Poster presented at 12TH INTERNATIONAL CONGRESS OF PLANT PATHOLOGY ICPP2023, Lyon, 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

Ludwiczewska, Mirella

Lysøe, Erik

Paluchowska, Paulina

Rossmann, Simeon L.

Yin, Zhimin

Śliwka, Jadwiga

English

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Phytophthora infestans causes late blight (Figure 1), a highly destructive potato diseasewhich has challenged global agriculture for centuries. Genes involved in resistance to P.infestans (Rpi genes) have been discovered in wild potato species (Solanum spp.), andsome of these Rpi genes have been introduced into potato cultivars. All known genesconferring resistance to P. infestans belong to the coiled coil nucleotide-binding siteleucine-rich repeat (CC-NBS-LRR) gene family. Currently, more than 70 Rpi genes havebeen identified in Solanum species and nearly 50 Rpi genes have been cloned. However,we do not know which Rpi genes are present in the many different potato genotypes,especially since the resistance conferred by many of them has been overcome by therapidly evolving pathogen.MATERIALS AND METHODS• 335 potato genotypes (183 potato cultivars grown in Poland and Norway (Figure 2), 98 breeding lines and 54 genotypes of wild potato species)• Detection of Rpi genes fragments using 1-3 PCR primer pairs per gene (Table 1). • Amplification of entire coding sequences of the Rpi genes, barcoding andsequencing using the PacBio single-molecule real-time (SMRT) circularconsensus sequencing (CCS) method.RESULTS▪ Using PCR primers the presence of R3a, R3b, R1, Rpi-phu1, R2, Rpi-ber1 and Rpi-blb1 genes were detected, so far in 69, 45, 40, 40, 24, 18 and 7 potato genotypes,respectively.▪ The AmpSeq strategy is efficient, each of two libraries (120 and 123 PCR products; 7 Rpi genes) yielded more than 4 million reads with an average length of 4.086 bp.Sequencing of the next 4 Rpi genes is in progress.▪ The presence of Rpi genes in different potato genotypes was confirmed and newvariants of these genes were detected using PacBio sequencing (Figure 3).▪ From 2 to 14 amplicon sequence variants (ASVs) per Rpi gene (includingpseudogenes) were detected in 243 potato genotypes (Figure 3).▪ The highest number of ASVs was detected in wild potato species (26), the lowestnumber of ASVs were detected in breeding lines (14; Figure 4).▪ From 1 to 5 ASVs were obtained per potato genotype.▪ The highest number of ASVs per gene was obtained for R3a, in total 14, while thelowest number of ASVs was obtained for the R1 gene (Figure 3).▪ Highest numbers of different Rpi genes were detected in potato cultivars Escort (R1, Rpi-abptT86, R3a, R3b, R3bG3111), Klepa (R1, R2-like, R3a-like, Rpp13-like, R3bG3111)and Rudawa (Rpi-abptT86, R3a, R3a-like, R3b, R3bG3111).# Rpi gene Gene source Reference sequence1 R1 Solanum demissum AF4474892 R2/Rpi-blb3 Solanum bulbocastanum FJ5363463 R3a Solanum tuberosum AY8493824 R3b Solanum demissum JF900492.15 R8 Solanum demissum KU5301536 R9 Solanum demissum Armstrong et al. 20197 Rpi-phu1 (Rpi-vnt1) Solanum phureja/Solanum venturii FJ423044.18 Rpi-blb1 Solanum bulbocastanum AY426259.19 Rpi-blb2 Solanum bulbocastanum DQ12212510 Rpi-ber1 Solanum berthaultii MW390806.111 Rpi-chc1.1 Solanum chacoense MW383255Rpi-chc1.2 MW410797Table 1 List of Rpi genes detected in potato genotypes2 212 211328950246810121416R1 R2 R3a R3b Rpi-phu1 Rpi-ber1 Rpi-blb1Number of detected ASVsRpi genesNumber of new gene variantsNumber of genes variants previously detected in other studies23648181395112361024681012141618R1 R2 R3a R3b Rpi-phu1 Rpi-ber1 Rpi-blb1Number of detected ASVs Rpi genesPotato cultivars Wild potato species Breeding linesFigure 4 Number of detected ASVs in potato cultivars, wild potato species and breeding linesCONCLUSION / FUTURE PLANS• The most frequently detected Rpi gene was R3a, found in 69 potatogenotypes. This was also the most diverse gene.• The least frequently detected Rpi gene found in 7 potato genotypes was Rpi-blb1.• The least diverse gene among the 7 sequenced was the R1 gene.• Data on the distribution and sequence diversity of the Rpi genes can lead todiscovery of new Rpi variants and facilitate breeding.• Further sequencing of the Rpi genes is in progress..The research leading to these results has received funding from the Norwegian Financial Mechanism 2014-2021, project DivGene: UMO-2019/34/H/NZ9/00559Figure 1 The symptoms of late blight (Phytophthorainfestans) infection in potato leavesHigh-throughput analysis of Rpi genes in potato cultivars, breeding lines and wild Solanum speciesPaulina Paluchowska1, Zhimin Yin1, Erik Lysøe2, Simeon L. Rossmann2, Mirella Ludwiczewska1, Marta Janiszewska1, May Bente Brurberg2,3 and Jadwiga Śliwka11Plant Breeding and Acclimatization Institute - National Research Institute (IHAR-PIB) in Radzików, Młochów Divison, Poland2Norwegian Institute of Bioeconomy Research (NIBIO), Ås, Norway3Norwegian University of Life Sciences (NMBU), Ås, NorwayFigure 2 Origin of the potato genotypes used in this studyFigure 3 Number of detected ASVs, using PacBio single-molecule real-time(SMRT) circular consensus sequencing (CCS) methodContact: p.paluchowska@ihar.edu.pl325016Breeding lines, Σ=98 452415519321153222Poland Norway Germany the Netherlands DenmarkHungary France Romania Scotland UKUSA Unknown SwedenPotato cultivars, Σ=18354Wild potato species, Σ=54 IHAR-PIB collectionAIM: Diversity analysis of 11 potato Rpi genes (Table 1) in potatoes varying inploidy and origin using an Amplicon Sequencing (AmpSeq) approach.

High throughput analysis of Rpi genes in potato cultivars, breeding lines and wild Solanum species

https://zenodo.org/record/8369228/files/ICPP%202023%20PaluchowskaPaulina.pdf

High throughput analysis of Rpi genes in potato cultivars, breeding lines and wild Solanum species

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