Identification of candidate tolerance genes to low-temperature during maize germination by GWAS and RNA-seqapproaches

Maize (Zea mays L.) is one of the main agricultural crops with the largest yield and acreage in the world. However, maize germplasm is very sensitive to low temperatures, mainly during germination, and low temperatures significantly affect plant growth and crop yield. Therefore, the identification of genes capable of increasing tolerance to low temperature has become necessary.

In this study, fourteen phenotypic traits related to seed germination were used to assess the genetic diversity of maize through genome-wide association study (GWAS). A total of 30 single-nucleotide polymorphisms (SNPs) linked to low-temperature tolerance were detected (-log10(P) > 4), fourteen candidate genes were found to be directly related to the SNPs, further additional 68 genes were identified when the screen was extended to include a linkage disequilibrium (LD) decay distance of r² ≥ 0.2 from the SNPs. RNA-sequencing (RNA-seq) analysis was then used to confirm the linkage between the candidate gene and low-temperature tolerance. A total of ten differentially expressed genes (DEGs) (|log₂ fold change (FC)| ≥ 0.585, P < 0.05) were found within the set distance of LD decay (r² ≥ 0.2). Among these genes, the expression of six DEGs was verified using qRT-PCR. Zm00001d039219 and Zm00001d034319 were putatively involved in 'mitogen activated protein kinase (MAPK) signal transduction' and 'fatty acid metabolic process', respectively, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Thus, these genes appeared to be related to low-temperature signal transduction and cell membrane fluidity.

Overall, by integrating the results of our GWAS and DEG analysis of low-temperature tolerance during germination in maize, we were able to identify a total of 30 SNPs and 82 related candidate genes, including 10 DEGs, two of which were involved in the response to tolerance to low temperature. Functional analysis will provide valuable information for understanding the genetic mechanism of low-temperature tolerance during germination in maize.