Poster presented at 2023 IS-MPMI Congress, Molecular Plant-Microbe Interactions, Providence, USA

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 is an oomycete responsible for potato late blight (Figure 1), adevastating disease that caused the Great Irish Famine in the 19th century. Nowadaysthe disease can be controlled by pesticides, but host resistance gains importance as anenvironment friendly alternative in disease management. All known genes conferringresistance to P. infestans (Rpi genes) belong to the coiled coil nucleotide-binding siteleucine-rich repeat (CC-NBS-LRR) gene family. Currently, more than 70 Rpi geneshave been identified in Solanum species and, nearly 50 Rpi genes have been cloned. However, we do not know which Rpi genes are present in potato cultivars, especiallysince the resistance conferred by many of them has been overcome by the rapidlyevolving pathogen.Diversity analysis of 11 potato Rpi genes (Table 1) in potatoes varying in ploidy and originusing an Amplicon Sequencing (AmpSeq) approach.Aim of studyMaterials and Methods• 335 potato genotypes (183 potato cultivars grown in Poland and Norway, 98 breedinglines 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 and sequencingusing the PacBio single-molecule real-time (SMRT) circular consensus sequencing(CCS) methodResults▪ Using PCR primers the presence of R3a, R3b, R1, Rpi-phu1, R2, Rpi-ber1 andRpi-blb1 genes were detected, so far in 69, 45, 40, 40, 24, 18 and 7 potatogenotypes, respectively.▪ The AmpSeq strategy is efficient, a library of, so far 243 different PCR products (7Rpi 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 2).▪ From 2 to 14 amplicon sequence variants (ASVs) per Rpi gene (includingpseudogenes) were detected in 243 potato genotypes (Figure 2).▪ The highest number of ASVs were detected in wild potato species (26), the lowestnumber of ASVs were detected in breeding lines (14; Figure 3).▪ 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 2).▪ The highest numbers of ASVs of different Rpi genes were detected in potatocultivars 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 previouslydetected in other studiesFigure 2 Number of detected ASVs, using PacBio single-molecule real-time(SMRT) circular consensus sequencing (CCS) method23648181395112361024681012141618R1 R2 R3a R3b Rpi-phu1Rpi-ber1 Rpi-blb1Number of detected ASVs Rpi genesPotato cultivars Wild potato species Breeding linesFigure 3 Number of detected ASVs in potato cultivars, wild potato speciesand breeding lines• The most frequently detected Rpi gene was R3a, foundin 69 potato genotypes. This was also the most diversegene.• The least frequently detected Rpi gene found in 7potato genotypes was Rpi-blb1.• The least diverse gene among the 7 sequenced wasthe R1 gene.• Data on the distribution and sequence diversity of theRpi genes can lead to discovery of new Rpi variants and facilitate breeding.• Further sequencing of the Rpi genes is in progress.Conclusions/Future plansAmplicon sequencing of NBS-LRR genes conferring resistance to Phytophthora infestans in potatoPaulina 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, Platanowa 19 Młochów, Poland2Norwegian Institute of Bioeconomy Research (NIBIO), Ås, Norway3Norwegian University of Life Sciences (NMBU), Ås, NorwayThe research leading to theseresults has received fundingfrom the Norwegian FinancialMechanism 2014-2021,project DivGene:UMO-2019/34/H/NZ9/00559Figure 1 The symptoms of late blight(Phytophthora infestans) infection in potato leavesCorresponding author Paulina Paluchowska, p.paluchowska@ihar.edu.pl 

Amplicon sequencing of NBS LRR genes conferring resistance to Phytophthora infestans in potato

https://zenodo.org/record/8369843/files/IS-MPMI_P.Paluchowska.pdf

Amplicon sequencing of NBS LRR genes conferring resistance to Phytophthora infestans in potato

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