Identification of Salt Tolerance-Related <i>NAC</i> Genes in Wheat Roots Based on RNA-Seq and Association Analysis

Excavating new salt tolerance genes and utilizing them to improve salt-tolerant wheat varieties is an effective way to utilize salinized soil. The <i>NAC</i> gene family plays an important role in plant response to salt stress. In this study, 446 <i>NAC</i> sequences were isolated from the whole genome of common wheat and classified into 118 members based on subgenome homology, named <i>TaNAC1</i> to <i>TaNAC118</i>. Transcriptome analysis of salt-tolerant wheat breeding line CH7034 roots revealed that 144 of the 446 <i>TaNAC</i> genes showed significant changes in expression levels at least two time points after NaCl treatment. These differentially expressed <i>TaNACs</i> were divided into four groups, and Group 4, containing the largest number of 78 genes, exhibited a successive upregulation trend after salt treatment. Single nucleotide polymorphisms (SNPs) of the <i>TaNAC</i> gene family in 114 wheat germplasms were retrieved from the public database and were subjected to further association analysis with the relative salt-injury rates (RSIRs) of six root phenotypes, and then 20 SNPs distributed on chromosomes 1B, 2B, 2D, 3B, 3D, 5B, 5D, and 7A were correlated with phenotypes involving salt tolerance (<i>p</i> < 0.0001). Combining the results of RT-qPCR and association analysis, we further selected three <i>NAC</i> genes from Group 4 as candidate genes that related to salt tolerance, including <i>TaNAC26-D3.2</i>, <i>TaNAC33-B</i>, and <i>TaNAC40-B</i>. Compared with the wild type, the roots of the <i>tanac26-d3.2</i> mutant showed shorter length, less volume, and reduced biomass after being subjected to salt stress. Four SNPs of <i>TaNAC26-D3.2</i> formed two haplotypes, Hap1 and Hap2, and germplasms with Hap2 exhibited better salt tolerance. Snp3, in exon 3 of <i>TaNAC26-D3.2,</i> causing a synonymous mutation, was developed into a Kompetitive Allele-Specific PCR marker, K3, to distinguish the two haplotypes, which can be further used for wheat germplasm screening or marker-assisted breeding. This study provides new genes and molecular markers for improvement of salt tolerance in wheat.