Identification of chilling-tolerant genes in maize via bulked segregant analysis sequencing

Maize (Zea mays L.) is a thermophilic crop that is commonly affected by chilling stress, which adversely affects growth and yield. However, the genetic bases underlying the cold response and acclimation mechanisms remain unclear. Here, we crossed two maize inbred lines that exhibit opposite chilling tolerance at the seedling stage, Dan9046 (D9046; extremely chilling-tolerant) and Jingnuo 6 (JN6; extremely chilling-sensitive). From the 564 F2 individuals evaluated, the 50 most chilling-tolerant and the 50 most chilling-sensitive lines were selected to construct the chilling-tolerant and -sensitive pools, respectively. The two pools and the parental lines were then analyzed via bulked segregant analysis sequencing (BSA-seq). Loci linked to cold tolerance were identified in two regions of chromosome 5 based on single nucleotide polymorphism (SNP)-index and Δ(SNP-index) values. These two genomic intervals contained a total of 70 candidate genes. Of those genes, 49 had homologous genes that previous research has indicated are involved in stress tolerance. Metabolic pathways were enriched according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Between D9046 and JN6, there were a total of 921 SNPs and insertion/deletion mutations (Indels) that either caused a non-synonymous mutation or were located in a non-coding regions. Kompetitive Allele-Specific PCR (KASP) assays revealed that 20 SNPs within six genes exhibited allelic polymorphisms bias among the chilling-tolerant or chilling-sensitive inbred lines, consistent with the BSA-seq data results. Reverse transcription quantitative PCR (RT-qPCR) results revealed differential expression patterns in five of those genes (Zm00001d014321, Zm00001d014324, Zm00001d014325, Zm00001d015212 and Zm00001d015223) between D9046 and JN6 after low-temperature treatment, suggesting that they may have been related to chilling tolerance. In summary, we identified allelic polymorphisms of candidate genes that were related to chilling tolerance, providing genetic resources and KASP markers for future improvement of maize cold tolerance and breeding via marker-assisted selection. Data will be made available on request.