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Cited by 103Wheat and Barley Genetics and PathologyPowdery Mildew Fungal DiseasesPlant Disease Resistance and Genetics

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Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69

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Li, Yinghui, Wei, Zhen-Zhen, Sela, Hanan, Govta, Liubov, Klymiuk, Valentyna, Roychowdhury, Rajib, Chawla, Harmeet Singh, Ens, Jennifer, Wiebe, Krystalee, Bocharova, Valeria, Ben-David, Roi, Pawar, Prerna B., Zhang, Yuqi, Jaiwar, Samidha, Molnár, István, Doležel, Jaroslav, Coaker, Gitta, Pozniak, Curtis J., Fahima, Tzion·2024·Plant Communications
DOI 10.1016/j.xplc.2023.100646PMC PMC10811346

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Abstract

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

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Figure 1 Fine mapping of Pm69 derived from WEW G305-3M. (A) Infection types (ITs) of the WEW accession G305-3M (0, highly resistant), the T. durum line LDN (4, highly susceptible), and their F1 progeny (0, highly resistant) to Bgt #70 at 7 days post-infection (dpi); DAB staining of leaves at 3 dpi was performed to detect ROS accumulation, visualized as reddish-brown coloration; trypan blue staining of leaves at 3 dpi was performed to detect fungal structures and plant cell death, visualized as blue coloration. MH, mature haustorium; HP, haustorial primordium; Hy, hyphae. Scale bars: 50 μm. (B) The Pm69 gene region on the long arm of wheat chromosome 6B. (C) Locations of Pm69-flanking genetic markers on the Zavitan WEW_v.2.0 reference genome. (D) Genetic map of Pm69. The green markers were developed using WEW_v.2.0 sequences, and the blue markers were developed using ONT contigs. (E) G305-3M ONT assembly contigs mapped to the Pm69 gene region. The physical interval of the Pm69 gene region on ONT contigs is marked in blue.
Figure 1 Fine mapping of Pm69 derived from WEW G305-3M. (A) Infection types (ITs) of the WEW accession G305-3M (0, highly resistant), the T. durum line LDN (4, highly susceptible), and their F1 progeny (0, highly resistant) to Bgt #70 at 7 days post-infection (dpi); DAB staining of leaves at 3 dpi was performed to detect ROS accumulation, visualized as reddish-brown coloration; trypan blue staining of leaves at 3 dpi was performed to detect fungal structures and plant cell death, visualized as blue coloration. MH, mature haustorium; HP, haustorial primordium; Hy, hyphae. Scale bars: 50 μm. (B) The Pm69 gene region on the long arm of wheat chromosome 6B. (C) Locations of Pm69-flanking genetic markers on the Zavitan WEW_v.2.0 reference genome. (D) Genetic map of Pm69. The green markers were developed using WEW_v.2.0 sequences, and the blue markers were developed using ONT contigs. (E) G305-3M ONT assembly contigs mapped to the Pm69 gene region. The physical interval of the Pm69 gene region on ONT contigs is marked in blue.CC BY-NC-ND · PubMed Central
Figure 2 Comparisons of the G305-3M ONT contigs with the 6B pseudomolecule of tetraploid wheat genomes around the Pm69 gene region. (A) Wild emmer wheat Zavitan reference genome WEW_v.2.0. (B) Durum wheat Svevo RefSeq Rel. 1.0. The x axis: cumulative length of G305-3M contigs; the y axis: physical location of 6B pseudomolecule in the reference genomes. Contigs utg380–utg17163 marked with different colors belong to the ONT assembly of G305-3M.
Figure 2 Comparisons of the G305-3M ONT contigs with the 6B pseudomolecule of tetraploid wheat genomes around the Pm69 gene region. (A) Wild emmer wheat Zavitan reference genome WEW_v.2.0. (B) Durum wheat Svevo RefSeq Rel. 1.0. The x axis: cumulative length of G305-3M contigs; the y axis: physical location of 6B pseudomolecule in the reference genomes. Contigs utg380–utg17163 marked with different colors belong to the ONT assembly of G305-3M.CC BY-NC-ND · PubMed Central
