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The aim of these studies was to characterize nucleotide substitutions leading to the phenotype of brassinosteroid-insensitive, semi-dwarf barley mutant 093AR. Two substitutions in the sequence of barley HvBRI1 gene, encoding leucine-rich repeats receptor kinase (LRR-RK), which participates in brassinosteroid (BR) signalling, were identified in this chemically-induced barley mutant of the cv. Aramir. The LRR-RK is a transmembrane protein phosphorylating downstream components. The identified substitutions CC>AA at positions 1760 and 1761 in the HvBRI1 gene of this mutant led to a missense mutation, causing the Thr-573 to Lys-573 replacement in the protein sequence. The threonine residue is situated in the distal part of a 70-amino acids island responsible for binding of BR molecules. As this residue is conserved among BRI1 protein homologs in Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Hordeum vulgare, it was postulated that this residue is crucial for the protein function. The genetic analyses indicated that the mutant 093AR was allelic to the spontaneous, semi-dwarf mutant uzu which carries A>G substitution at position 2612 of the HvBRI1 gene (GenBank acc. no. AB088206). A comparison of the genomic sequence of HvBRI1 in the mutants uzu, 093AR and in the cv. ‘Aramir’ confirmed the presence of the single-nucleotide A>G substitution at position 2612 in the sequence encoding kinase domain of HvBRI1 polypeptide in uzu, but not in 093AR mutant, indicating that a new allele of the HvBRI1 gene was identified.
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Gene segregation in a barley DH population

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A large population of anther culture-derived barley regenerants and their progeny was tested for allele segregation at 1 isozyme and 8 morphological marker loci. The segregation of genetic markers was examined separately for haploid, diploid and polyploid regenerants. All the 9 analysed genes except al (albino lemma) on chromosome 3 segregated according to the expected 1:1 ratio in the microspore-derived barley population. There was no difference in allele distribution between haploid and diploid regenerants. Among the limited number of 34 analysed tetraploids a significant excess of the dominant allele at locus о (orange lemma) of chromosome 6 was also observed. The recombination frequency between linked genes (n - lk2 on chromosome 1 and r - s on chromosome 7) estimated in the DH population did not differ significantly from recombination rates calculated in F₂ progeny or presented in barley chromosome maps. The phenomenon of gametic selection is discussed in relation to the genotype dependency of anther culture response and procedures used for DH production in barley.
Gametoclonal variation among anther culture-derived plants of three barley genotypes was estimated on the basis of cytological analysis (DH1, DH2 generation), observation of morphological variants (DH2, DH3) and chlorophyll mutation test (DH2, DH3). Individual head rows were grown in the field to detect possible chimeric structure of regenerants and to assess the number of variants and mutations in each line. Spontaneously doubled plants were the most frequent class (70%) among regenerants and almost 90% of them were completely fertile. There was a difference in proportion of haploids produced by different genotypes, but the highest frequency observed did not exceed 21%. The remaining regenerants were tetraploid, and contained chromosomal mutations or chimeras. In total, there were about 15% of polyploids and plants carrying chromosomal aberrations (translocations, inversions) among DH1 individuals. The changes in chromosome number and structure were the main source of observed variation. The level of gene mutation induced in vitro was relatively low. No more than 1% of microspore-derived plants expressed visible morphological changes in DH2 progeny. Only two morphological variants derived from the Bruce cultivar proved to be homozygous mutants (dwarf type) stable up the to third generation. The frequency of DH plants carrying chlorophyll mutation was 5.8%, but most of them (82%) were chimeric and had only a small mutation sector. The level of gametoclonal variation depended on the donor plant genotype. The highest proportion of variants and mutations was observed among DH plants derived from the Bruce cultivar, while the lowest was recorded among plants regenerated from anther culture of the doubled haploid line H930-36. Mechanisms leading to the observed variation and implications resulting from the presented experiments concerning implementation of anther culture in barley breeding were discussed. It was concluded that this method resulted in a high frequency of spontaneous doubling, a low frequency of genetic changes, and being less time and effort-consuming than the ’Bulbosum’ technique, can be applied to most barley breeding programs.
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