Poster presented at EAPR Pathology & Pests Section Meeting, Arras, France

The research leading to these results has received funding from the Norwegian Financial Mechanism 2014-2021, project DivGene: UMO2019/34/H/NZ9/0055

Brurberg, May Bente

Janiszewska, Marta

Lysøe, Erik

Michalak, Krystyna

Paluchowska, Paulina

Rossmann, Simeon L.

Yin, Zhimin

Śliwka, Jadwiga

English

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PacBio amplicon sequencing of Rysto homologues in wild potato speciesPaulina Paluchowska1, Erik Lysøe2, Simeon Lim Rossmann2, Marta Janiszewska1, Krystyna Michalak1, May Bente Brurberg2,3, Jadwiga Śliwka1, Zhimin Yin11Plant Breeding and Acclimatization Institute - National Research Institute (IHAR-PIB) in Radzikow, Młochów Divison, Poland2Norwegian Institute of Bioeconomy Research (NIBIO), Ås, Norway3Norwegian University of Life Sciences (NMBU), Ås, NorwayFig. 1 Solanum stoloniferum donor of gene Rysto(https://ics.hutton.ac.uk/germinate-cpc/#/home)The research leading to these results has received funding from the Norwegian Financial Mechanism 2014-2021, project DivGene: UMO-2019/34/H/NZ9/00559Potato virus Y (PVY) is among the top ten economically important plant viruses. It causes potato tuber necrotic ringspot disease, leading todecreased yield and tuber quality. Wild relatives of potato are valuable sources of genes involved in resistance to many pathogens thatattack potatoes, including PVY. The gene Rysto derived from Solanum stoloniferum (Fig. 1) confers extreme resistance to PVY.Fig.5 Phylogenetic tree of Rysto homologous proteins. Rysto protein and the obtained Rysto-like proteins with 100% identity are marked in bold (red). The Rysto-like homologous proteins with 100% identity to each other within a branch are marked in bold (blue). Other proteins from Solanaceae are marked with green.Protein sequences were aligned using the ClustalW program. The phylogenetic tree is constructed using FastTree2 tool used minimum-evolution subtree-pruning-regrafting (SPRs) and maximum-likelihood nearest-neighbor interchanges (NNIs). XP016552770.2 - Capsicum annum /disease resistance proteinRoq1-like; XP006367311.2 - S. tuberosum/TMV resistance protein-like; XP027769684.1 - S. pennellii/TMV resistance protein-like; KAH0736372.1 S.tuberosum/hypothetical protein; KAG5573837.1 - S. commersonii/hypothetical protein; XP049377448.1- S. stenotomum/disease resistance protein Roq1-like;QEL52751.1/QEL52752.1- S. stoloniferum / Rysto protein – dominant/less abundant isoform.AIM:To screen Rysto homologues and to analyze their diversity in wild relatives of potato.PLANT MATERIAL: 298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species, IHAR-PIB’s collection.RESULTS:• A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of ten tuber-bearing Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363) (Figs. 1 and 3). • From 1 to 13 Rysto-like sequences were detected per Solanum sp. accession,between 1 and 8 Rysto-like variants were detected per potato genotype. In resistantcontrols, the number of Rysto-like sequences varied from 1 to 3.• Nucleotide identity of the obtained 55 Rysto-like sequences to that of the referenceRysto gene (MN393235.1, Grech-Baran et al. 2020) ranged from 89.87% to 99.98%.The Rysto-like sequences originated from primer pair T showed higher levels ofnucleotide diversity than that originated from primer pairs V and U, with the greatestdiversity found in the third intron (Fig. 4 ).• In total 45 unique protein sequences were predicted (Fig. 5). Two of them, Rysto-like26 identified in potato cultivars Alicja, Bzura, White Lady and Rysto-like16 inpotato clone PW363, showed 100% identity to the Rysto reference protein. Identity ofthe remaining 43 predicted Rysto-like proteins to the reference protein ranged from76.41 to 99.92 %. TIR and NB-ARC domains showed the highest level ofconservation, while LRR and C-JID domains were found to be most variable.CONCLUSION:• This study confirmed the presence of Rysto gene in potato cultivars Alicja, Bzura, White Lady and breeding linePW363. Two variants were found in Alicja in addition to variant described by Grech-Baran et al. (2020).• Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in thetested potato clone and cultivars.• Inter-species and inter-genotype variability of the Rysto-like sequences in the studied tuber-bearing Solanumspecies were observed, in respect to the number of variants identified, composition of the variants and contentof individual variants.METHOD: PacBio amplicon sequencing of the full coding sequences of Rysto homologues using barcoded primer pairs V, U and T (Fig. 2).The sequencing service was provided by the Norwegian Sequencing Centre (www.sequencing.uio.no).Fig. 2 PCR products obtained with primer pairs V, U and T covering Rysto geneReferences:Grech-Baran M, Witek K, Szajko K, Witek AI, Morgiewicz K, Wasilewicz-Flis I, Jakuczun H, Marczewski W, Jones JDG, Hennig J. (2020) Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor. Plant Biotechnol J. 18:655-667. Fig. 3 Resistance of tuber-bearing Solanum species to potato virus Y (PVY). aem, S. aemulans; ant, S.antipovichii; arp, S. arrac-papa; fen, S. fendleri; hou, S. hougasii; nnt, S. neoantipovichii; pta, S. papita;plt, S. polytrichon; ver, S. verrucosum.Fig. 4 Nucleotide diversity (Pi) of the Rysto homologues. Pi values rangebetween 1 (very diverse) and 0 (conserved). (a) 25 variants obtained withU and V primers; (b) 30 variants obtained with T primer (without the ATGstart codon). N-terminal domain homologous to the Drosophila Tolldomain and human interleukin-1 receptor (TIR; blue); leucine-richrepeats (LRR; purple); nucleotide-binding domain (NB-ARC; green); C-terminal jelly roll/Ig-like domain (C-JID; orange). START (2518) STOP (7279)(5065 bp)(4942 bp)(4772 bp)

PacBio amplicon sequencing of Rysto homologues in wild potato species

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PacBio amplicon sequencing of Rysto homologues in wild potato species

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