Positional cloning identified HvTUBULIN8 as the candidate gene for round lateral spikelet (RLS) in barley (Hordeum vulgare L.).

KEY MESSAGE: Map-based cloning, subcellular localization, virus-induced-gene-silencing and transcriptomic analysis reveal HvTUB8 as a candidate gene with pleiotropic effects on barley spike and leaf development via ethylene and chlorophyll metabolism. Barley lateral spikelet morphology and grain shape play key roles in grain physical quality and yield. Several genes and QTLs for these traits have been cloned or fine mapped previously. Here, we report the phenotypic and genotypic analysis of a barley mutant with round lateral spikelet (rls) from cv. Edamai 934. rls had round lateral spikelet, short but round grain, shortened awn, thick glume and dark green leaves. Histocytologic and ultrastructural analysis revealed that the difference of grain shape of rls was caused by change of cell arrangement in glume, and the dark leaf color resulted from enlarged chloroplast. HvTUBULIN8 (HvTUB8) was identified as the candidate gene for rls by combination of RNA-Seq, map-based-cloning, virus-induced-gene-silencing (VIGS) and protein subcellular location. A single G-A substitution at the third exon of HvTUB8 resulted in change of Cysteine 354 to tyrosine. Furthermore, the mutant isoform Hvtub8 could be detected in both nucleus and cytoplasm, whereas the wild-type protein was only in cytoplasm and granular organelles of wheat protoplasts. Being consistent with the rare phenotype, the "A" allele of HvTUB8 was only detected in rls, but not in a worldwide barley germplasm panel with 400 accessions. VIGS confirmed that HvTUB8 was essential to maintain spike integrity. RNA-Seq results suggested that HvTUB8 may control spike morphogenesis via ethylene homeostasis and signaling, and control leaf color through chlorophyll metabolism. Collectively, our results support HvTUB8 as a candidate gene for barley spike and leaf morphology and provide insight of a novel mechanism of it in barley development.

Characterization and fine mapping of a novel barley Stage Green-Revertible Albino Gene (HvSGRA) by Bulked Segregant Analysis based on SSR assay and Specific Length Amplified Fragment Sequencing.

BACKGROUND: Leaf color variations are common in plants. Herein we describe a natural mutant of barley cultivar Edamai No.6, whs18, whose leaf color showed stable and inheritable stage-green-revertible-albino under field condition. METHODS: Bulked Segregant Analysis (BSA) based on SSR assay and Specific Length Amplified Fragment Sequencing (SLAF-seq) was used to map the candidate gene for this trait. RESULTS: We found that leaf color of whs18 was green at seedling stage, while the seventh or eighth leaf began to show etiolation, and albino leaves emerged after a short period. The newly emerged leaves began to show stripe white before jointing stage, and normal green leaves emerged gradually. The duration of whs18 with abnormal leaf color lasted for about 3 months, which had some negative impacts on yield-related-traits. Further investigations showed that the variation was associated with changes in chlorophyII content and chloroplast development. Genetic analysis revealed that the trait was controlled by a single recessive nuclear gene, and was designed as HvSGRA in this study. Based on the F2 population derived from Edamai No.9706 and whs18, we initially mapped the HvSGRA gene on the short arm of chromosome 2H using SSR and BSA. GBMS247 on 2HS showed co-segregation with HvSGRA. The genetic distance between the other marker GBM1187 and HvSGRA was 1.2 cM. Further analysis using BSA with SLAF-seq also identified this region as candidate region. Finally, HvSGRA interval was narrowed to 0.4 cM between morex_contig_160447 and morex_contig_92239, which were anchored to two adjacent FP contigs, contig_34437 and contig_46434, respectively. Furthermore, six putative genes with high-confidence in this interval were identified by POPSEQ. Further analysis showed that the substitution from C to A in the third exon of fructokinase-1-like gene generated a premature stop codon in whs18, which may lead to loss function of this gene. CONCLUSIONS: Using SSR and SLAF-seq in conjunction with BSA, we mapped HvSGRA within two adjacent FP contigs of barley. The mutation of fructokinase-1-like gene in whs18 may cause the stage green-revertible albino of barley. The current study lays foundation for hierarchical map-based cloning of HvSGRA and utilizing the gene/trait as a visualized maker in molecular breeding in future.