Genotipagem molecular de híbridos de Pancirus trifoliate e Citrus sunki, por meio de marcadores TRAP – PCR (Target Region Amplification – Polymorphism- Polymerase Chain Reaction).

Abstract

The citrus breeding programs have been researched alternatives to control phytosanitary problems concerning to citriculture, by using the Poncirus trifoliata rootstock as a disease resistant source, among these the gummosis disease, caused by the Phytophthora parasitica Oomycete. Genotyping of individuals using DNA-associated molecular markers provides the possibility for screening of individuals that carries interesting traits in a fast and efficient manner. The general aim of this work was to perform the molecular genotyping of a F1 population obtained of Citrus sunki versus Poncirus trifoliata cross, by using TRAP-PCR (Target Region Amplification Polymorphism-Polymerase Chain Reaction) markers, focusing the mapping of QTls associated with P. parasitica gummosis resistance. Based on previous information about citrus expressed genes (from CitEST) and contrasting phenotype hybrids for resistance and susceptibility to P. parasitica infection, we performed bulk segregant analyses for DNAs from resistant genitor (P. trifoliata), susceptible genitor (C. sunki), pool of DNAs from resistant hybrids and pool of DNAs from susceptible hybrids to test 12 primers combinations, two fixed versus six arbitrary primers. As results, the bulk segregant analyses revealed that the SRG1/A6 (fixed primer for the SRG1 (Senescence Related Gene 1 gene)/Arbitrary primer number 6) primer combination produced 02 polymorphic fragments, that were present in both resistant genitor and F1 hybrids. This selected primer combination was used for genotyping a population of 161 hybrids, and among them 93 showed the two TRAP markers. The two TRAP markers were individually analyzed for the Mendelian segregation pattern, in a 1:1 ratio, by using the Qui-Square test. Only one TRAP marker was integrated into the P. trifoliata genetic map, at the linkage group C4, using the JoinMap v.3.0 program. QTLs detection was perfomed using the QTLCartographer v.1.25 program and compost interval mapping, that indicated the presence of a QTL between 71 and 79 centiMorgans with position for the marker at 79 centiMorgans (LOD > 1,6). In relation at the citrus genetic mapping and the QTLs identification, new primer combinations need to be evaluated in order to generate TRAP markers to saturate the the P. trifoliata genetic map with a more accurate estimative.