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

Abstract

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.

Fig. 1

Seedling and spike morphology of rls. a Seedling of E934 and rls; b Wild-type (MM), medium mutant (mM) type and mutant (mm) type spikes; c central spikelets; d lateral spikelet; e bilateral empty glumes; f, g: seeds of E934 and rls

Fig. 2

Immature spike and ultrastructure of chloroplast from E934 and rls. a, b 5-mm spike of E934 and rls; c, d lateral spikelet of E934 and rls; e, f ultrastructure of leaf cell from E934 and rls; g, h chloroplast of E934 and rls; i, j: horizontal section of lemma from E934 and rls (bar = 100 μm). SC, silicified cells; OPC, outer parenchyma cells; IPC, inner parenchyma cells

Fig. 3

Map-based cloning of HvRLS. a Initial mapping; b, c fine mapping; d phenotype and genotype of recombinant lines. Note: A, B and H in Fig. 3d stand for homologous wild-type allele MM, homologous mutant allele mm and heterozygous allele mM, respectively

Fig. 4

Sequence, expression and functional analysis of HvTUB8. a Gene structure of HvTUB8 (Red indicates the G-A and C-Y substitution on the third exon); b phylogenetic analysis of HvTUB8 and its homologue genes; c partial alignment of HvTUB8 and its homologue genes (red star indicates the C-Y substitution); d spatio-temporal expression of HvTUB8 in E934; e spikes of BSMV:HvTUB8-infected and non-infected E934 (WT) (color figure online)

Fig. 5

Subcellular localization of HvTUB8 protein. 35S::eGFP indicates the expression of GFP protein in wheat protoplasts as the negative control. 35S::HvTUB8-eGFP indicates the cytoplasm localization of wild-type HvTUB8 protein in wheat protoplasts. Green is GFP signal. Bright is the bright light. Merged indicates the confused with the green signal and the bright light. Bars: 10 μm

Fig. 6

Expression of DEGs in ethylene and chloroplast pathways