Background: WD40 transcription factors, a large gene family in eukaryotes, are involved in a variety of growth regulation and development pathways. WD40 plays an important role in the formation of MYB-bHLH-WD (MBW) complexes associated with anthocyanin synthesis, but studies of Qingke barley are lacking. Results: In this study, 164 barley HvWD40 genes were identified in the barley genome and were analyzed to determine their relevant bioinformatics. The 164 HvWD40 were classified into 11 clusters and 14 subfamilies based on their structural and phylogenetic protein profiles. Co-lineage analysis revealed that there were 43 pairs between barley and rice, and 56 pairs between barley and maize. Gene ontology (GO) enrichment analysis revealed that the molecular function, biological process, and cell composition were enriched. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that the RNA transport pathway was mainly enriched. Based on the identification and analysis of the barley WD40 family and the transcriptome sequencing (RNA-seq) results, we found that HvWD40-140 (WD40 family; Gene ID: r1G058730), HvANT1 (MYB family; Gene ID: HORVU7Hr1G034630), and HvANT2 (bHLH family; Gene ID: HORVU2Hr1G096810) were important components of the MBW complex related to anthocyanin biosynthesis in Qingke, which was verified via quantitative real-time fluorescence polymerase chain reaction (qRT-PCR), subcellular location, yeast two-hybrid (Y2H), and bimolecular fluorescent complimentary (BiFC) and dual-luciferase assay analyses. Conclusions: In this study, we identified 164 HvWD40 genes in barley and found that HvnANT1, HvnANT2, and HvWD40-140 can form an MBW complex and regulate the transcriptional activation of the anthocyanin synthesis related structural gene HvDFR. The results of this study provide a theoretical basis for further study of the mechanism of HvWD40-140 in the MBW complex related to anthocyanin synthesis in Qingke.

Colored hulless barley are more suitable in food processing compared to normal (yellow) varieties because it is rich in bioactive compounds and produces higher extraction pearling fractions. Therefore, seed coat color is an important agronomic trait for the breeding and study of hulless barley. Genotyping-by-sequencing single-nucleotide polymorphism (GBS-SNP) analysis of a doubled haploid (DH) mapping population (Nierumuzha × Kunlun10) was conducted to map the purple seed coat color genes (Psc). A high-density genetic map of hulless barley was constructed, which contains 3662 efficient SNP markers with 1129 bin markers. Seven linkage groups were resolved, which had a total length of 645.56 cM. Chromosome length ranged from 60.21 cM to 127.21 cM, with average marker density of 0.57 cM. A total of five loci accounting for 3.79% to 23.86% of the observed phenotypic variation for Psc were detected using this high-density map. Five structural candidate genes (F3’M, HID, UF3GT, UFGT and 5MAT) and one regulatory factor (Ant1) related to flavonoid or anthocyanin biosynthesis were identified.. Five structural candidate genes and one regulatory factor related to flavonoid or anthocyanin biosynthesis have been identified using a high-density genetic map of hulless barley. This study lays the foundation for map-based cloning of Psc but provides a valuable tool for studying marker-trait associations and its application to marker-assisted breeding of hulless barley.

Highland barley has a different composition and structure to other crops. It has higher contents of total polyphenol (TPC), total flavonoid (TFC) and β-glucan, which can be supplemented to improve the nutrition of wheat-flour-based food. In this study, the flours of three different grain-colored highland barley varieties Beiqing 6 (BQ), Dulihuang (DLH), and Heilaoya (HLY), were added to Jimai60 (JM, a wheat variety with medium gluten) wheat flour at different substitution levels to investigate their effects on the unextractable polymeric protein (UPP) content, micro-structure, rheological properties and mixing properties of dough, and the color, texture, flavor, and in vitro digestion of Chinese steam bread (CSB). The results showed that the moderate substitution of highland barley (20%) increased the UPP%, optimized the micro-structure of gluten, and improved its rheological properties by increasing dough viscoelasticity. The CSBs made from the composite flours exhibited a similar specific volume, cohesiveness, springiness and resilience to wheat CSB, while the firmness of composite CSBs (particularly JM-HLY-20) was delayed during storage. Importantly, the addition of highland barley increased the contents of TPC, TFC and β-glucan, but decreased the in vitro starch digestibility of CSBs. A sensory evaluation showed that JM-HLY CSB was the most preferable. Taken together, highland barley can be used as a fine supplement to food products, with health-promoting properties.

To assess the genetic diversity of the primary Qingke cultivars and establish their unique genetic profiles, 837 barley simple sequence repeat (SSR) primers were screened across 12 cultivars. The selection process involved the utilization of polyacrylamide gel electrophoresis and capillary electrophoresis technology, to identify primers exhibiting desirable characteristics, such as polymorphism, stability, and reproducibility. Subsequently, we analyzed the genetic diversity of the primary Qingke cultivars to for DNA fingerprints. A total of 18 pairs of SSR markers were selected as the optimal markers for constructing fingerprints of major Qingke cultivars. These included 83 observed alleles (N_a), ranging from there to 11, with an average of 4.61 per pair. Notably, Bmag0496 and Scssr04163 exhibited higher allelic diversity, with 11 and 8 loci, respectively. The polymorphism information content (PIC) ranged from 0.36 to 0.74, with an average of 0.52. The expected heterozygosity (H_e) ranged from 0.4031 to 0.7682, with an average of 0.59, and the observed heterozygosity (H_o) varied between 0.13 and 0.67, with an average of 0.32. The outcomes obtained through phylogenetic tree analysis, population structure assessment and principal component analysis demonstrated that the primary Qingke cultivars could be classified into three distinct groups: group I primarily originated from Xizang and Qinghai provinces; group II mainly consisted of cultivars from Yunnan and Heilongjiang provinces; and group III predominantly comprised cultivars originating from Qinghai and Gansu provinces. Interestingly, the Sichuan cultivars were distributed across all three groups without any clear tendency toward a specific cluster or subgroup. These findings indicated that the genetic distance among Qingke cultivars was significantly correlated with geographic location but not exclusively determined by it. The construction of DNA fingerprints for the primary Qingke cultivars used these identified sets of SSR primers (18 pairs) laid a solid foundation for cultivar identification, conservation and utilization efforts related to this crop.

To investigate the role of the HvnAnt2 gene in the formation of different qingke barley grain colours, HvnAnt2 was isolated from the leaves of the White 91-97-3 (white), Blue qingke (blue), Kunlun 17 (black), and Purple qingke (purple). The HvnAnt2 gene encodes a hydrophilic unstable protein consisting of 561 amino acids, without a transmembrane structure and without a signal peptide, with one bHLH-MYC_N and one HLH domain at amino acids 22-204 and 387-436, respectively. The HvnAnt2 of qingke barley was most closely related to Hordeum vulgare and most distantly related to Triticum monococcum; it was mainly related to light responsiveness and methyl jasmonate responsiveness. The Ant2 gene was highly expressed in Kunlun 17 and Purple qingke barley grains during the later stage of development. The protein interaction prediction showed that flavonoid 3'-monooxygenase, anthocyanin biosynthesis gene regulators, and key enzymes in folate metabolism interacted with the Ant2 protein. This study provides a reference for further analysis of the Ant2 gene in the anthocyanin synthesis pathways of qingke barley with different grain colours.