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

Dandan Qin, Jing Dong, Fuchao Xu, Ganggang Guo, Shuangtao Ge, Qing Xu, Yuxin Xu, Meifang Li

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. 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. 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. 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.