Identification of T-DNA structure and insertion site in transgenic crops using targeted capture sequencing

The commercialization of GE crops requires a rigorous safety assessment, which includes a precise DNA level characterization of inserted T-DNA. In the past, several strategies have been developed for identifying T-DNA insertion sites including, Southern blot and different PCR-based methods. However, these methods are often challenging to scale up for screening of dozens of transgenic events and for crops with complex genomes, like potato. Here, we report using target capture sequencing (TCS) to characterize the T-DNA structure and insertion sites of 34 transgenic events in potato. This T-DNA is an 18 kb fragment between left and right borders and carries three resistance (R) genes (RB, Rpi-blb2 and Rpi-vnt1.1 genes) that result in complete resistance to late blight disease. Using TCS, we obtained a high sequence read coverage within the T-DNA and junction regions. We identified the T-DNA breakpoints on either ends for 85% of the transgenic events. About 74% of the transgenic events had their T-DNA with 3R gene sequences intact. The flanking sequences of the T-DNA were from the potato genome for half of the transgenic events, and about a third (11) of the transgenic events have a single T-DNA insertion mapped into the potato genome, of which five events do not interrupt an existing potato gene. The TCS results were confirmed using PCR and Sanger sequencing for 6 of the best transgenic events representing 20% of the transgenic events suitable for regulatory approval. These results demonstrate the wide applicability of TCS for the precise T-DNA insertion characterization in transgenic crops

Genetic diversity assessment of farmers’ and improved potato (Solanum tuberosum) cultivars from Eritrea using simple sequence repeat (SSR) markers

Sixty three potato clones (51 farmers’ and 12 varieties) from Eritrea, 18 and 12 varieties from Kenya and Rwanda, respectively were characterized using 12 highly polymorphic simple sequence repeat (SSR) markers. The study was designed to assess the genetic diversity and varietal distinctness among the different samples. In total, 91 alleles ranging between 2 (STM1053) to 13 (STM0031) alleles per marker were scored. All but 97.8 SSR markers were highly polymorphic with an average PIC value of 0.87 (0.51 to 0.98). All of the 51 farmers’ cultivars were clearly distinct from each other. Samples from Eritrea showed the highest genetic diversity as explained by the diversity index (h). The principal coordinate analysis (PCoA) revealed that the local farmers’ Eritrean samples are different from the Kenyan, Rwandese and even the imported varieties. Genetic distance analysis generated three clusters correlating with the PCoA findings. Cluster I consisted of 45 samples with 6 sub-clusters; Cluster II consisted of 29 samples with a majority (26) from Eritrea while cluster III consisted of 19 samples. Potato materials from Eritrea appeared to cluster separately from the other samples, which reflects a contribution from the Tuberosum germplasm prominent in temperate regions, unlike from the Andigenum germplasm for Kenyan and Rwandan potato materials. Most of the Eritrean samples in cluster I are farmers’ cultivars with intermediate maturity, good performance and better tuber quality characteristics. Cluster II contains mainly the imported variety from Eritrea characterized by late emergence and late maturity. The Kenyan and Rwandese were grouped mainly in Cluster III. In summary, the farmers’ cultivars are distinct from the Kenyan and Rwandese materials and represent more genetic diversity than the varieties imported into Eritrea. This finding is of interest to national breeding program to use the farmer’s materials as source of genetic variation for traits of interest.Keywords: Potato, simple sequence repeat (SSR), principal coordinate analysis (PCoA), cluster analysis, Eritrea, multivariat

Comparative Phenotypic and Agronomic Assessment of Transgenic Potato with 3R-Gene Stack with Complete Resistance to Late Blight Disease

Transgenic potato event Vic.172, expressing three naturally occurring resistance genes (R genes) conferring complete protection against late blight disease, was evaluated for resistance to late blight, phenotypic characterization, and agronomic performance in field conditions at three locations during three seasons in Uganda. These trials were conducted by comparison to the variety Victoria from which Vic.172 derives, using identical fungicide treatment, except when evaluating disease resistance. During all seasons, the transgenic event Vic.172 was confirmed to have complete resistance to late blight disease, whereas Victoria plants were completely dead by 60–80 days after planting. Tubers from Vic.172 were completely resistant to LB after artificial inoculation. The phenotypic characterization included observations of the characteristics and development of the stems, leaves, flowers, and tubers. Differences in phenotypic parameters between Vic.172 and Victoria were not statistically significant across locations and seasons. The agronomic performance observations covered sprouting, emergence, vigor, foliage growth, and yield. Differences in agronomic performance were not statistically significant except for marketable yield in one location under high productivity conditions. However, yield variation across locations and seasons was not statistically significant, but was influenced by the environment. Hence, the results of the comparative assessment of the phenotype and agronomic performance revealed that transgenic event Vic.172 did not present biologically significant differences in comparison to the variety Victoria it derives from