Enhancing barley yield potential and germination rate: gene editing of HvGA20ox2 and discovery of novel allele sdw1.ZU9.

Several dwarf and semi-dwarf genes have been identified in barley. However, only a limited number have been effectively utilized in breeding programs to cultivate lodging resistant varieties. This is due to the common association of dwarf and semi-dwarf traits with negative effects on malt quality. In this study, we employed gene editing to generate three new haplotypes of sdw1/denso candidate gene gibberellin (GA) 20-oxidase2 (GA20ox2). These haplotypes induced a dwarfing phenotype and enhancing yield potential, and promoting seed dormancy, thereby reducing pre-harvest sprouting. Moreover, ß-amylase activity in the grains of the mutant lines was significantly increased, which is beneficial for malt quality. The haplotype analysis revealed significant genetic divergence of this gene during barley domestication and selection. A novel allele (sdw1.ZU9), containing a 96-bp fragment in the promoter region of HvGA20ox2, was discovered and primarily observed in East Asian and Russian barley varieties. The 96-bp fragment was associated with lower gene expression, leading to lower plant height but higher germination rate. In conclusion, HvGA20ox2 can be potentially used to develop semi-dwarf barley cultivars with high yield and improved malt quality.

HvBGlu3, a GH1 ß-glucosidase enzyme gene, negatively influences ß-glucan content in barley grains.

KEY MESSAGE: HvBGlu3, a ß-glucosidase enzyme gene, negatively influences ß-glucan content in barley grains by mediating starch and sucrose metabolism in developing grains. Barley grains are rich in ß-glucan, an important factor affecting end-use quality. Previously, we identified several stable marker-trait associations (MTAs) and novel candidate genes associated with ß-glucan content in barley grains using GWAS (Genome Wide Association Study) analysis. The gene HORVU3Hr1G096910, encoding ß-glucosidase 3, named HvBGlu3, is found to be associated with ß-glucan content in barley grains. In this study, conserved domain analysis suggested that HvBGlu3 belongs to glycoside hydrolase family 1 (GH1). Gene knockout assay revealed that HvBGlu3 negatively influenced ß-glucan content in barley grains. Transcriptome analysis of developing grains of hvbglu3 mutant and the wild type indicated that the knockout of the gene led to the increased expression level of genes involved in starch and sucrose metabolism. Glucose metabolism analysis showed that the contents of many sugars in developing grains were significantly changed in hvbglu3 mutants. In conclusion, HvBGlu3 modulates ß-glucan content in barley grains by mediating starch and sucrose metabolism in developing grains. The obtained results may be useful for breeders to breed elite barley cultivars for food use by screening barley lines with loss of function of HvBGlu3 in barley breeding.

Calmodulin and calmodulin-like gene family in barley: Identification, characterization and expression analyses.

Calmodulin (CaM) and calmodulin-like (CML) proteins are Ca2+ relays and play diverse and multiple roles in plant growth, development and stress responses. However, CaM/CML gene family has not been identified in barley (Hordeum vulgare). In the present study, 5 HvCaMs and 80 HvCMLs were identified through a genome-wide analysis. All HvCaM proteins possessed 4 EF-hand motifs, whereas HvCMLs contained 1 to 4 EF-hand motifs. HvCaM2, HvCaM3 and HvCaM5 coded the same polypeptide although they differed in nucleotide sequence, which was identical to the polypeptides coded by OsCaM1-1, OsCaM1-2 and OsCaM1-3. HvCaMs/CMLs were unevenly distributed over barley 7 chromosomes, and could be phylogenetically classified into 8 groups. HvCaMs/CMLs differed in gene structure, cis-acting elements and tissue expression patterns. Segmental and tandem duplication were observed among HvCaMs/CMLs during evolution. HvCML16, HvCML18, HvCML50 and HvCML78 were dispensable genes and the others were core genes in barley pan-genome. In addition, 14 HvCaM/CML genes were selected to examine their responses to salt, osmotic and low potassium stresses by qRT-PCR, and their expression were stress-and time-dependent. These results facilitate our understanding and further functional identification of HvCaMs/CMLs.

Genome-Wide Association Study on Total Starch, Amylose and Amylopectin in Barley Grain Reveals Novel Putative Alleles.

The content and composition of starch in cereal grains are closely related to yield. Few studies have been done on the identification of the genes or loci associated with these traits in barley. This study was conducted to identify the genes or loci controlling starch traits in barley grains, including total starch (TS), amylose (AC) and amylopectin (AP) contents. A large genotypic variation was found in all examined starch traits. GWAS analysis detected 13, 2, 10 QTLs for TS, AC and AP, respectively, and 5 of them were commonly shared by AP and TS content. qTS-3.1, qAC-6.2 and qAP-5.1 may explain the largest variation of TS, AC and AP, respectively. Four putative candidate genes, i.e., HORVU6Hr1G087920, HORVU5Hr1G011230, HORVU5Hr1G011270 and HORVU5Hr1G011280, showed the high expression in the developing barley grains when starch accumulates rapidly. The examined 100 barley accessions could be divided into two groups based on the polymorphism of the marker S5H_29297679, with 93 accessions having allele GG and seven accessions having AA. Moreover, significantly positive correlation was found between the number of favorable alleles of the identified QTLs and TS, AC, AP content. In conclusion, the identified loci or genes in this study could be useful for genetic improvement of grains starch in barley.

Identification of genetic loci and candidate genes related to ß-glucan content in barley grain by genome-wide association study in International Barley Core Selected Collection.

ß-glucan is an important trait to be improved in barley breeding programs, as it greatly affects the quality of the end products when barley grains are used as raw material of feed or malt production or consumed as food for human. Although the genes associated with ß-glucan synthesis have been identified, genetic regulation of ß-glucan accumulation in barley grains is still completely unclear. In this study, 100 accessions from International Barley Core Selected Collection (BCS) were planted in two environments for two consecutive years to determine the genotypic variation of grain ß-glucan content. A genome-wide association study (GWAS) identified 14 stable marker-trait associations (MTAs) (-Log10(P)> 4) for grain ß-glucan content. Significantly positive correlation was found between grain ß-glucan content and the number of favorable alleles of 14 stable MTAs. Seven putative candidate genes encoding some enzymes in glucose metabolism were found to be associated with ß-glucan content. One of the putative genes, HORVU6Hr1G088380, could be an important gene controlling barely ß-glucan content, with the SNPs being closely linked in all tested accessions and divided into two haplotypes. High resolution melting (HRM) analysis of the first SNP suggested that the HRM-SNP marker is valid for marker-assisted selection in barley breeding. This study provides useful information for the genes and markers related to grain ß-glucan content in barley. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-020-01199-5.