Joint GWAS and WGCNA uncover the genetic control of calcium accumulation under salt treatment in maize seedlings.

Soil salinization is an important factor threatening the yield and quality of maize. Ca2+ plays a considerable role in regulating plant growth under salt stress. Herein, we examined the shoot Ca2+ concentrations, root Ca2+ concentrations, and transport coefficients of seedlings in an association panel composed of 305 maize inbred lines under normal and salt conditions. A genome-wide association study was conducted by using the investigated phenotypes and 46,408 single-nucleotide polymorphisms of the panel. As a result, 53 significant SNPs were specifically detected under salt treatment, and 544 genes were identified in the linkage disequilibrium regions of these SNPs. According to the expression data of the 544 genes, we carried out a weighted coexpression network analysis. Combining the enrichment analyses and functional annotations, four hub genes (GRMZM2G051032, GRMZM2G004314, GRMZM2G421669, and GRMZM2G123314) were finally determined, which were then used to evaluate the genetic variation effects by gene-based association analysis. Only GRMZM2G123314, which encodes a pentatricopeptide repeat protein, was significantly associated with Ca2+ transport and the haplotype G-CT was identified as the superior haplotype. Our study brings novel insights into the genetic and molecular mechanisms of salt stress response and contributes to the development of salt-tolerant varieties in maize.

GWAS and WGCNA uncover hub genes controlling salt tolerance in maize (Zea mays L.) seedlings.

KEYMESSAGE: Two hub genes GRMZM2G075104 and GRMZM2G333183 involved in salt tolerance were identified by GWAS and WGCNA. Furthermore, they were verified to affect salt tolerance by candidate gene association analysis. Salt stress influences maize growth and development. To decode the genetic basis and hub genes controlling salt tolerance is a meaningful exploration for cultivating salt-tolerant maize varieties. Herein, we used an association panel consisting of 305 lines to identify the genetic loci responsible for Na+- and K+-related traits in maize seedlings. Under the salt stress, seven significant single nucleotide polymorphisms were identified using a genome-wide association study, and 120 genes were obtained by scanning the linkage disequilibrium regions of these loci. According to the transcriptome data of the above 120 genes under salinity treatment, we conducted a weighted gene co-expression network analysis. Combined the gene annotations, two SNaC/SKC (shoot Na+ content/shoot K+ content)-associated genes GRMZM2G075104 and GRMZM2G333183 were finally identified as the hub genes involved in salt tolerance. Subsequently, these two genes were verified to affect salt tolerance of maize seedlings by candidate gene association analysis. Haplotypes TTGTCCG-CT and CTT were determined as favorable/salt-tolerance haplotypes for GRMZM2G075104 and GRMZM2G333183, respectively. These findings provide novel insights into genetic architectures underlying maize salt tolerance and contribute to the cultivation of salt-tolerant varieties in maize.

GWAS with a PCA uncovers candidate genes for accumulations of microelements in maize seedlings.

Microelements are necessary for plant growth and development, they control key processes of physiological metabolism. Herein, we evaluated three accumulation-related performances for each of the four microelements (Fe, Zn, Cu, and Mn) among 305 inbred maize lines. Quantification of these microelements in maize roots and shoots revealed abundant phenotypic variations in the association panel, with the variation coefficients ranging from 0.31 to 0.76. Principal component analysis (PCA) of the three related traits (concentration in root, concentration in shoot, and transport coefficient) showed that PC1 and PC2 explained >95% of phenotypic variations for each element. The scores of PC1 and PC2 were thereby used for a genome-wide association study by combining 44,134 SNPs of this panel. A total of 27, 1, 5, and 3 SNPs were significantly (P < .05) associated with Zn-PC1, Zn-PC2, Cu-PC1, and Mn-PC2, respectively, with 11 genes closely linked (r2 > 0.8) to these SNPs. Of them, GRMZM2G142870, GRMZM2G045531, and GRMZM2G143512 were individually annotated as ABC transporter C family member 14, zinc transporter 3, and heavy metal ATPase10. A candidate gene association analysis further verified that GRMZM2G142870 and GRMZM2G045531 affect Zn and Mn accumulations, respectively. Evaluation of contrasting allele ratios in elite lines indicated that the majority of the alleles correlating with higher Zn or Cu had not been utilized in maize breeding. Integration of more "higher-accumulation" alleles in the elite lines will be practical for improving Zn and Cu accumulations in maize. Our findings contribute to genetic revelation and molecular marker-assisted selection of microelement accumulations in maize.

Comparative transcriptome analyses of maize seedling root responses to salt stress.

Salt stress affects crop yield by limiting growth and delaying development. In this study, we constructed 16 transcriptome libraries from maize seedling roots using two maize lines, with contrasting salt tolerance, that were exposed to salt stress for 0, 6, 18 and 36 h. In total, 6,584 differential expression genes (DEGs; 3,669 upregulated, 2,915 downregulated) were induced in the salt-sensitive line and 6,419 DEGs (3,876 upregulated, 2,543 downregulated) were induced in the salt-tolerant line. Several DEGs common to both lines were enriched in the ABA signaling pathway, which was presumed to coordinate the process of maize salt response. A total of 459 DEGs were specifically induced in the salt-tolerant line and represented candidate genes responsible for high salt-tolerance. Expression pattern analysis for these DEGs indicated that the period between 0 and 6 h was a crucial period for the rapid response of the tolerant genes under salt stress. Among these DEGs, several genes, Aux/IAA, SAUR, and CBL-interacting kinase have been reported to regulate salt tolerance. In addition, the transcription factors WRKY, bZIP and MYB acted as regulators in the salt-responsive regulatory network of maize roots. Our findings will contribute to understanding of the mechanism on salt response and provide references for functional gene revelation in plants.

Genetic Dissection of Maize Embryonic Callus Regenerative Capacity Using Multi-Locus Genome-Wide Association Studies.

The regenerative capacity of the embryonic callus, a complex quantitative trait, is one of the main limiting factors for maize transformation. This trait was decomposed into five traits, namely, green callus rate (GCR), callus differentiating rate (CDR), callus plantlet number (CPN), callus rooting rate (CRR), and callus browning rate (CBR). To dissect the genetic foundation of maize transformation, in this study multi-locus genome-wide association studies (GWAS) for the five traits were performed in a population of 144 inbred lines genotyped with 43,427 SNPs. Using the phenotypic values in three environments and best linear unbiased prediction (BLUP) values, as a result, a total of 127, 56, 160, and 130 significant quantitative trait nucleotides (QTNs) were identified by mrMLM, FASTmrEMMA, ISIS EM-BLASSO, and pLARmEB, respectively. Of these QTNs, 63 QTNs were commonly detected, including 15 across multiple environments and 58 across multiple methods. Allele distribution analysis showed that the proportion of superior alleles for 36 QTNs was <50% in 31 elite inbred lines. Meanwhile, these superior alleles had obviously additive effect on the regenerative capacity. This indicates that the regenerative capacity-related traits can be improved by proper integration of the superior alleles using marker-assisted selection. Moreover, a total of 40 candidate genes were found based on these common QTNs. Some annotated genes were previously reported to relate with auxin transport, cell fate, seed germination, or embryo development, especially, GRMZM2G108933 (WOX2) was found to promote maize transgenic embryonic callus regeneration. These identified candidate genes will contribute to a further understanding of the genetic foundation of maize embryonic callus regeneration.