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Leaf color is an important trait for not only controlling crop yield but also monitoring plant status under temperature stress. In this study, a thermo-inducible chlorophyll-deficient mutant, named V-V-Y, was identified from a gamma-radiated population of the barley variety Vlamingh. The leaves of the mutant were green under normal growing temperature but turned yellowish under high temperature in the glasshouse experiment. The ratio of chlorophyll a and chlorophyll b in the mutant declined much faster in the first 7-9 days under heat treatment. The leaves of V-V-Y turned yellowish but took longer to senesce under heat stress in the field experiment. Genetic analysis indicated that a single nuclear gene controlled the mutant trait. The mutant gene (vvy) was mapped to the long arm of chromosome 4H between SNP markers 1_0269 and 1_1531 with a genetic distance of 2.2 cM and a physical interval of 9.85 Mb. A QTL for grain yield was mapped to the same interval and explained 10.4% of the yield variation with a LOD score of 4. This QTL is coincident with the vvy gene interval that is responsible for the thermo-inducible chlorophyll-deficient trait. Fine mapping, based on the barley reference genome sequence, further narrowed the vvy gene to a physical interval of 0.428 Mb with 11 annotated genes. This is the first report of fine mapping a thermo-inducible chlorophyll-deficient gene in barley. [ABSTRACT FROM AUTHOR]

Background: The dwarfing gene sdw1 has been widely used throughout the world to develop commercial barley varieties. There are at least four different alleles at the sdw1 locus. Results: Mutations in the gibberellin 20-oxidase gene (HvGA20ox2) resulted in multiple alleles at the sdw1 locus. The sdw1.d allele from Diamant is due to a 7-bp deletion in exon 1, while the sdw1.c allele from Abed Denso has 1-bp deletion and a 4-bp insertion in the 5' untranslated region. The sdw1.a allele from Jotun resulted from a total deletion of the HvGA20ox2 gene. The structural changes result in lower gene expression in sdw1.d and lack of expression in sdw1.a. There are three HvGA20ox genes in the barley genome. The partial or total loss of function of the HvGA20ox2 gene could be compensated by enhanced expression of its homolog HvGA20ox1and HvGA20ox3. A diagnostic molecular marker was developed to differentiate between the wild-type, sdw1.d and sdw1.a alleles and another molecular marker for differentiation of sdw1.c and sdw1.a. The markers were further tested in 197 barley varieties, out of which 28 had the sdw1.d allele and two varieties the sdw1.a allele. To date, the sdw1.d and sdw1.a alleles have only been detected in the modern barley varieties and lines. Conclusions: The results provided further proof that the gibberellin 20-oxidase gene (HvGA20ox2) is the functional gene of the barley sdw1 mutants. Different deletions resulted in different functional alleles for different breeding purposes. Truncated protein could maintain partial function. Partial or total loss of function of the HvGA20ox2 gene could be compensated by enhanced expression of its homolog HvGA20ox1 and HvGA20ox3. [ABSTRACT FROM AUTHOR]

Background: Barley semi-dwarf genes have been extensively explored and widely used in barley breeding programs. The semi-dwarf gene ari-e from Golden Promise is an important gene associated with some agronomic traits and salt tolerance. While ari-e has been mapped on barley chromosome 5H using traditional markers and next-generation sequencing technologies, it has not yet been finely located on this chromosome. Results: We integrated two methods to develop molecular markers for fine-mapping the semi-dwarf gene ari-e: (1) specific-length amplified fragment sequencing (SLAF-seq) with bulked segregant analysis (BSA) to develop SNP markers, and (2) the whole-genome shotgun sequence to develop InDels. Both SNP and InDel markers were developed in the target region and used for fine-mapping the ari-e gene. Linkage analysis showed that ari-e cosegregated with marker InDel-17 and was delimited by two markers (InDel-16 and DGSNP21) spanning 6.8 cM in the doubled haploid (DH) Dash × VB9104 population. The genetic position of ari-e was further confirmed in the Hindmarsh × W1 DH population which was located between InDel-7 and InDel-17. As a result, the overlapping region of the two mapping populations flanked by InDel-16 and InDel-17 was defined as the candidate region spanning 0.58 Mb on the POPSEQ physical map. Conclusions: The current study demonstrated the SLAF-seq for SNP discovery and whole-genome shotgun sequencing for InDel development as an efficient approach to map complex genomic region for isolation of functional gene. The ari-e gene was fine mapped from 10 Mb to 0.58 Mb interval. [ABSTRACT FROM AUTHOR]

Recalcitrance to tissue culture and genetic transformation is the major bottleneck for gene manipulation in crops. In barley, immature embryos of Golden Promise have typically been used as explants for transformation. However, the genotype dependence of this approach limits the genetic modification of commercial varieties. Here, we developed an anther culture-based system that permits the effective creation of transgenic and gene-edited plants from commercial barley varieties. The protocol was tested in Golden Promise and four Australian varieties, which differed in phenology, callus induction, and green plant regeneration responses. Agrobacterium -mediated transformation was performed on microspore-derived callus to target the HvPDS gene, and T0 albinos with targeted mutations were successfully obtained from commercial varieties. Further editing of three targets was achieved with an average mutation rate of 53% in the five varieties. In 51 analyzed T0 individuals, Cas9 induced a large proportion (69%) of single-base indels and two-base deletions in the target sites, with variable mutation rates among targets and varieties. Both on-target and off-target activities were detected in T1 progenies. Compared with immature embryo protocols, this genotype-independent platform can deliver a high editing efficiency and more regenerant plants within a similar time frame. It shows promise for functional genomics and the application of CRISPR technologies for the precise improvement of commercial varieties., (© 2020 Murdoch University.)

Background: Acid soil is a serious limitation to crop production all over the world. Toxic aluminium (Al) cations in acid soil inhibit root growth and reduce yield. Although a gene tolerant to acid soil has been identified, it has not been used in malting barley breeding, which is partly due to the acid soil tolerance gene being linked to unfavorable malting quality traits., Results: A Brazilian malting barley variety Br2 was identified as tolerant to acid soil. A doubled haploid (DH) population was developed from a cross between Br2 and the Australian acid-sensitive cultivar Hamelin. The DH population was tested for acid soil tolerance in native acid soil and a hydroponic system with pH 4.2, pH 4.2 + Al or pH 6.5, and genotyped using SSR, DArT and gene-specific markers. A single QTL was detected for all parameters related to acid soil tolerance. The QTL was mapped to the known HvMATE location on chromosome 4H. Sequence alignment of the HvMATE gene identified 13 INDELs and 87 SNPs, where one SNP coded for a single amino acid difference between the two varieties. A gene-specific marker was developed to detect the single nucleotide polymorphism between Hamelin and Br2. This marker co-segregated with aluminium tolerance and accounted for 79 % of phenotypic variation for acid soil tolerance., Conclusion: The present study identified a novel source of acid soil/Al tolerance from a Brazilian malting barley cultivar Br2. This variety tolerated Al toxicity but was sensitive to low pH which is similar to most other Al-tolerant varieties. A gene-specific marker Cit7 was developed based on the HvMATE gene sequence. Cit7 will improve the efficiency of molecular-assisted selection of new barley varieties with tolerance to acid soil. Multiple alleles exist for the acid soil tolerance gene on chromosome 4H, so a malting barley variety that tolerates acid soil could be developed by selecting suitable tolerant alleles. Tolerance to low pH may play an important role for barley to adapt to acid soils.