A Strategy for Cloning Apomixis-Associated cDNA Markers from Buffelgrass

A strategy to select apomixis-associated cDNA sequences was partially tested in buffelgrass (Pennisetum ciliare (L.) Link, = Cenclrrns ciliaris L.). The dominant A gene allele which confers apomixis would likely be expressed as mRNA only in reproductive tissue undergoing megasporogenesis. Sexual lines lack the apomixis allele. Poly A+ mRNA was isolated from meiotic florets of obligately apomictic line Higgins and sexual line B-11-7 and cloned into lwnbda gl23A. cDNA sequences associated with the npomictic phenotype were enriched by subtracting sequences common lo both plants as follows. Insert DNA sequences of sexual and apomictic cDNAs were released with Notl and Sall. Single­stranded ends of sexual cDNA inserts were removed with SI-nuclease and then the insert cDNA was digested with A/111 and Rsal to produce blunt-ended sexual cDNA fragments. Denatured apomictlc cDNA (not blunt-ended) was hybridised with a 50- to 100-fold excess of denatured sexual cDNA fragments. Sequences common to both plants hybridised to produce double-stranded, blunt-ended sequences that were non­clonable. Sequences specific lo the apomictic plant had sticky ends and were cloned into lambda gt22A. Apomixis-associated clones were obtained after several selection cycles in which plaque lifts were probed with biotinylated mRNA pooled from either sexual or apomictic lines. At each cycle, purified apomixis-associated plaques also co-cultured with a much lower number of plaques that hybridised with a sexual probe. Experiments are underway to improve the selectivity of this method.