Genome-Wide Association Study and RNA-Seq Elucidate the Genetic Mechanisms Behind Aphid (Rhopalosiphum maidis F.) Resistance in Maize

Abstract

Maize is a crucial food crop and industrial raw material, significantly contributing to national food security. Aphids are one of the most prevalent and destructive pests in maize production, necessitating the exploration of pest-resistant germplasm and the development of resistant varieties as the most fundamental and effective strategy for mitigating aphid-induced damage. This study established an aphid resistance evaluation system and identified 17 elite resistant inbred lines through multi-year screening. A genome-wide association study (GWAS) revealed 22 significant single-nucleotide polymorphisms (SNPs) associated with aphid resistance, including genes involved in benzoxazinoid (Bx) biosynthesis (such as Bx2), insect resistance-related transcription factors (such as WRKY23), plant lectins, and other resistance pathways. RNA-seq analysis of the samples before and after aphid infestation detected 1037 differentially expressed genes (DEGs) in response to aphid infestation, with KEGG enrichment highlighting benzoxazinoid biosynthesis and starch/sucrose metabolism as primary response pathways. Integrating GWAS and RNA-seq results revealed the presence of several benzoxazinoid synthesis-related genes on the short arm of chromosome 4 (Chr4S). FMqRrm1, a Kompetitive Allele-Specific PCR (KASP) marker, was derived from the Chr4S region. We subsequently utilized this marker for marker-assisted selection (MAS) to introgress the Chr4S region from the aphid-resistant inbred line into two aphid-susceptible inbred lines. The results demonstrated that the Chr4S favorable allele significantly reduced aphid occurrence by 1.5 to 2.1 grades. This study provides a critical theoretical foundation and practical guidance for understanding the molecular mechanism of aphid resistance in maize and molecular breeding for aphid resistance.

Keywords: RNA-seq; aphid; association analysis; maize; molecular breeding.

Figure 1

The phenotype for aphid resistance in the GWAS population. (a) The typical aphid resistance score (ARS) grades 0 to 5. (b) Distribution and correlation of aphid resistance score across environments. ** p < 0.01.

Figure 2

GWAS results for aphid resistance using the MLM statistical approach. (a) Manhattan plot displaying GWAS results. (b) QQ plot of GWAS results. (c) Bubble maps for GO enrichment analysis of significant loci. BP: Biological processes; MF: molecular function; CC: cellular component.

Figure 3

Analysis of the differentially expressed genes (DEGs) following aphid infestation. (a) Heatmap for correlation analysis of gene expression levels across samples. (b) The number of genes that show differential expression after aphid infection. (c) A Venn diagram of DEGs at four sampling times following aphid infestation.

Figure 4

GO (a) and KEGG (b) pathway enrichment of genes that consistently respond to aphid infestation.

Figure 5

Development of aphid-resistant markers and their application in marker-assisted breeding (MAS). (a) Development of molecular marker, FMqRrm1, for MAS for aphid resistance. The blue dots signify the favorable allele AA associated with aphid resistance, the green dots denote the standard allele aa linked to aphid susceptibility, the red fluorescence represents the heterozygous Aa, and the gray area is the negative control. (b) Resistance comparison after introducing aphid-resistant chromosomal fragments into the HL170 background. (c) Resistance comparison after introducing aphid-resistant chromosomal fragments into the 12H691 background. Data are means ± SD from three biological replicates. ** p < 0.01, *** p < 0.001, **** p < 0.0001.