Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum

PODIO, M.; RODRÍGUEZ, M.P.; FELITTI, S.A.; STEIN, J.; MARTÍNEZ, E.J.; SIENA, L.A.; QUARIN, C.; PESSINO, S.C.; ORTIZ, J.P.A.

GENETICS AND MOLECULAR BIOLOGY

SOC BRASIL GENETICA

Año: 2012 vol. 35 p. 827 - 837

1415-4757

In previous studies we reported the identification of several AFLP, RAPD and RFLPmolecular markers linked to apospory in Paspalum notatum. The objective of this workwas to sequence these markers, obtain their flanking regions by chromosome walking,and perform an in silico mapping analysis in rice and maize. Moreover, methylationsensitive RFLP experiments were carried out to investigate the methylation status oftwo apospory-related sequences. Fourteen molecular markers were analyzed. Severalprotein-coding sequences were identified. Copy number estimations and RFLP linkageanalysis showed that sequence PnMAI3 displayed 2-4 copies per genome and linkage toapospory. Extension of this marker by chromosome walking revealed an additionalprotein-coding sequence mapping in silico at the apospory-syntenic regions of rice andmaize. Through the global sequencing procedure, ~ 5 kb corresponding to differentmarkers were characterized. Sequence information allowed a more refined syntenyanalysis with model species. Results indicate synteny with segments of chromosomes 2and 12 of rice, as well as 3 and 5 of maize. Two apomixis-associated loci were tested inmethylation-sensitive RFLP experiments on genomic DNA extracted from leaves.Although both target sequences were methylated, no methylation polymorphismsassociated with the mode of reproduction were detected.