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Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing

Abstract

Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow1,2,3. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine resistance (R) gene sequence capture (RenSeq)4 with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone Rpi-amr3i. This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.

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Figure 1: Comparison of MiSeq and PacBio RenSeq read-based assemblies.
Figure 2: The phylogenetic tree of SP2271 NLRs.
Figure 3: Candidate gene Rpi-amr3i confers full resistance against P. infestans in transient complementation assays in N. benthamiana and in stable transgenic potato plants.

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Primary accessions

European Nucleotide Archive

NCBI Reference Sequence

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Acknowledgements

We would like to thank S. Knapp, S. Kamoun, B. Wulff, B. Steuernagel, G. Rallapalli, O. Furzer and N. Champouret for useful discussions, The Genome Analysis Centre (TGAC) for excellent PacBio service, L. Tomlinson and W. Barrett for technical assistance, P. Lindhout for Solynta potato lines, S. Perkins and J. Campling for maintenance of plants in the greenhouse, M. Smoker and J. Pike for plant transformation, V. Vleeshouwers for the R2 construct, TSL Bioinformatics Support Team and specifically C. Schudoma for bioinformatics assistance, and A. Davis for photography. This work was financed from BBSRC grants nos BB/H019820/1, BB/G02197X/1, BB/L011794/1, BB/L009757/1, BB/J010375/1 and the Gatsby Charitable Foundation. This research was supported in part by the NBI Computing infrastructure for Science (CiS) group through the provision of a High Performance Computing Cluster. P. infestans isolate MP324 (ref. 25) was provided by J. Śliwka from the Plant Breeding and Acclimatization Institute – National Research Institute, Młochów Centre (Poland).

Author information

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Authors

Contributions

K.W., F.J. and J.D.G.J. designed the study. K.W. and A.I.W. performed the experiments. K.W., F.J. and A.I.W. analyzed the data. M.D.C. and D.B. contributed to SMRT RenSeq protocol development. K.W., F.J. and J.D.G.J. wrote the manuscript. K.W. and F.J. made equivalent contributions and should be considered joint first authors.

Corresponding authors

Correspondence to Kamil Witek or Jonathan D G Jones.

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Competing interests

Kamil Witek and Jonathan D.G. Jones have filed a US patent application 62/159,240 based on this work. Matthew D. Clark owns shares in Pacific Biosciences of California.

Integrated supplementary information

Supplementary Figure 1 Resistance in S. americanum accession SP1102 is physically linked to the R2/Rpi-blb3, C17 and C18 DM reference genome loci

(a) 10-week-old S. americanum plants were inoculated with P. infestans strain 88069. Top to bottom: accession 954750186 (working name SP2271) is susceptible to the tested isolates; accessions 954750174 (SP2272), 954750184 (SP2273) and 944750095 (SP1102) are resistant. Each leaf was inoculated with 4-6 droplets containing 500 zoospores each; photographs were taken 7 dpi. Scale bar indicates 1cm. This experiment was repeated at least three times with similar results (b) Bulked segregant analysis coupled with RenSeq enables positioning of Rpi-amr3 on DM reference genome chromosome 4. 50 susceptible F2 and F3 plants were genotyped with markers derived from Whole Genome Shotgun sequencing (WGS) data. Two flanking markers (WGS_1_4 at 3.59Mb and WGS_2_4 at 8.69Mb), and eight co-segregating with resistance (WGS markers), were identified. Physical positions of NLR genes and markers are given in Mb, based on the reference genome. Solid areas mark NLR cluster locations. (c) Genotyping and phenotyping of 405 plants from a selfed backcross population (BC1F2) confirmed the location of Rpi-amr3 within the C18 locus (10 NLRs in reference genome), flanked by markers WGS_2_5 (6.61Mb) and WGS_1_11 (7.99Mb), Numbers in brackets indicate number of recombinants out of all screened plants. (d) Sequence comparisons of PacBio-RenSeq derived de novo assembly with members of the C18 locus identified four homologous sub groups, designated Rpi-amr3 candidates a-n. Asterisk marks expressed candidates.

Supplementary Figure 2 An example of chimeric MiSeq assembly of 2x250bp SP1102 RenSeq data

SPAdes assembled MiSeq sequence contigs were aligned to PacBio derived contigs with high stringency (Geneious). Top (red) and bottom (black) – PacBio contigs, middle – chimeric MiSeq contig with marked regions fitting PacBio contigs (black and red). Above and below SPAdes NODE_643 identity to PacBio Contig_105 and Contig_238 are shown, respectively. Grey area in SPAdes contig marks region of NB-ARC domain with 99% identity to both PacBio contigs (240nt), which is a part of larger fragment (650nt) displaying 96% identity between PacBio contigs. Overall identity between PacBio contigs (ORF region) is 85% on nucleotide level. ORFs are marked with arrows; NLR typical protein domains: coiled-coil (CC; purple box), nucleotide binding site (NB-ARC; blue box) and leucine-rich repeats (LRR; green box). Fused protein domain DUF 3542 is shown as light purple box. Drawn to scale.

Supplementary Figure 3 Confirmation of continuity and accuracy of the PacBio ROI assembled Contig_7

Whole Genome Shotgun (WGS) Illumina 100bp paired-end data were mapped to Contig_7 (Supplementary File 2) using BWA with default settings and then filtered with SAM tools for correctly mapped read pairs. Mapping results were visualized in Savant Genome Browser and inspected manually. No signatures of misassembly or indels larger than a single nucleotide (typically errors in homopolymeric regions) were noticed. Single visible SNPs (up to 50% in nucleotide ratio) in the ORF result from non-specific read mapping from close paralogs. From top to bottom: graphical representation of Contig_7 reference sequence as assembled from PacBio ROI data; ORF predicted with Geneious R8 (blue bars); WGS coverage data, blue color = perfect coverage, various color bars = SNPs or single indels, black line marks 30x coverage; WGS data read pairing, blue color = proper pairing, no discordant, everted or unmapped pairs present.

