Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.
In Portugal, the Cova da Beira region is well-known for the production of Prunus spp. and is considered the main peach production area in the country. In the spring of 2021 and 2022, field surveys in peach and nectarine orchards showed symptoms of decline such as cankers, gummosis, dry branches, abortion of flowers, mummified fruits and the partial or total death of some plants. Brown rot is caused by three species of the genus Monilinia, M. fructigena, M. laxa and M. fructicola, the last is an OEPP/EPPO A2 quarantine organism on peach trees. Brown rot disease had previously been described in the Cova da Beira region, however, the recent high mortality and severity of symptoms raised doubts as to the species involved. Symptomatic plant material was collected from thirteen orchards and used for fungal isolation and molecular detection according to the OEPP/EPPO standard. M. fructicola was confirmed morphologically and molecularly in two orchards, and molecularly (duplex real-time PCR) detected in two others. Whole genome sequencing using Oxford Nanopore MinION was also carried out to confirm the identification. Pathogenicity tests were performed on peach, nectarine and sweet cherry fruit according to Koch’s postulates. Based on all the results obtained, we report the first detection of M. fructicola in P. persica in Portugal.
Background Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies. However, some plant tissues are recalcitrant to the successful preparation of cDNA libraries thus impairing further analyses. Results A detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® was developed. Through this method, 8 barcoded cDNA libraries from two tamarillo (Solanum betaceum Cav.) types of callus (friable and compact) and with distinct embryogenic competencies (embryogenic non-embryogenic) have been successfully prepared and sequenced. Conclusions An efficient method for preparing barcoded cDNA libraries from plant callus is presented. Long-read sequencing using these libraries allows a comprehensive analysis of plant transcriptomes.
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