Citrus clementina pistils cDNA-AFLP library

Vector: pGEM-T easy; Site_1: EcoRI; Site_2: MseI; Total RNA was extracted using Plant RNA Reagent (Invitrogen) from unpollinated and self-pollinated pistils of Comune clementine and its self-compatible natural mutation Monreal, grown at the experimental station of Catania University. About 100 ug total RNA was used to isolate mRNA by PolyATract mRNA Isolation System III (Promega). cDNA synthesis and cDNA-AFLP analysis were carried according to Bachem et al (1996). Differentially expressed and polymorphic sequences were visually screened and excised from gels, eluted overnight with water, reamplified with the same conditions used for selective amplifications and electrophoresed in 1% agarose gel. All the successfully reamplified TDFs were excised from agarose, purified with QIAquick gel extraction kit (Qiagen), cloned into pgem-T Easy vector (Promega), and sequenced using an ABI310 genetic analyzer (Applied Biosystems). Seqman software (DNAstar, Madison,WI, USA) was used to remove vector sequences and to find possible overlaps among the transcript derived fragments.