Expression of Specific Alleles of Zinc-Finger Transcription Factors, HvSAP8 and HvSAP16, and Corresponding SNP Markers, Are Associated with Drought Tolerance in Barley Populations

Abstract

Two genes, HvSAP8 and HvSAP16, encoding Zinc-finger proteins, were identified earlier as active in barley plants. Based on bioinformatics and sequencing analysis, six SNPs were found in the promoter regions of HvSAP8 and one in HvSAP16, among parents of two barley segregating populations, Granal × Baisheshek and Natali × Auksiniai-2. ASQ and Amplifluor markers were developed for HvSAP8 and HvSAP16, one SNP in each gene, and in each of two populations, showing simple Mendelian segregation. Plants of F₆ selected breeding lines and parents were evaluated in a soil-based drought screen, revealing differential expression of HvSAP8 and HvSAP16 corresponding with the stress. After almost doubling expression during the early stages of stress, HvSAP8 returned to pre-stress level or was strongly down-regulated in plants with Granal or Baisheshek genotypes, respectively. For HvSAP16 under drought conditions, a high expression level was followed by either a return to original levels or strong down-regulation in plants with Natali or Auksiniai-2 genotypes, respectively. Grain yield in the same breeding lines and parents grown under moderate drought was strongly associated with their HvSAP8 and HvSAP16 genotypes. Additionally, Granal and Natali genotypes with specific alleles at HvSAP8 and HvSAP16 were associated with improved performance under drought via higher 1000 grain weight and more shoots per plant, respectively.

Keywords: ASQ and Amplifluor markers; SNP plant genotyping; barley; drought; gene expression; grain yield and yield components; shoots number per plant; stress-associated genes; thousand grain weight; zinc-finger transcription factors.

Figure 1

Molecular phylogenetic dendrogram based on amino acid sequences of proteins SAP8 and SAP4 (a), and SAP16 (b) among monocot species. Sub-clades A1 and A2 designate SAP8 and SAP4 peptides among cereals, and Clade B shows other monocots. Barley HvSAP protein accession IDs were retrieved from the NCBI database and all homologous protein sequences from other monocot plant species were identified in KEGG (Kyoto Encyclopedia of Genes and Genomes). The botanical names of the presented species are as follows, in anti-clockwise order as in the panel (a) with the addition of panel (b): Clade A: Sorghum bicolor, Zea mays, Brachypodium distachyon, Hordeum vulgare, Oryza brachyantha, Oryza sativa ssp. japonica, Aegilops tauschii, and Setaria italica; Clade B: Musa acuminata, Elaeis guineensis, Phoenix dactylifera, Phalaenopsis equestris, and Asparagus officinalis. The corresponding protein sequence accession IDs were added after the species names. The unrooted Consensus tree with Equal angle dendrogram was generated by the program SplitsTree4 (http://www.splitstree.org) (accessed on 18 June 2021).

Figure 2

Comparison of sequence fragments of promoter regions of HvSAP8 (a) and HvSAP16 (b) in parents of barley hybrid populations, cultivars Granal, Baisheshes, Natali and Auksiniai-2, respectively. SNPs are indicated by arrows with corresponding colour. Order numbers were used for two SNPs in HvSAP8 while only a single SNP in HvSAP16 was found.

Figure 3

Allele discrimination scores based on SNPs in HvSAP8 for genotyping of the hybrid population Granal × Baisheshek (a) and in HvSAP16 for genotyping of the hybrid population Natali × Auksiniai-2 (b). Relative fluorescence units (RFU) for fluorophores FAM and HEX were transformed automatically into genotyping of alleles 1 and 2, respectively, using a Thermo Fisher Scientific QuantStudio-7 Real-Time PCR instrument (a) and a Bio-Rad CFX96 Real-Time PCR instrument (b). Homozygotes for allele 1 (FAM) are designated by red dots and red circles; homozygotes for allele 2 (HEX) are shown in blue dots and blue squares; green dots and green triangles represent heterozygotes. Numbers of studied plants were n = 42 and n = 50, for Granal × Baisheshek and Natali × Auksiniai-2 hybrid populations, respectively. The normalisation was made by the qPCR software with the comparison of fluorescence data to No-template control (NTC, sterile water) shown as black squares in the Thermo Fisher Scientific instrument only.

Figure 4

Expression analysis of HvSAP8 (a,b) and HvSAP16 (c,d) genes in two hybrid populations of barley, Granal × Baisheshek (G×B) (a,c), and in Natali × Auksiniai-2 (N×A) (b,d). Each experiment contains eight genotypes including both parents and six breeding lines of each hybrid population. Three breeding lines were selected by SNP genotypes identical to each parent and are separated by a dashed line in the figure panels. Leaf samples were collected from soil-grown plants at four consecutive time-points, where Relative expression at ‘day 0′ (well-watered Controls) was set as unit level 1 for all genotypes and experiments. Three collection time-points were at day 8, 12 and 16, from when water was withdrawn, corresponding to mild, moderate and strong drought treatment, respectively. Expression data were normalised using two Reference genes, HvADP (ADP-ribosylation factor 1-like protein) and HvGAPD (Glycolytic glyceraldehyde-3-phosphate dehydrogenase), and are presented as the average ± SE of three biological replicates (individual plants) and two technical repeats for each genotype and treatment. Significant differences (p < 0.05) from relative level 1 are designated by asterisks (*) above the bars, for each genotype and experiment, calculated using ANOVA.

Figure 5

Grain yield and components in barley plants from two hybrid populations, Granal × Baisheshek (G×B) (a,c,e), and Natali × Auksiniai-2 (N×A) (b,d,f), grown in pots with soil under moderate drought until harvest. Eight genotypes including both parents and six breeding lines for the hybrid population identified in this study are separated by a dashed line in the figure panels. Grain yield per plant (a,b), number of shoots per plant (c,d), and 1000 grain weight (e,f), are presented as mean bars ± SE for 15 harvested plants (5 plants × 3 pots) for each genotype. Significant differences (p < 0.05) are indicated by asterisks (*) between average values for corresponding groups of genotypes, calculated using ANOVA.

Figure 6

ASQ primer design in the fragment of HvSAP8 gene promoter sequence in barley parents, Granal and Baisheshek. The targeted SNP is designated by red and other SNP are shown by degenerative nucleotides in the sequence in Bold. Two Forward allele-specific primers (F1 and F2) have a common part indicated in yellow but differ by nucleotides at the SNP position underlined and highlighted in red. At the 5′-end, the 13-bp fragment is common and indicated in lower case letters, while the 6-bp fragment in the middle of both Forward primers is a specific ‘tag’ shown in Bold-Italic case. The common reverse primer is highlighted in pink, with ‘reverse-complement’ order indicated by black and white fonts of letters in Reverse primer and in the sequence, respectively.