Genome-Wide Association Study Identifies Quantitative Trait Loci and Candidate Genes Involved in Deep-Sowing Tolerance in Maize (Zea mays L.)

Abstract

Deep sowing is an efficient strategy for maize to ensure the seedling emergence rate under adverse conditions such as drought or low temperatures. However, the genetic basis of deep-sowing tolerance-related traits in maize remains largely unknown. In this study, we performed a genome-wide association study on traits related to deep-sowing tolerance, including mesocotyl length (ML), coleoptile length (CL), plumule length (PL), shoot length (SL), and primary root length (PRL), using 255 maize inbred lines grown in three different environments. We identified 23, 6, 4, and 4 quantitative trait loci (QTLs) associated with ML, CL, PL, and SL, respectively. By analyzing candidate genes within these QTLs, we found a γ-tubulin-containing complex protein, ZmGCP2, which was significantly associated with ML, PL, and SL. Loss of function of ZmGCP2 resulted in decreased PL, possibly by affecting the cell elongation, thus affecting SL. Additionally, we identified superior haplotypes and allelic variations of ZmGCP2 with a longer PL and SL, which may be useful for breeding varieties with deep-sowing tolerance to improve maize cultivation.

Keywords: ZmGCP2; deep sowing; genome-wide association study; maize; plumule length; quantitative trait locus; shoot length.

Figure 1

Linkage disequilibrium (LD) and structure analysis of the association population. (A) Values of ΔK plotted against K = 1–10 based on the structure analysis of the accessions population. (B) Population structure with K  =  3, 5, and 7. (C) Phylogenetic tree of the entire panel for K  =  5. (D) LD decay across the 10 maize chromosomes. Chr1–Chr10 represent chromosomes 1–10.

Figure 2

Circular Manhattan plot of the genome-wide association study (GWAS) for deep-sowing tolerance-related traits. (A–D) Manhattan plots of ML, CL, PL, and SL, respectively. ML, mesocotyl length; CL, coleoptile length; PL, plumule length; SL, shoot length. I–III represent different environments: (I) 2020 Chongzhou, Sichuan; (II) 2020 Xishuangbanna, Yunnan; (III) 2021 Chongzhou, Sichuan. Chr1–Chr10 represent chromosomes 1–10. The red line represents the significance threshold of p = 1 × 10⁻⁵. The red dots indicate single nucleotide polymorphisms (SNPs) significantly associated with deep-sowing tolerance-related traits.

Figure 3

Chromosomal distributions of the quantitative trait loci (QTLs) for deep-sowing tolerance-related phenotypes in the association population. Chr1–10 represent chromosomes 1–10. The color of the chromosome represents the density of SNPs. The QTL names are indicated according to the following rules: the chromosome number is indicated by the first number following the phenotype name, and the number following the dash is used to identify different QTLs found on the same chromosome for the same trait.

Figure 4

Mutation and phenotypes of zmgcp2 compared to wild-type B73. (A) Schematic charts of ZmGCP2 gene structure; asterisk stands for stop codon. The red arrowhead indicates the mutation position in zmgcp2. (B) Sequencing results of B73 and zmgcp2 at the mutation position. (C–E) Comparison of ML, PL, and SL between B73 and zmgcp2. (F) Seedling phenotypes of B73 and zmgcp2; scale bars = 2 cm. (G–H) Plumule cells of B73 and zmgcp2 under blue excitation light; scale bars = 50 μm. (I) Comparison of plumule cell length between B73 and zmgcp2; n ≥ 90 cells from 3 seedlings. Student’s t-test; ***, p < 0.001; ns, not significant.

Figure 5

Association analysis of the candidate gene ZmGCP2. (A) Candidate gene association analysis of ZmGCP2 with PL-best linear unbiased estimate (BLUE). (B) Candidate gene association analysis of ZmGCP2 with SL-BLUE. (C) Pairwise LD between the markers. (D) Details of four haplotypes (Hap1, Hap2, Hap3, and Hap4); n represents the inbred line number of each haplotype. (E,F) Comparison of PL and SL between different haplotypes. Student’s t-test; *, p < 0.05; ***, p < 0.001; ns, not significant. (G) Distribution of inbred lines from different groups in the four haplotypes.