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Improved chilling tolerance is important for maize production. Previous efforts in transgenics and marker-assisted breeding have not achieved practical results. In this study, the antifreeze protein (AnAFP) from the super-xerophyte Ammopiptanthus nanuswas aligned to KnS-type dehydrins. Phosphorylation in vitroand subcellular localization showed that AnAFP was phosphorylated by maize casein kinase II and translocated from nucleus to cytoplasm under chilling stress. AnAFP also increased lactate dehydrogenase activity. A parent line of commercial maize hybrids was transformed with the AnAFPgene. Based on thermal asymmetric interlaced PCR, one hemizygous and two homozygous integration sites were identified in one T1line. Ectopic expression of AnAFPin transgenic lines was confirmed by quantitative real-time PCR, RNA-seq, and Western blotting. After chilling treatment, the transgenic lines showed significantly improved phenotype, higher kernel production, survival rate and biomass, and lower relative electrolyte leakage and superoxide dismutation than the untransformed line. Thus, ectopic expression of AnAFPgene improved chilling tolerance in the transgenic lines, which could be used to apply for further safety assessment for commercial breeding.

Fusarium ear rot (FER) is a destructive maize fungal disease worldwide. In this study, three tropical maize populations consisting of 874 inbred lines were used to perform genome-wide association study (GWAS) and genomic prediction (GP) analyses of FER resistance. Broad phenotypic variation and high heritability for FER were observed, although it was highly influenced by large genotype-by-environment interactions. In the 874 inbred lines, GWAS with general linear model (GLM) identified 3034 single-nucleotide polymorphisms (SNPs) significantly associated with FER resistance at the P-value threshold of 1 × 10−5, the average phenotypic variation explained (PVE) by these associations was 3% with a range from 2.33% to 6.92%, and 49 of these associations had PVE values greater than 5%. The GWAS analysis with mixed linear model (MLM) identified 19 significantly associated SNPs at the P-value threshold of 1 × 10−4, the average PVE of these associations was 1.60% with a range from 1.39% to 2.04%. Within each of the three populations, the number of significantly associated SNPs identified by GLM and MLM ranged from 25 to 41, and from 5 to 22, respectively. Overlapping SNP associations across populations were rare. A few stable genomic regions conferring FER resistance were identified, which located in bins 3.04/05, 7.02/04, 9.00/01, 9.04, 9.06/07, and 10.03/04. The genomic regions in bins 9.00/01 and 9.04 are new. GP produced moderate accuracies with genome-wide markers, and relatively high accuracies with SNP associations detected from GWAS. Moderate prediction accuracies were observed when the training and validation sets were closely related. These results implied that FER resistance in maize is controlled by minor QTL with small effects, and highly influenced by the genetic background of the populations studied. Genomic selection (GS) by incorporating SNP associations detected from GWAS is a promising tool for improving FER resistance in maize.

The current assembled maize genomes cannot represent the broad genetic diversity of maize germplasms. Acquiring more genome sequences is critical for constructing a pan-genome and elucidating the linkage between genotype and phenotype in maize. Here we describe the genome sequence and annotation of A188, a maize inbred line with high phenotypic variation relative to other lines, acquired by single-molecule sequencing and optical genome mapping. We assembled a 2210-Mb genome with a scaffold N50 size of 11.61 million bases (Mb), compared to 9.73 Mb for B73 and 10.2 Mb for Mo17. Based on the B73_RefGen_V4 genome, 295 scaffolds (2084.35 Mb, 94.30% of the final genome assembly) were anchored and oriented on ten chromosomes. Comparative analysis revealed that ∼30% of the predicted A188 genes showed large structural divergence from B73, Mo17, and W22 genomes, which causes high protein divergence and may lead to phenotypic variation among the four inbred lines. As a line with high embryonic callus (EC) induction capacity, A188 provides a convenient tool for elucidating the molecular mechanism underlying the formation of EC in maize. Combining our new A188 genome with previously reported QTL and RNA sequencing data revealed eight genes with large structural variation and two differentially expressed genes playing potential roles in maize EC induction.