Figure 3 Workflow for identification of Pm69 on the ONT contigs by MutRNA-seq. (A) Phenotypes of EMS-derived mutants (M-40, M-22, M-12, and M-18) and the wild type (G305-3M) infected with Bgt #70. M-40, M-22, M-12, M-18, and G305-3M were analyzed by RNA-seq to identify SNVs among the three ONT contigs. (B) Expressed genes in the Pm69 gene region were identified by aligning the RNA-seq reads to G305-3M ONT contigs. Vertical red lines: the locations of SNVs in the susceptible mutants identified after mapping RNA-seq reads to G305-3M ONT contigs. (C) Distribution of independent mutations. M-3 was identified by Sanger sequencing. (D) Predicted structure of the PM69 protein with the coiled-coiled Rx N-terminal domain (Rx_N), nucleotide-binding adaptor shared by Apaf-1, R proteins, and Ced-4 (NB-ARC) domain, leucine-rich repeat (LRR) domain, and precise location of each identified mutation.
Figure 3 Workflow for identification of Pm69 on the ONT contigs by MutRNA-seq. (A) Phenotypes of EMS-derived mutants (M-40, M-22, M-12, and M-18) and the wild type (G305-3M) infected with Bgt #70. M-40, M-22, M-12, M-18, and G305-3M were analyzed by RNA-seq to identify SNVs among the three ONT contigs. (B) Expressed genes in the Pm69 gene region were identified by aligning the RNA-seq reads to G305-3M ONT contigs. Vertical red lines: the locations of SNVs in the susceptible mutants identified after mapping RNA-seq reads to G305-3M ONT contigs. (C) Distribution of independent mutations. M-3 was identified by Sanger sequencing. (D) Predicted structure of the PM69 protein with the coiled-coiled Rx N-terminal domain (Rx_N), nucleotide-binding adaptor shared by Apaf-1, R proteins, and Ced-4 (NB-ARC) domain, leucine-rich repeat (LRR) domain, and precise location of each identified mutation.CC BY-NC-ND · PubMed Central
Figure 4 Functional validation of the Pm69 candidate gene by virus-induced gene silencing (VIGS). (A) VIGS of the Pm69 candidate gene in G305-3M. (B) VIGS of the Pm69 candidate gene in the introgression line (Ruta+Pm69). BSMV:GFP was used as a negative control. BSMV:wild type, the wild type of the pCa-BSMV-γ vector, was used as a negative control. BSMV:PDS is the PDS gene-silencing construct used to check the efficiency of the VIGS system; BMSV:Pm69-1 and Pm69-2 are the constructs that targeted the Rx N-terminal domain and the NB-ARC domain, respectively. Asterisks indicate significant differences in Pm69 expression levels in the 4th leaf segments of target plants compared with the negative control (BSMV:wild type) one week post-virus infection, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t-test). (C) Histopathology characterization of Pm69 wild-type plants (BSMV:GFP) and Pm69-silenced plants (BSMV:Pm69-1 and BSMV:Pm69-2). DAB and Coomassie brilliant blue staining of Pm69- and GFP-silenced leaves of G305-3M at 7 dpi with Bgt #70. Scale bars: 100 μm.
Figure 4 Functional validation of the Pm69 candidate gene by virus-induced gene silencing (VIGS). (A) VIGS of the Pm69 candidate gene in G305-3M. (B) VIGS of the Pm69 candidate gene in the introgression line (Ruta+Pm69). BSMV:GFP was used as a negative control. BSMV:wild type, the wild type of the pCa-BSMV-γ vector, was used as a negative control. BSMV:PDS is the PDS gene-silencing construct used to check the efficiency of the VIGS system; BMSV:Pm69-1 and Pm69-2 are the constructs that targeted the Rx N-terminal domain and the NB-ARC domain, respectively. Asterisks indicate significant differences in Pm69 expression levels in the 4th leaf segments of target plants compared with the negative control (BSMV:wild type) one week post-virus infection, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t-test). (C) Histopathology characterization of Pm69 wild-type plants (BSMV:GFP) and Pm69-silenced plants (BSMV:Pm69-1 and BSMV:Pm69-2). DAB and Coomassie brilliant blue staining of Pm69- and GFP-silenced leaves of G305-3M at 7 dpi with Bgt #70. Scale bars: 100 μm.CC BY-NC-ND · PubMed Central
Figure 5 Geographic distribution of the Pm69 allele in WEW populations. (A) Geographic distribution of WEW collections (black circles) that were screened for the presence of the Pm69 functional allele. (B) Geographic distribution of individual WEW plants collected near the recorded G305-3M collection site (marked by a blue triangle, south of Kadita, Northern Israel). (C) A submap of (B) shows that only three accessions contained Pm69. The maps were obtained from Google Maps. (D) Phenotypes in response to Bgt #70 and agarose gel electrophoresis of PCR products amplified by markers uhw403 (549 bp for Pm69) and M-Pm60-S1 (831 bp for TdPm60) from representative WEW accessions.
