HvNCX, a prime candidate gene for the novel qualitative locus qS7.1 associated with salinity tolerance in barley

Abstract

A major QTL (qS7.1) for salinity damage score and Na+ exclusion was identified on chromosome 7H from a barley population derived from a cross between a cultivated variety and a wild accession. qS7.1 was fine-mapped to a 2.46 Mb physical interval and HvNCX encoding a sodium/calcium exchanger is most likely the candidate gene. Soil salinity is one of the major abiotic stresses affecting crop yield. Developing salinity-tolerant varieties is critical for minimizing economic penalties caused by salinity and providing solutions for global food security. Many genes/QTL for salt tolerance have been reported in barley, but only a few of them have been cloned. In this study, a total of 163 doubled haploid lines from a cross between a cultivated barley variety Franklin and a wild barley accession TAM407227 were used to map QTL for salinity tolerance. Four significant QTL were identified for salinity damage scores. One (qS2.1) was located on 2H, determining 7.5% of the phenotypic variation. Two (qS5.1 and qS5.2) were located on 5H, determining 5.3-11.7% of the phenotypic variation. The most significant QTL was found on 7H, explaining 27.8% of the phenotypic variation. Two QTL for Na⁺ content in leaves under salinity stress were detected on chromosomes 1H (qNa1.1) and 7H(qNa7.1). qS7.1 was fine-mapped to a 2.46 Mb physical interval using F₄ recombinant inbred lines. This region contains 23 high-confidence genes, with HvNCX which encodes a sodium/calcium exchanger being most likely the candidate gene. HvNCX was highly induced by salinity stress and showed a greater expression level in the sensitive parent. Multiple nucleotide substitutions and deletions/insertions in the promoter sequence of HvNCX were found between the two parents. cDNA sequencing of the HvNCX revealed that the difference between the two parents is conferred by a single Ala77/Pro77 amino acid substitution, which is located on the transmembrane domain. These findings open new prospects for improving salinity tolerance in barley by targeting a previously unexplored trait.

Fig. 1

Comparison of two parental varieties Franklin and Tam407227 under control and salinity stress

Fig. 2

QTL mapping of locus for salinity damage score and leaf Na + content. Black line: QTL for salinity damage score under salinity stress; Red line: QTL for Na + contents under salinity stress (color figure online)

Fig. 3

Fine mapping of qS7.1. A: Mapping of qS7.1 and qNa7.1, the green line represents QTL for salinity damage score, the red line represents QTL for Na⁺ content; B: Marker names are indicated at the top of the column and the genotypes of the selected recombinants and the phenotype of lines of the F_3:4 progenies derived from F₃ plants are presented (salinity tolerance: T; salinity sensitivity: S). White rectangles indicate the homozygotes with the resistance allele of Tam, and gray rectangles indicate the homozygotes with the susceptibility allele of Franklin. The qS7.1 was delimited to a 2464-kb region between the M85905568 and M88369902 markers. C:High-confidence annotated genes in this region. The promising candidate gene HORVU.MOREX.r3.7HG0665750.1 was marked using red color and others were marker with blue color (color figure online)

Fig. 4

Transcription analysis of HvNCX response to salt stress in root and leaves after 24 h and 48 h salinity treatment. 24 h-C: 24 h under control conditions; 24 h-S: 24 h under salinity stress conditions; 48 h-C: 48 h under control conditions; 48 h-S: 48 h under salinity stress conditions

Fig. 5

Sequencing analysis of HvNCX