An apomixis-linked non coding allele of the origin of recognition complex subunit 3 down-regulates the functional alelle by an antisense-mediated mechanism in Paspalum

SIENA LA; ORTIZ JPA; PAOLOCCI F; CÁCERES ME; CALDERINI O; PESSINO SC; KAUSHAL P; PUPILLI F

Bahía Blanca

Workshop; IV Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis en gramíneas forrajeras; 2014

CERZOS-CONICET

Apomixis in several Paspalum species is controlled by a complex locus (ACL) located in a genomic region showing repression of recombination and synteny with segments of chromosomes 12 and 2 of rice. Several candidate genes mapping at ACL have been identified in P. simplex and P. notatum and functional studies of most interesting ones are ongoing. One of these genes presents homology with the subunit 3 of the Origin of Recognition Complex, a protein aggregate that has important role in DNA replication and cell differentiation. Southern blot analysis with PsORC3 sequence showed 100% linkage in cis with apomixis in several Paspalum spp. Three copies of PsORC3 were detected in P. simplex: one called PsORC3a is apomixis specific. It presents several non-sense mutations and stops codons that predicted a non-functional protein. Another one, PsORCsb, likely represents a functional allele present in both apomictic and sexual genotypes, and a third one, PsORC3c, expressing a truncated protein, segregated independently from apomixis. Allele-specific real-time PCR showed that PsORC3a is poorly expressed in all developmental stages only in flowers of apomictic genotypes while PsORC3b is expressed only in sexual flowers with a significant increment after anthesis until the early phases of seed development. Conversely, the expression of the same allele (PsORC3b) was low in all developmental stages in apomictic flowers, suggesting it is down regulated. ISH analysis showed that at anthesis ORC3 alleles were expressed in the nucellus, central cell and egg cells in both sexual and apomictic flowers. Signal intensity increased significantly at early phases of seed development in sexual flowers, while in apomictic ones it remained low. Curiously enough, the ?sense? probe yielded a strong hybridising signal in all developmental stages of apomictic flowers while no signals were observed in sexual ones. Reverse genetic analyses carried out in rice and Arabidopsis showed that homozygous defective mutants at the ORC3 showed characteristic sporophytic lethal phenotypes in which embryos and/or endosperms arrested at early developmental stages. Gametophyte development was unaffected. To sum up we believe that PsORC3a in apomicts silences its sexual allelic counterpart by an antisense mediated mechanism. The biological consequence of this silencing is probably related to the repression of the sexual (fertilizationmediated) development in flowers of the apomictic genotype, indicating that parthenogenesis does not arise from a silencing of sexuality but rather, apomixis and sexuality of the embryo development are two independent processes.