Genetic basis of kernel starch content decoded in a maize multi-parent population

Abstract

Starch is the most abundant storage carbohydrate in maize kernels and provides calories for humans and other animals as well as raw materials for various industrial applications. Decoding the genetic basis of natural variation in kernel starch content is needed to manipulate starch quantity and quality via molecular breeding to meet future needs. Here, we identified 50 unique single quantitative trait loci (QTLs) for starch content with 18 novel QTLs via single linkage mapping, joint linkage mapping and a genome-wide association study in a multi-parent population containing six recombinant inbred line populations. Only five QTLs explained over 10% of phenotypic variation in single populations. In addition to a few large-effect and many small-effect additive QTLs, limited pairs of epistatic QTLs also contributed to the genetic basis of the variation in kernel starch content. A regional association study identified five non-starch-pathway genes that were the causal candidate genes underlying the identified QTLs for starch content. The pathway-driven analysis identified ZmTPS9, which encodes a trehalose-6-phosphate synthase in the trehalose pathway, as the causal gene for the QTL qSTA4-2, which was detected by all three statistical analyses. Knockout of ZmTPS9 increased kernel starch content and, in turn, kernel weight in maize, suggesting potential applications for ZmTPS9 in maize starch and yield improvement. These findings extend our knowledge about the genetic basis of starch content in maize kernels and provide valuable information for maize genetic improvement of starch quantity and quality.

Keywords: association mapping; linkage analysis; pathway-driven; starch content; trehalose-6-phosphate synthase.

Figure 1

Phenotypic variation in starch content and summary of single QTLs for starch content identified by SLM analysis in six RIL populations. (a) Phenotypic variation in starch content among the six RIL populations. The short, horizontal bars of different colours indicate the starch content values for the seven parental lines. (b) Distribution of single QTLs on chromosomes. QTL regions across the maize genome are represented by confidence intervals, and LOD values are scaled by colour. (c) Broad‐sense heritability (h ²) and total PVE for single QTLs for starch content in each population. (d) Effect size and the origin of the increasing alleles of the identified single QTLs. Orange and blue bars indicate that increasing alleles come from the parents with high and low starch content, respectively, in a given population.

Figure 2

Overview of QTLs for starch content identified by JLM and GWAS methods. (a) Manhattan plot resulting from the JLM results for starch content in maize kernels. The horizontal dashed line shows the threshold of the likelihood ratio test (LRT = 2.99). (b) Distribution of allelic effects on starch content from seven founders at the QTLs identified by JLM. The columns show the number of alleles from the seven founders ordered according to the values of starch content. The red circles represent the starch content of each parent. The dark green and blue shadings show the number of alleles from a founder with the largest positive and negative effects at a given locus, respectively, whereas the light green and blue shadings show the number of alleles from a founder with moderate positive and negative effects, respectively. (c) Chromosome distribution of significant SNPs via GWAS. The orange upward‐facing triangles indicate that the major allele increased starch content relative to the minor allele, the blue downward‐facing triangles indicate the opposite effect, and the black dots indicate the candidate SNPs identified by the backward regression model. (d) Venn diagram of co‐localization between QTLs detected by the three models. Orange, blue and green numbers represent the number of QTLs detected by SLM, JLM and GWAS, respectively.

Figure 3

Associations between starch content and ZmABCI6, ZmACP3, and ZmCPR. (a) Manhattan plot of the results from the regional association mapping for starch content in AMP508. The black horizontal dashed line indicates the Bonferroni‐adjusted significance threshold (P = 1.0 × 10^–4). The red dots indicate the lead SNPs at each significant locus; the plausible biological candidate gene at each of these loci is shown. (b–d) Associations between the SNPs at the ZmABCI6 (b), ZmACP3 (c) and ZmCPR (d) loci and starch content. The red dots show the lead SNP with the most significant association. Colour coding of the remaining SNPs reflects their extent of linkage disequilibrium (r ²) with the lead SNP. The black and grey boxes above the x axis represent exons and UTRs, respectively. The InDels (orange triangles) and nonsynonymous SNPs (blue vertical lines) in the promoter, UTRs and exons are shown. (e–g) Co‐localization of candidate genes and the QTL or SNPs identified via SLM and RIL‐GWAS. Blue and green lines show the LOD profile of the QTL identified via SLM for the indicated RILs, whereas the blue dots represent the RIL‐GWAS results. The red dashed vertical lines indicate the position of candidate genes. (h–j) Genetic effects of the lead SNPs at the ZmABCI6 (h), ZmACP3 (i) and ZmCPR (j) loci on starch content. The P‐values were calculated based on a two‐tailed Student’s t‐test.

Figure 4

ZmTPS9 affected starch content and kernel weight. (a) LOD profile of qSTA4−2 in the K22/CI7 RIL population, JLM and RIL‐GWAS results at this locus. The red dashed lines indicate the position of ZmTPS9. The blue shading depicts the support interval from JLM with its height indicating the LRT score. (b) Gene structure of ZmTPS9 and sequence comparisons between K22 and CI7. Black and grey boxes represent exons and UTRs, respectively. The grey vertical lines show SNPs in non‐coding regions and synonymous SNPs in exons; the green segments show InDels; the red lines show nonsynonymous SNPs in exons. The red star indicates the mutation position of tps9. The red nucleotides indicate nonsynonymous SNPs between K22 and CI7. (c) Expression pattern of ZmTPS9 in developing kernels at 20 DAP. (d) Constructs used to test the effect of InDel283 on ZmTPS9 expression in transient expression assays in maize leaf protoplasts. K22, K22⁻²⁸³, CI7 and CI7⁺²⁸³ constructs harbour the promoter and 5’UTR of different ZmTPS9 alleles, including 1290 bp from K22, 1007 bp from K22 without a 283‐bp insertion, 1019 bp from CI7 and 1302 bp from CI7 with a 283‐bp insertion. (e) The alleles without a 283‐bp insertion are associated with increased LUC activity in comparison with the alleles with a 283‐bp insertion. The data were normalized with respect to the average values of the K22 construct. (f) Ear and kernel morphologies and genotype of wild type (WT) and tps9. Scale bars: 1 cm. The blue shading indicates the mutated nucleotide. (g–h) Quantification of starch content and hundred kernel weight between WT and tps9. 19SY: Sanya in 2019; 19BN: Bayan Nur in 2019. In c, e, g and h, the P‐values were based on a two‐tailed Student’s t‐test.