Defining the ‘HoneySweet’ insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica)

Abstract

‘HoneySweet’ plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, ‘HoneySweet’ DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum ‘Improved French’. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the ‘HoneySweet’ transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.

References

[1] Scorza, R. et al. ‘HoneySweet’(C5), the first genetically engineered plum pox virus-resistant Plum (Prunus domestica L.) cultivar. HortScience 51, 601-603 (2016).

[2] García, J. A., Glasa, M., Cambra, M. & Candresse, T. Plum pox virus and sharka: a model potyvirus and a major disease. Mol. Plant Pathol. 15, 226-241 (2014).

[3] Scorza, R. et al. Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene. Plant Cell Rep. 14, 18-22 (1994).

[4] Scorza, R. et al. Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene. Transgenic Res. 10, 201-209 (2001).

[5] Scorza, R. et al. Hairpin Plum pox virus coat protein (hpPPV-CP) structure in ‘HoneySweet’C5 plum provides PPV resistance when genetically engineered into plum (Prunus domestica) seedlings. Jul.-K.ühn-Arch. 427, 141 (2010).

[6] Guttikonda, S. K. et al. Molecular characterization of transgenic events using next generation sequencing approach. PLoS One 11, e0149515 (2016).

[7] Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210-3212 (2015).

[8] Jung, S. et al. 15 years of GDR: new data and functionality in the Genome Database for Rosaceae. Nucleic Acids Res. 47, D1137-D1145 (2018).

[9] Verde, I. et al. The Peach v2.0 release: high-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity. BMC Genomics 18, 225 (2017).

[10] Callahan, A. M., Dardick, C. D. & Scorza, R. Multilocation comparison of fruit composition for ‘HoneySweet’, an RNAi based plum pox virus resistant plum. PloS One 14, e0213993 (2019).

[11] Schnell, J. et al. A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments. Transgenic Res 24, 1-17 (2015).

[12] Li, R. et al. Molecular characterization of genetically-modified crops: challenges and strategies. Biotechnol. Adv. 35, 302-309 (2017).

[13] Pauwels, K. et al. Next-generation sequencing as a tool for the molecular characterisation and risk assessment of genetically modified plants: Added value or not? Trends Food Sci. Tech. 45, 319-326 (2015).

[14] Shirasawa, K. et al. Phased genome sequence of an interspecific hybrid flowering cherry, Somei-Yoshino (Cerasus x yedoensis). DNA Res. 26, 379-389 (2019).

[15] Siddique, K., Wei, J., Li, R., Zhang, D. & Shi, J. Identification of T-DNA insertion site and flanking sequence of a genetically modified maize event IE09S034 using next-generation sequencing technology. Mol. Biotechnol. 61, 694-702 (2019).

[16] Kawai, Y., Ono, E. & Mizutani, M. Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants. Plant J. 78, 328-343 (2014).

[17] Scorza, R., Callahan, A. M., Damsteegt, V., Levy, L. & Ravelonandro, M. Transferring potyvirus coat protein genes through hybridization of transgenic plants to produce plum pox virus resistant plums (Prunus domestica L.). Acta Hortic. 472, 421-428 (1997).

[18] Ravelonandro, M., Briard, P., Monsion, M. & Scorza, R. Stable transfer of the plum pox virus (ppv) capsid transgene to seedlings of two french cultivars ‘Prunier D’ente 303’ and ‘Quetsche 2906’, and preliminary results of PPV challenge assays. Acta Hortic. 577, 91-96 (2001).

[19] Crane, M. & Lawrence, W. Genetics of Garden Plants. 236 (The MacMillan Co, London, 1934).

[20] Rybin, W. A. Spontaneous and experimentally produced hybrids between blackthorn and cherry plum and the descent problem of the cultivated plum. Planta 25, 22-58 (1936).

[21] Zhebentyayeva, T. et al. Genetic characterization of worldwide Prunus domestica (plum) germplasm using sequence-based genotyping. Hortic. Res. 6, 12 (2019).

[22] Reales, A., Sargent, D. J., Tobutt, K. R. & Rivera, D. Phylogenetics of Eurasian plums, Prunus L. section Prunus (Rosaceae), according to coding and non-coding chloroplast DNA sequences. Tree Genet. Genomes 6, 37-45 (2010).

[23] Eryomine, G. V. New data on origin of Prunus domestica L. Acta Hortic. 283, 27-30 (1990).

[24] Kubisiak, T. L. et al. A transcriptome-based genetic map of Chinese chestnut (Castanea mollissima) and identification of regions of segmental homology with peach (Prunus persica). Tree Genet. Genomes 9, 557-571 (2013).

[25] Humann, J. L., Lee, T., Ficklin, S. & Main, D. in Gene Prediction: Methods and Protocols.(ed Martin Kollmar). Structural and Functional Annotation of Eukaryotic Genomes with GenSAS. (Humana Press, New York, 2019).

[26] Lowe, T. M. & Chan, P. P. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res. 44, W54-W57 (2016).

[27] Lagesen, K. et al. RNammer: consistent annotation of rRNA genes in genomic sequences. Nucleic Acids Res. 35, 3100-3108 (2007).

[28] Mitchell, A. L. et al. InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 47, D351-D360 (2018).

[29] Collum, T. D., Lutton, E., Raines, C. D., Dardick, C. & Culver, J. N. Identification of phloem-associated translatome alterations during leaf development in Prunus domestica L. Hortic. Res. 6, 16 (2019).

[30] Collum, T. D. et al. Translatome profiling of plum pox virus infected leaves in European plum reveals temporal and spatial coordination of defense responses in phloem tissues. Mol. Plant Microbe Interact. 36, 66-77 (2020).

[31] Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd edn, (Cold Spring Harbor Laboratory Press, NY USA, 1989).

[32] Verde, I. et al. The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat. Genet. 45, 487 (2013).

[33] Doyle, J. J. & Doyle, J. L. Isolation of plant DNA from fresh tissue. Focus 12, 39-40 (1990).

[34] Callahan, A. M., Morgens, P. H., Wright, P. & Nichols, K. E. Comparison of pch313 (pTOM13 homolog) RNA accumulation during fruit softening and wounding of two phenotypically different peach cultivars. Plant Physiol. 100, 482-488 (1992).