Poster75: Quantitative real time PCR assessment of cassava transgenic plants: copy number estimate and quantification of gene expression

Quantitative real time PCR assessment of cassava transgenic plants: Copy number estimate and quantification of gene expressionBeltrán J1., Chavarriaga P1., Echeverry M2., Jaimes H1., Ladino J1., López D 1., Duque M1 and Tohme J1.1. Conservation and Use of Tropical Genetic Resources, CIAT, AA 6713 , Cali, Colombia 2. Universidad de Puerto RicoINTRODUCTION• Southern blot and qPCR, for copy number estimationTo improve the conventional molecular analysis of cassava transgenic The analysis revealed different integration patterns, confirmingplants, we developed qPCR methods to estimate copy number and that the lines arose from independent transformation events. quantify mRNA levels of transgenes in cassava lines obtained through M 11 27 42 43 53 54 90 NT P M 11 27 42 43 53 54 90 NT P hptIIGUSPlusAgrobacterium-mediated transformation. Six lines were also analyzed by Southern blot. The copy number was concordant in most cases with those estimated by qPCR. Most of the transgenic events (86.6%) had low copy number (1 or 2), thus 0.5 kb 0.5 kb corroborating that Agrobacterium generally inserts a low copy number of transgenes into plants.Of the six lines evaluated, five coincided exactly with the qPCRestimates indicating an 83.3% agreement between the two Quantitative mRNA expression data grouped the transgenic lines into methodologies: three expresser categories —high, medium, and low— for hygromycinTable 2. Comparison of copy numbers for two transgenes, as estimated by Southern phosphotransferase (hptII) and ß-glucuronidase (GUSPlus) genes.blot and qPCR for six transgenic cassava lines GUSPlus mRNA data agreed with results from histochemical GUS Copy number for gene: staining.GUSPlus hptII LineqPCR Southern blot qPCR Southern blot 112 2 2 2 METHODS272 2 2 2 422 3 2 3 432 2 2 2 533 3 3 3 901 1 1 1 Transformed cassava lines• Quantifying transgene expression using qRT-PCRDNARNABy comparing the highest and lowest expresser lines, we calculated differences of up to 380 times for GUSPlus and 3000 times for hptII:Transgene Detection by melting cDNA synthesiscurve analysis3.5Transgene mRNA detection by A3Nmelting curve analysisCopy number estimation by mR 2.5 ofreal-time PCRQuantification of mRNA levels 2untoGusplusby real-time RT-PCR1.5m ahpt IIe1tivCopy number by Southern blotGus testlae 0.5R01129424352546090131RESULTS AND DISCUSSIONTransgenic lines• Copy number estimation by real-time PCRRT-PCR results agreed with the qRT-PCR data for most samples and for both transgenes:M 11 29 42 43 52 54 60 90 131 NT H2O Copy numbers for the genes GUSPlus and hptII were estimated in 15 181 bpGUSPlustransgenic lines 182 bphptII169 bp18STable 1. Copy number for the GUSPlus and hptII genes, estimated by qPCR for 15 transgenic cassava lines • GUS test Copy number for gene: A differential pattern of intensities for GUS can be seen. TheseLine GUSPlus hptII 2 1 1 intensities mirrored the data obtained with qRT-PCR:11 2 2 27 2 2 29 90 43 11 52 54 42 60 131 NT29 1 2 42 2 2 43 2 2 46 1-2 1-2 50 2 2 51 2 1 • Summarizing, qPCR was more efficient than the laborious techniques 52 1 1 conventionally used to detect transgene copy number and expression.53 3 3 55 3 3 60 1 1 • Results suggest that, in 3-year-old transgenic cassava plants, high and 90 1 1 131 1 1 stable expression of transgenes can be found. PERSPECTIVESMost lines contained one or two copies of each gene; in some, the copy number was different for the two genes, suggesting rearrangements of •Use qPCR for future characterization of transgenic events in cassava, the T-DNAwhich could facilitate the selection of the most promising events.