We used the C38 EST assembly (http://harvest.ucr.edu/) and OligoSpawn software (http://138.23.178.42) to design the overgo probes employed to screen a Citrus sinensis Vaniglia BAC library (19X). For BAC library screening we selected 81 overgo probes associated with unigenes that putatively code for enzymes relevant to fruit quality (flavonol, anthocyanin, carotenoid, chlorophyll, cellulose, starch, ascorbic acid, aromatic amino acid and lignin biosynthesis; sucrose catabolism; glycolysis; oxidative/nonoxidative pentose phosphate pathway; fatty acid biosynthesis and oxidation; Krebs cycle). A two-dimensional 9 X 9 overgo pooling strategy was used. Hybr idizat ion probes were pooled and hybridized in groups of intersecting rows and columns to high-density BAC filters, followed by a deconvolution process that established BAC-probe addresses. BAC addresses were obtained for 75 of the 81 overgo probes initially selected, for a total of 1018 BAC clones, a number consistent with the depth of coverage of the BAC library. BAC end sequencing was carried out and sequence similarity searches (BLAST) were conducted against draft assemblies of the Citrus clementina genome (http://phytozome.net). The BAC clones corresponding to each probe were located within the same scaffold as the target gene, demonstrating that the approach we used was successful in isolating the target genes.
Utilizzando la EST assembly C38 (http://harvest.ucr.edu/) e il software "OligoSpawn" (http://138.23.178.42), sono state selezionate 81 sonde overgo associate con unigene che codificano per gli enzimi strutturali delle vie biosintetiche aventi un ruolo rilevante nel determinare la qualità dei frutti. Con le sonde selezionate è stato effettuato lo screening di una BAC library (19x) di Citrus sinensis cv Vaniglia per individuare i cloni contenenti i corrispondenti geni. Degli 81 geni selezionati, 75 sono stati individuati, corrispondenti ad un totale di 1018 cloni BAC. La mappatura dei BACs individuati contro il genoma di Citrus clementina (http://phytozome.net) ha dimostrato che i cloni corrispondenti a ciascuna sonda sono posizionati all'interno dello stesso scaffold contenente il gene target, confermando che la metologia seguita è stata vincente nell'isolare i geni oggetto del presente studio.
Identificazione di cloni bac di Citrus sinensis contenenti geni rilevanti per la qualità dei frutti
2013
Abstract
We used the C38 EST assembly (http://harvest.ucr.edu/) and OligoSpawn software (http://138.23.178.42) to design the overgo probes employed to screen a Citrus sinensis Vaniglia BAC library (19X). For BAC library screening we selected 81 overgo probes associated with unigenes that putatively code for enzymes relevant to fruit quality (flavonol, anthocyanin, carotenoid, chlorophyll, cellulose, starch, ascorbic acid, aromatic amino acid and lignin biosynthesis; sucrose catabolism; glycolysis; oxidative/nonoxidative pentose phosphate pathway; fatty acid biosynthesis and oxidation; Krebs cycle). A two-dimensional 9 X 9 overgo pooling strategy was used. Hybr idizat ion probes were pooled and hybridized in groups of intersecting rows and columns to high-density BAC filters, followed by a deconvolution process that established BAC-probe addresses. BAC addresses were obtained for 75 of the 81 overgo probes initially selected, for a total of 1018 BAC clones, a number consistent with the depth of coverage of the BAC library. BAC end sequencing was carried out and sequence similarity searches (BLAST) were conducted against draft assemblies of the Citrus clementina genome (http://phytozome.net). The BAC clones corresponding to each probe were located within the same scaffold as the target gene, demonstrating that the approach we used was successful in isolating the target genes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
