Isolation of cDNA clones differentially expressed in flowers of apomictic and sexual Paspalum notatum

Abstract

Paspalum notatum is a subtropical forage grass, which reproduces by either sexuality or aposporous gametophytic apomixis. The objective of this work was to identify and isolate mRNA transcripts differentially expressed during the development of the megagametophyte from spikelets of apomictic and sexual P. notatum. Crossing of a sexual mother plant with an apomictic pollen donor generated a progeny family segregating for reproductive mode. Individuals from this F1 family were cytoembryologically classified as sexual or apomictic. Spikelet mRNA compositions from both groups of plants were compared by differential display using an RNA-bulked procedure. Fifty primer combinations were assayed to generate nearly 2,500 total bands in the fingerprints. Three transcripts expressed at higher levels in apomictic plants (apo417, apo398, and apo396) were identified, isolated and cloned. Sequencing showed a high level of homology among the isolated clones. Analysis by RT-PCR Southern blots followed by densitometric studies confirmed that expression reached a level around 30 times higher in apomictic than in sexual individuals and was probably induced at early stages of the megagametophyte development. Genomic DNA from the parental and the F1 progeny plants showed 4-5 bands when hybridised with apo417 in Southern blots. Comparisons to the sequence data banks revealed no identities to genes of known function. However, a putative deduced 3' protein fragment showed homology to the well-characterised KSP multi-phosphorylation domain previously detected in several cdc2-regulated cytoskeletal proteins.