Zinc finger knuckle genes are associated with tolerance to drought and dehydration in chickpea (Cicer arietinum L.)

Abstract

Chickpea (Cicer arietinum L.) is a very important food legume and needs improved drought tolerance for higher seed production in dry environments. The aim of this study was to determine diversity and genetic polymorphism in zinc finger knuckle genes with CCHC domains and their functional analysis for practical improvement of chickpea breeding. Two CaZF-CCHC genes, Ca04468 and Ca07571, were identified as potentially important candidates associated with plant responses to drought and dehydration. To study these genes, various methods were used including Sanger sequencing, DArT (Diversity array technology) and molecular markers for plant genotyping, gene expression analysis using RT-qPCR, and associations with seed-related traits in chickpea plants grown in field trials. These genes were studied for genetic polymorphism among a set of chickpea accessions, and one SNP was selected for further study from four identified SNPs between the promoter regions of each of the two genes. Molecular markers were developed for the SNP and verified using the ASQ and CAPS methods. Genotyping of parents and selected breeding lines from two hybrid populations, and SNP positions on chromosomes with haplotype identification, were confirmed using DArT microarray analysis. Differential expression profiles were identified in the parents and the hybrid populations under gradual drought and rapid dehydration. The SNP-based genotypes were differentially associated with seed weight per plant but not with 100 seed weight. The two developed and verified SNP molecular markers for both genes, Ca04468 and Ca07571, respectively, could be used for marker-assisted selection in novel chickpea cultivars with improved tolerance to drought and dehydration.

Keywords: CCHC domain; DArT analysis; SNP; chickpea; drought and dehydration; gene expression; seed yield; zinc finger knuckle gene.

Figure 1

Molecular-phylogenetic tree of zinc finger proteins containing CCHC domains in chickpea and other legume plant species. The chickpea proteins are coded as Ca, Cicer arietinum with the following species in alphabetical order: A.duranensis, Arachis duranensis (wild ancestor peanut, A genome); A.ipaensis, Arachis ipaensis (wild ancestor peanut, B genome); Ca.cajan, Cajanus cajan (pigeon pea); G.max, Glycine max (soybean); G.soja, Glycine soja (wild soybean); Lo.japonicus, Lotus japonicus (wild model legume); Lu.angustifolius, Lupinus angustifolius (narrowleaf, blue lupine); M.truncatula, Medicago truncatula (barrelclover); P.vulgaris, Phaseolus vulgaris (common bean); V.angularis, Vigna angularis (adzuki bean); V.radiata, Vigna radiata (mung bean). The proteins were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov), and chickpea proteins were converted into those annotated in LIS, the Legume Information System database (https://www.legumeinfo.org). The full sequence of the chickpea and other legume proteins are presented in Supplementary Material S2 .

Figure 2

SNP identified in the promoter regions of two ZF-CCHC genes, Ca04468 (A) and Ca07571 (B), in chromosomes Ca4 and Ca5, respectively. The position of each SNP is indicated by the number of nucleotides before the Start-codon. Full description of the identified SNP, corresponding sequences from two reference genomes, cv. Frontier and accession ICC-4958, and their comparisons are present in Supplementary Material S3 .

Figure 3

Fragments of Sanger sequencing with two SNPs selected for further study. (A) SNP1 [C/T] in gene Ca04468 in the parents of hybrid population 1, ♀ Krasnokutsky-123 and ♂ ICC-12654, and (B) SNP4 [A/G] in gene Ca07571 in the parents of hybrid population 2, ♀ ICC-10945 and ♂ Looch. The SNP is designated by arrows in the corresponding color.

Figure 4

Example of genotyping for Ca04468 gene in parents, Krasnokutsky-123 and ICC-12654, with FAM (A) and HEX (B), respectively, using the ASQ method. Examples of allele discrimination for genes Ca04468 (C) and Ca07571 (D), respectively, in two hybrid populations, 1 and 2, respectively.

Figure 5

Validation of SNP genotyping for Ca07571 gene using CAPS marker Ca07571-SNP4-MnlI separated in 12% polyacrylamide gel, in parents (lanes 1 and 2) and selected breeding lines (lanes 3–10) from hybrid population 1 [♀ ICC-10945 × ♂ Looch]. Fragments of 77 bp after digestion with MnlI are indicated by arrows.

Figure 6

Distributions of DArT haplotype blocks in chromosomes Ca4 (A) and Ca5 (B) in parents and selected breeding lines from hybrid population 1 (Krasnokutsky-123 and ICC-12654) and hybrid population 2 (ICC-10945 and Looch), respectively. Five identified haplotype blocks with numbers above are designated by green and brown lines according to the position on the physical map of chickpea cv. Frontier, 50M bp in each chromosome. Green and brown dots correspond to SNPs found in parents and their breeding lines. Red arrows indicate positions of the target genes Ca04468 in chromosome Ca4 (A) and Ca07571 in chromosome Ca5 (B). Representative “zoom” of these genetic regions with both target genes (C) shows the distribution of eight surrounding DArT markers in parents and hybrid breeding lines. Maternal and paternal alleles are designated by red and blue boxes, respectively. Sequences and genetic positions of the used DArT markers are present in Supplementary Material S5 .

Figure 7

RT-qPCR expression analysis of Ca04468 under drought stress (A) and Ca07571 after dehydration treatment (B) in chickpea plants of parents and selected breeding lines from two hybrid populations. On the left-hand side of each panel, parents Krasnokutsky-123 and ICC-12654 and three breeding lines H18 represent hybrid population 1, while parents ICC-10945 and Looch and four breeding lines H35, on the right-hand side of the panels, belong to hybrid population 2. Plants of ICC-4958 were included as a reference genotype with a fully sequenced genome and are indicated in pink. For drought, leaf samples were collected from soil-grown plants when water was withdrawn, whereas detached leaves were exposed to dehydration on paper-towel at room temperature. Four consecutive time-points were used for sampling, and “point 0” was arranged as controls in both treatments. For all genotypes and experiments, the expressions of controls were set as unit level 1, indicated by dashed lines. Expression data were normalized using two reference genes, CaELF1α (elongation factor 1-alfa) and CaHSP90 (heat shock protein 90), and are present as the average ± SE of three biological replicates (individual plants) and two technical repeats for each genotype and treatment. Significant differences (*p < 0.05 and **p< 0.01) from level 1 were calculated using two-way ANOVA with post-hoc Tukey test.

Figure 8

Seed-related traits: seed weight per plant (A) and hundred seed weight (B) in parents and selected breeding lines from hybrid populations 1 and 2. Data were calculated as the average of the plants grown in the field experiments, 1 m² plot (n = 10), with three replicates, in the Akmola region, Northern Kazakhstan, over 2 years, 2021 (lighter color) and 2022 (darker color) with mild and strong drought, respectively. Parents and breeding lines that carried unfavorable alleles of Ca04468 and Ca07571 genes are shown in bars with stripes and dots. Error bars represent standard errors. Significant differences (*p < 0.05) are shown for genotypes compared to another parent and breeding lines in each hybrid population, and were calculated using two-way ANOVA with post-hoc Tukey test.