Hordeum vulgare Transcriptome or Gene expression. Hordeum vulgare

Barley uniculm2 (cul2) plants generally do not produce tillers, the modified lateral branches growing from axillary meristems located at the base of plants. We conducted a genetic suppressor screen to recover cul2 suppressors. A near-isogenic cul2 line in the cultivar Bowman was mutagenized with sodium azide. This line also carried the tightly linked marker orange lemma (rob1). Two suppressors were identified after screening over 15,000 F2 families, and allelism tests determined that the suppressors were allelic. In addition to suppressing the cul2 low-tillering trait, the suppressor mutation gave short plants with liguleless leaves that resembled the previously described eligulum-a (eli-a) mutant. Three eli-a alleles were then tested. All three alleles suppressed the tillering phenotype of cul2 and complemented our suppressor in allelism tests. To clone the gene, RNAseq was performed on the backcross parent Bowman, the Bowman-cul2; rob1 progenitor line, the related Bowman-cul2 line, the two suppressor lines in the Bowman-cul2; rob1 background, and the two suppressors in the Bowman background. RNA was isolated from crown tissue from 14-day old seedlings, three replicates of six plants each and replicates were pooled for RNA preps. Sequencing for 76 nucleotide paired end reads was performed on a Illumina HiSeq GAIIx machine, 10 million reads per sample. Sequencing reads were assembled, and SNPs were called. One candidate transcript was identified with a unique SNP in each suppressor. Sanger sequencing confirmed the SNPs, and in the two original suppressor mutations and the three eli-a alleles there were two nonsense mutations and three missense mutations. ELI-A encodes a previously unknown peptide containing a putative RNaseH-like domain.