Supplementary Figure 4 Error rate of ROI reads and resulting assemblies.

(a) Box plot showing accuracy of ROI reads divided into bins based on their length (500 bp intervals). ROI reads that were used to de novo assemble the C18 clusters of resistant and susceptible parents, were re-aligned to the error corrected contigs. The percentage of accuracy was calculated for each individual ROI read based on the pairwise identity. (b) Bar graph showing total number of errors per ROI read coverage over de novo assembled contigs. WGS data was mapped to de novo assembled contigs of the C18 cluster and the mapping data was checked for errors. Colors represent the four bases.

Supplementary Figure 5 Transient complementation assays in Nicotiana benthamiana with six Rpi-amr3 candidates

Third leaves of N. benthamiana plants were infiltrated with the binary vector pICSLUS0003::35S overexpressing either the late blight resistance gene R2 (positive control), six Rpi-amr3 candidates, Rpi-amr3i-S cloned from the susceptible accession 2271 or GFP (negative control). Leaves are 24 hours later inoculated with P. infestans strain 88069. Only leaves infiltrated with R2 and Rpi-amr3i remained infection free, while P. infestans was able to proliferate on the remaining Rpi-amr3 candidates, the susceptible allele of Rpi-amr3i-S as well as the GFP control. Scale bar indicates 1cm. These experiments were repeated twice with similar results.

Supplementary Figure 6 Genomic construct with Rpi-amr3i confers resistance against P. infestans in a transient complementation assay in N. benthamiana.

The Rpi-amr3i construct with native promoter and terminator, restricts P. infestans growth to the same level as under control of the 35S promoter. A vector overexpressing GFP was used as a negative control. The experiment was performed as described previously; photographs were taken after 6 days. Scale bar indicates 1cm. This experiment was repeated twice with similar results.

Supplementary Figure 7 Stable transgenic plants carrying Rpi-amr3i under the regulation of a 35S promoter display resistance to all tested isolates

Transgenic diploid potato “Line 26” (Solynta B.V.) that express Rpi-amr3i under the 35S promoter are resistant to P. infestans isolates 88069 (upper), 06_3928A (middle) and EC3527 (bottom; right panel). The transgenic line displays no to weak HR at the place of inoculation. In contrast, transgenic plants carrying the non-functional candidate Rpi-amr3a (left panel) showed large necrotic lesions and sporulation. Each leaflet was inoculated with a droplet containing 500 spores; photographs were taken 6 dpi. Scale bar indicates 1cm. These experiments were repeated twice with similar results.

Supplementary Figure 8 Relative levels of Rpi-amr3i expression under native regulatory elements in transgenic potato plants.

Relative copy number of Rpi-amr3i mRNAs per 1 million copies of EF1 mRNA internal control. Expression levels similar to wild-type Rpi-amr3i mRNA (lines 3-7) correspond to full resistance. Level of expression was measured in fully grown leaves from 10 and 14 week-old plants for Solynta transgenic and WT lines and in 12-week-old SP1102. Errors bars show standard deviation based on two time points. Primers show high specificity, as no amplification was observed in Solynta WT plants.

Supplementary Figure 9 Amino acid sequence of Rpi-amr3i with highlited conserved domains.

Conserved domains within the sequence are highlighted; coiled-coil, purple; NB-ARC, blue; leucine-rich repeats, green.

Supplementary Figure 10 Rpi-amr3i establishes a new branch in the phylogeny of cloned functional Solanaceae R genes

(a) The Rpi-amr3 gene family forms a separate branch among previously cloned R genes. Gpa2/Rx represent the closest clade, members share however less than 35% amino-acid sequence identity. This maximum likelihood tree is based on the alignment of the full length amino-acid sequences of various functional Solanaceae NLR resistance genes, and Caenorhabditis elegans protein CED4 as outgroup. The numbers at nodes represent their bootstrap support (% support out of 100 bootstraps). (b) The phylogenetic tree of C18 cluster of R and S parents was constructed as described above, using all NLRs mapping to this cluster. C18 cluster members of resistant parent are named Rpi-amr3a-n, names of paralogs from susceptible parent start with NLR followed by number (see Supplementary File 7 for full NLR list).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–9 (PDF 4536 kb)

Supplementary Dataset 1

Fasta-file containing all marker sequences used to position and map the Rpi-amr3 gene. (TXT 23 kb)

Supplementary Dataset 2

Fasta file containing Contig_7. (TXT 7380 kb)

Supplementary Dataset 3

Fasta-file containing NB-ARC domains of all genes used for phylogenetic tree construction. (TXT 121 kb)

Supplementary Dataset 4

Fasta file containing complete NLRs of SP2271 accession annotated with MAST search output. (TXT 4962 kb)

Supplementary Dataset 5

MAST search result for SP2271 NLRs loci. (TXT 55 kb)

Supplementary Dataset 6

Annotated sequence of the pICSLUS0003 vector in GeneBank format. (TXT 10 kb)

Supplementary Dataset 7

Annotated sequence of the pICSLUS0001 vector in GeneBank format. (TXT 8 kb)

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Witek, K., Jupe, F., Witek, A. et al. Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing. Nat Biotechnol 34, 656–660 (2016). https://doi.org/10.1038/nbt.3540

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