- Quinn et al. - If reads from B73 alleles preferentially aligned to the B73 genome relative to reads from Mo17 alleles, RILs with B73 alleles at cis-eQTL would have higher transcript abundances than RILs with Mo17 alleles.. - Using the B73 genome as a reference, we detected 9306 cis- eQTL out of 22,408 variable genes. - In contrast, using Mo17 the reference genome detected 7985 cis- eQTL from 16,983 variable genes. - B73 cis-eQTL detected when reads were aligned to the B73 reference genome were not detected when Mo17 was used as a reference genome. - of Mo17 positive cis-eQTL detected when reads were aligned to the Mo17 reference genome were not de- tected when B73 was used as the reference genome.. - Total cis-eQTL c 9306 7985. - B73 positive cis-eQTL Mo17 positive cis-eQTL RNA-seq were mapped to the B73 a or Mo17 b reference using STAR with the default criteria as given in Supplemental Information.. - c The numbers of genes with cis-eQTL were recorded excluding genes with both cis and trans eQTL. - At 212 genes, B73 alleles significantly increased transcript expression resulting in cis-eQTL. - 2 The relationship between the proportion of cis-eQTL that had positive B73 effects when aligned to the B73 reference genome and the number of SNPs between B73 and Mo17 genes. - The line plots are a lowess smooth of the number of SNPs per gene per 2 Mb of chromosome 3 sequence and the proportion of B73 positive cis-eQTL per gene per 2 Mb of chromosome 3 sequence. - The left y-axis is the average number of SNPs within cis-eQTL genes in the intervals. - The right y-axis is proportion of B73 positive cis-eQTL out of the total cis-eQTL in the interval. - Among the genes with positive B73 cis-eQTL effects, a median of 15.7% of reads mapped to UTRs. - In contrast, among the genes with negative B73 cis-eQTL effects, the median was 13.4% (Wilcoxon rank sum test, W P <. - a Expression differences between B73 and Mo17 were highly correlated with numbers of SNPs per 1 kb exon for the 5559 B73 positive cis-eQTL genes with SNP information. - b Expression level differences between Mo17 and B73 were weakly correlated with numbers of SNPs per 1 kb exon for the 2561 Mo17 positive cis-eQTL genes. - Similarly, among the 6005 cis-eQTL detected in analyses of both the default and more relaxed alignment datasets had greater B73 effects in the default ana- lysis (Fig. - 4 Comparison of alignment criteria on cis-eQTL effect size. - Each cis-eQTL for which the B73 allele increased a target gene ’ s expression was labelled “ yes ” if its effect estimated using the most relaxed alignment criteria was lower than its effect using the default alignment criteria. - The cis-eQTL effect was estimated as the average of the mean expression of RILs with the B73 genotype minus the mean expression of RILs with the Mo17 genotype. - Among the 28,289 genes with the most variable expression, 11,908 were significantly affected by one cis-eQTL and no trans-eQTL (Table 4).. - For of these genes, the B73 cis-eQTL up- regulated transcript levels (Table 4). - Alignment bias affected genes in addition to those with cis eQTL. - of 8999) of the B73 positive cis- eQTL detected from analyses of gene expression from both the default and the most relaxed alignment criteria had higher effects in the former. - Re- ducing stringency reduced the total number of genes with cis eQTL by only 2% (265 genes) and the propor- tion of B73 positive eQTL by only 1.6% (Table 4).. - About 50% of cis-eQTL that were detected depended on the reference genome template that was used (Table 1), and many genes not associated with cis-eQTL likely had. - Li et al. - Wang et al. - cis-eQTL only default criteria relaxed criteria most relaxed criteria default criteria relaxed criteria most relaxed criteria default criteria. - B73 positive cis-eQTL Mo17 positive cis-eQTL . - Cho et al. - eQTL linked to the target gene were deemed cis-eQTL [54], and all other eQTLs were termed trans-eQTL. - The average gene expression of lines carrying the B73 and Mo17 cis-eQTL alleles were estimated using the effectplot function in R/qtl.. - Cis-eQTL magnitude was expressed as half of the differ- ence between the two alleles.. - www.panzea.org/genotypes), and extracted the B73 and Mo17 SNPs. - For each cis- eQTL gene, we calculated the ratio between the number of reads mapping to UTRs and the number of reads mapping to all exons for B73 derived reads and Mo17 derived reads [58]. - The median difference in splice junction read ratio and the median difference in UTR read ratio between the B73 inbred and the Mo17 inbred were calculated and the signifi- cance evaluated using the Wilcoxon signed rank test for genes with cis-eQTL. - A similar analysis was done on the subset of eQTL genes for which the B73 cis- eQTL allele was positive and the subset for which the Mo17 cis-eQTL allele was positive. - The significances of the median differences in splice junction and UTR read ratios between B73 cis-eQTL genes and Mo17 cis-eQTL genes were calculated using the Wilcoxon rank sum test.. - cis-eQTL: Cis-acting expression quantitative trait loci. - org/10.1186/s . - S1 The relationship between the proportion of cis-eQTL for which B73 alleles had positive effects on gene expression when aligned to the B73 reference genome and the number of SNPs be- tween B73 and Mo17 genes. - proportion of B73 positive cis-eQTL per gene in 2 Mb of chromosome se- quence. - The left y-axis is the average number of SNPs within cis-eQTL genes in 2 Mb intervals. - The right y-axis is the proportion of B73 positive cis-eQTL out of all cis-eQTL in a 2 Mb interval. - S2 Comparison of alignment criteria on cis-eQTL effect size. - Cis-eQTL effect is estimated as the average of the mean expression of genotype B73 minus the mean expression of genotype Mo17. - S3 For genes with B73 positive cis-eQTL when aligned to the B73 genome by TopHat2 with the default parameters, the expression level differences of homozygous B73 and Mo17 lines were plotted relative to SNP frequency. - Allelic expression differences between B73 and Mo17 were highly correlated with numbers of SNPs per 1 kb exon for the 8794 B73 positive cis-eQTL genes with SNP informa- tion. - 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