Anther culture and molecular characferization of a floralderived gene in the diploid species Hordeum Bulbosum L.

Abstract

The present study involved two aspects of the self-incompatible (SI) diploid species H. bulbosum. First, the production of homozygous lines via anther culture and secondly, the use of molecular techniques to investigate the developmental regulation, cloning and characterization of the pistil cDNA clone, Hs-6. Anther culture was successfully used to bypass the SI mechanism and to produce homozygous doubled . ,, haploid lines from a dip,loid species of H. bulbosum. Green plants were regenerated from H. bulbosum accession-GlsCt? using 50 mg/L PAA in FHG anther culture media. Two of the regenerants were shown to be homozygous for the Hs-6 gene and therefore should be homozygous for all loci, making them useful in genetic ~d molecular studies. Homologous sequences to the Hs-6 gene were fo.~undin barley, wheat, rye and H. . bulbosum. It is exclusively expressed at high levels in the pistils of SI cereals (rye and H. bulbosum) in a developmental and organ-specific manner typical of the SI genes and thus might perform a function unique to SI cereals including recognition of self pollen. Hs-6 is expressed at high levels in fertile, but at low levels in sterile florets and may indicate that this gene participates in maturation and the normal function of the pistil. ... Although, it is not known which specific pistil tissues express this gene, evidence for its possible involvement in the SI reaction and other pistil functions as well as developmental and organ-specific regulation in H. bulbosum is presented. The production and sequencing of a complete (or near full-length) cDNA sequence (Hbc 8) homologous to Hs-6 proved to be very difficult but revealed very unusual characteristics. The Hbc 8 encodes a 206 a.a polypeptide without a signal peptide sequence or a potential N-glycosylation site typical of extracellular and SI proteins. The predicted protein sequence does not match any sequence in the protein databanks. Quite unexpected was the finding that approximately 288 bp at the 5' end is made up of 3' portions of coding and non-coding segments of psbA gene, fused in reverse orientation to the 3' segment of an unknown nuclear gene. It does not appear to be fragment mixtures arising from cDNA library construction as it occurred in 6 out 12 independent cDNA clones. Possible origins of this mRNA producing structure are discussed.