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Tunisian barley (Hordeum vulgare L.) landraces, representing the oldest cultivated accessions, are growing in scattered populations across drought‐ and salt‐stressed environments and constitute a precious reservoir of potentially useful traits for breeding programs. The objective of this study was to elucidate genetic diversity and population structure of barley landraces across the landscape of Tunisia. Populations from 11 geographic zones were genotyped using 21 nuclear microsatellites. A high level of genetic polymorphism with 170 detected alleles was recorded among the studied genotypes. The average allelic richness was 8.095 alleles per locus. The index of genetic diversity (He) showed an average of 0.741. Genetic diversity was very high within populations, whereas differences among populations were difficult to detect. Only 0.15% of the DNA variation was apportioned among landraces (P < 0.001), whereas 99.85% of the DNA variation was maintained within these landraces. A high gene flow (Nm) was revealed among the investigated populations, which has been facilitated by exchange of barley seeds between Tunisian cereal farmers of different regions. Genetic diversity within Tunisian barley landrace germplasms may help to maintain adaptation to a broad range of environmental conditions and provide genetically diverse resources for barley breeders. Both ex situ (seed banks) and in situ (on‐farm) conservation strategies may be required to maintain barley landrace genetic resources.

Hybridity and the genuineness of hybrids are prominent characteristics for quality control of seeds and thereby for varietal improvement. In the current study, the cross between two local barley genotypes (Ardhaoui: female; Testour: male) previously identified as susceptible/tolerant to salt stress in Tunisia was achieved. The hybrid genetic purity of the generated F putative hybrids and the fingerprinting of the parents along with their offspring were assessed using a set of 17 nuclear SSR markers. Among the analyzed loci, 11 nSSR were shown polymorphic among the parents and their offspring. Based on the applied 11 polymorphic SSR loci, a total of 28 alleles were detected with an average of 2.54 alleles per locus. The locus presented the highest number of alleles. The highest polymorphism information content value was detected for the locus (0.6713) whereas the lowest PIC value (0.368) was revealed by the loci , and with a mean value of 0.4619. The probabilities of identical genotypes PI for the 11 microsatellite markers were 8.63 × 10. Banding patterns among parents and hybrids showed polymorphic fragments. The 11 SSR loci had produced unique fingerprints for each analyzed genotype and segregate between the two parental lines and their four hybrids. Parentage analysis confirms the hybrid purity of the four analyzed genotypes. Six Tunisian barley accessions were used as an outgroup in the multivariate analysis to confirm the efficiency of the employed 11 nSSR markers in genetic differentiation among various barley germplasms. Thus, neighbor joining and factorial analysis revealed clearly the discrimination among the parental lines, the four hybrids and the outgroup accessions. Out of the detected polymorphic 11 nuclear SSR markers, a set of five markers (, ,, and) were shown to be sufficient and informative enough to discriminate among the six genotypes representing the two parental lines and the four hybrids from each others. These five nSSR markers presented the highest number of alleles per locus (), expected heterozygosity (), PIC values and the lowest probabilities of identity (PI). These nSSR loci may be used as referral SSR markers for unambiguous discrimination and genetic purity assessment in barley breeding programs.

Barley is characterized by a rich genetic diversity, making it an important model for studies of salinity response with great potential for crop improvement. Moreover, salt stress severely affects barley growth and development, leading to substantial yield loss. Leaf and root transcriptomes of a salt-tolerant Tunisian landrace (Boulifa) exposed to 2, 8, and 24 h salt stress were compared with pre-exposure plants to identify candidate genes and pathways underlying barley’s response. Expression of 3585 genes was upregulated and 5586 downregulated in leaves, while expression of 13,200 genes was upregulated and 10,575 downregulated in roots. Regulation of gene expression was severely impacted in roots, highlighting the complexity of salt stress response mechanisms in this tissue. Functional analyses in both tissues indicated that response to salt stress is mainly achieved through sensing and signaling pathways, strong transcriptional reprograming, hormone osmolyte and ion homeostasis stabilization, increased reactive oxygen scavenging, and activation of transport and photosynthesis systems. A number of candidate genes involved in hormone and kinase signaling pathways, as well as several transcription factor families and transporters, were identified. This study provides valuable information on early salt-stress-responsive genes in roots and leaves of barley and identifies several important players in salt tolerance.