Combined QTL Mapping across Multiple Environments and Co-Expression Network Analysis Identified Key Genes for Embryogenic Callus Induction from Immature Maize Embryos

Abstract

The ability of immature embryos to induce embryogenic callus (EC) is crucial for genetic transformation in maize, which is highly genotype-dependent. To dissect the genetic basis of maize EC induction, we conducted QTL mapping for four EC induction-related traits, the rate of embryogenic callus induction (REC), rate of shoot formation (RSF), length of shoot (LS), and diameter of callus (DC) under three environments by using an IBM Syn10 DH population derived from a cross of B73 and Mo17. These EC induction traits showed high broad-sense heritability (>80%), and significantly negative correlations were observed between REC and each of the other traits across multiple environments. A total of 41 QTLs for EC induction were identified, among which 13, 12, 10, and 6 QTLs were responsible for DC, RSF, LS, and REC, respectively. Among them, three major QTLs accounted for >10% of the phenotypic variation, including qLS1-1 (11.54%), qLS1-3 (10.68%), and qREC4-1 (11.45%). Based on the expression data of the 215 candidate genes located in these QTL intervals, we performed a weighted gene co-expression network analysis (WGCNA). A combined use of KEGG pathway enrichment and eigengene-based connectivity (KME) values identified the EC induction-associated module and four hub genes (Zm00001d028477, Zm00001d047896, Zm00001d034388, and Zm00001d022542). Gene-based association analyses validated that the variations in Zm00001d028477 and Zm00001d034388, which were involved in tryptophan biosynthesis and metabolism, respectively, significantly affected EC induction ability among different inbred lines. Our study brings novel insights into the genetic and molecular mechanisms of EC induction and helps to promote marker-assisted selection of high-REC varieties in maize.

Keywords: QTL mapping; WGCNA; candidate gene; embryogenic callus; immature embryo; maize.

Figure 1

The calli induced from different doubled haploid lines in the IBM Syn10 DH population. (a,c) show the embryogenic callus (EC). (b,d) exhibit the non-embryogenic callus (NEC). Bar scale is 1 cm in (a,b) and 3 mm in (c,d).

Figure 2

Phenotypic frequency distribution of the IBM Syn10 DH population across three environments. RSF, LS, DC, and REC represent the rate of shoot formation, length of shoot, diameter of callus, and rate of embryogenic callus induction. XSBN, YA, and CZ denote the environments Xishuangbanna, Ya’an, and Chongzhou, respectively. Mo17 and B73 show the two parents of the population.

Figure 3

Chromosomal distributions of identified QTLs under three environments in this study. The red, blue, green, and purple represent Xishuangbanna (XSBN), Ya’an (YA), Chongzhou (CZ), and the best linear unbiased prediction (BLUP), respectively. Squares, circles, triangles, and pentagrams represent diameter of callus (DC), rate of shoot formation (RSF), length of shoot (LS), and rate of embryogenic callus induction (REC), respectively.

Figure 4

Gene co-expression network and hub genes responsible for maize embryogenic callus induction. (a) Eigengene expression pattern of the embryogenic callus induction-associated module (blue). Upper panel shows the gene expression heat map; lower panel shows the eigengene expression histogram. (b) Top 20 enriched pathways in blue module. (c) Co-expression network of hub genes. The four hub genes are shown in pink and orange. (d) The expression patterns of the four hub genes during embryogenic callus induction. CN9802, ZM28, JS0251, and YA3237 represent four different maize inbred lines. The significance of difference between each pair of these lines was estimated by using a t-test (2-tailed), with n = 3 (three biological replicates). *, **, and *** represent significant difference at p = 0.05, p = 0.01, and p = 0.001 levels, respectively, at least between one pair of the four lines, for each stage. ns = not significant. The bars show the standard deviation (SD).

Figure 5

Hub gene-based association mapping. (a,f) Manhattan plots (top) and LD heat maps (bottom) of Zm00001d034388 and Zm00001d028477. The significance threshold is shown as a red dashed line (p < 0.05). The gene structure is shown in the middle. The exons are represented by filled red boxes. (b–e) Differences in (b) REC (rate of embryogenic callus), (c) DC (diameter of callus), (d) RSF (rate of shoot formation), and (e) LS (length of shoot) between alleles or haplotypes of Zm00001d034388. (g,h) Differences in (g) LS and (h) RSF between alleles or haplotypes of Zm00001d028477. Statistical significance is determined by two-tailed t-test: *, **, and *** denote significance at p = 0.05, p = 0.01, and p = 0.001 levels, respectively.