102°E) in 2009, and Wuhan (30°N, 114°E) and Kunming (25°N, 102°E) in 2010 []. The best linear unbiased prediction (BLUP) of KRN was estimated using a linear mixed model in SAS software (SAS Institute Inc., 2001) by previous study [, ]. The association of KRN4 with KRN (BLUP data) [] was established using Tassel v3.0 with a mixed linear model (MLM) approach considering varietal relatedness (K) and population structure (Q) (MLM K + Q) [, –]. The linkage disequilibrium among associated SNPs was estimated using Haploview v4.1 []. [...] To discover DNA sequence variation and putative causal polymorphisms in UB3 and KRN4, gene-specific primers () were designed to amplify UB3 and KRN4 in the association mapping panel [, ]. We genotyped 428 inbred lines using the 1.2-Kb PAV in KRN4 as a marker, and sequenced about 4.0 Kb of DNA from 5'-upstream of UB3 to its 3'-UTR and ~3 Kb containing KRN4 in 110 or 428 inbred lines of the AM panel, respectively (the line number of the lines that were sequenced is listed in the ). SNPs and indels with MAF > 0.05 were used to estimate pairwise LD and to evaluate the association between polymorphic sites and KRN under the MLM K+ Q model [–]. Conditional analysis was conducted using the associated sites as covariates under an MLM K + Q model in Tassel v3.0. The MLM K + Q model was also used for haplotype-based association analysis. The selfed progeny of heterozygous recombinants RL6-RL12 which are segregating at 1.2-Kb PAV, S35 and S23 were employed to evaluate the genetic effect of 1.2-Kb PAV, S35 and S23 (). The individuals, which harbored homozygous alleles of the three sites, were used to estimate the additive effects of in each segregating population ().The selection pressure on KRN4 during the domestication and improvement of maize was estimated using 36 randomly selected landraces () from 280 diverse maize landrace collections () [] and 29 Z. mays subsp. parviglumis teosinte () from 120 teosinte accessions (). The KRN4 genomic region was amplified and sequenced using primers listed in . Nucleotide diversity (π) and Tajima’s D were estimated using DnaSP ver. 5.0 []. The 1.2-Kb PAV was treated as single PAV when estimating the nucleotide diversity (π). Four neutral loci (adh1, adh2, fus6 and te1) [–] were used as controls for the HKA test [] using Zea diploperennis as the outgroup. The overall HKA P-value was obtained by summing the individual χ2 values of the four control genes. Another 88 teosinte accessions (including 35 Z. mays subsp. parviglumis and 54 Z. mays subsp. mexicana accessions, ) and 244 maize landraces were genotyped by a PCR marker for the 1.2-Kb PAV and a KASP marker (http://www.kbioscience.co.uk/) for S35, to estimate their frequency in teosinte accessions and maize landraces (Primers are listed in ). All of the sequences have been deposited in NCBI Genebank KT928654—KT931615. […]

Pipeline specifications

Software tools TASSEL, Haploview, DnaSP, HKA
Applications Population genetic analysis, GWAS
Organisms Zea mays