Figure 5 Geographic distribution of the Pm69 allele in WEW populations. (A) Geographic distribution of WEW collections (black circles) that were screened for the presence of the Pm69 functional allele. (B) Geographic distribution of individual WEW plants collected near the recorded G305-3M collection site (marked by a blue triangle, south of Kadita, Northern Israel). (C) A submap of (B) shows that only three accessions contained Pm69. The maps were obtained from Google Maps. (D) Phenotypes in response to Bgt #70 and agarose gel electrophoresis of PCR products amplified by markers uhw403 (549 bp for Pm69) and M-Pm60-S1 (831 bp for TdPm60) from representative WEW accessions.CC BY-NC-ND · PubMed Central
Figure 6 Microcollinearity analysis of the Pm69 NLR cluster region among different Triticeae genomes. Black lines connect genes with high collinearity, and blue lines connect Pm69 and its homologs. The numbers on the yellow NLRs represent different NLR clades based on the evolutionary analysis. NLRs with the same number probably evolved from duplication events. Dark blue indicates Pm69, and light blue indicates pm69 homologs. Red triangles mark the locations of Pm69 and its most similar homologs. Pink indicates OPR11 homologs that are more conserved than the Pm69 homologs among different genomes. Green indicates other non-NLR proteins. The NLRs show greater genetic diversity than their flanking non-NLR proteins.
Figure 6 Microcollinearity analysis of the Pm69 NLR cluster region among different Triticeae genomes. Black lines connect genes with high collinearity, and blue lines connect Pm69 and its homologs. The numbers on the yellow NLRs represent different NLR clades based on the evolutionary analysis. NLRs with the same number probably evolved from duplication events. Dark blue indicates Pm69, and light blue indicates pm69 homologs. Red triangles mark the locations of Pm69 and its most similar homologs. Pink indicates OPR11 homologs that are more conserved than the Pm69 homologs among different genomes. Green indicates other non-NLR proteins. The NLRs show greater genetic diversity than their flanking non-NLR proteins.CC BY-NC-ND · PubMed Central
Figure 7 Introgression of Pm69 into cultivated wheat and pyramiding with a yellow rust resistance gene. (A) Phenotypes of different wheat parental and introgression lines to Bgt #70 and Pst #5006. WEW: G305-3M contains Pm69 and Yr15, G25 contains Yr15. Durum wheat: LDN and Svevo. Common wheat: Ruta and AvocetYr15. (B) Whole-plant growth habits of G305-3M, Ruta, and the representative introgression line Ruta+Pm69. The length of the ruler is 32 cm.
Figure 7 Introgression of Pm69 into cultivated wheat and pyramiding with a yellow rust resistance gene. (A) Phenotypes of different wheat parental and introgression lines to Bgt #70 and Pst #5006. WEW: G305-3M contains Pm69 and Yr15, G25 contains Yr15. Durum wheat: LDN and Svevo. Common wheat: Ruta and AvocetYr15. (B) Whole-plant growth habits of G305-3M, Ruta, and the representative introgression line Ruta+Pm69. The length of the ruler is 32 cm.CC BY-NC-ND · PubMed Central

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  1. McIntosh, 2020, Catalogue of gene symbols for wheat: 2020 supplement, Annu. Wheat Newsl., 66, 116

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Metadata

Authors
Li, Y., Wei, Z.-Z., Sela, H., Govta, L., Klymiuk, V., Roychowdhury, R., Chawla, H. S., Ens, J., Wiebe, K., Bocharova, V., Ben-David, R., Pawar, P. B., Zhang, Y., Jaiwar, S., Molnár, I., Doležel, J., Coaker, G., Pozniak, C. J., & Fahima, T.
Affiliations
  • University of Haifa (IL)
  • Sichuan Academy of Agricultural Sciences (CN)
  • University of Saskatchewan (CA)
  • China Agricultural University (CN)
  • Czech Academy of Sciences, Institute of Experimental Botany (CZ)
  • University of California, Davis (US)
Corresponding author
Fahima, Tzion
ORCID
Venue
Plant Communications
Publisher
Elsevier BV
Published
2024-01
Volume
5
Issue
1
Pages
100646
ISSN
2590-3462 (ISSN-L)
DOI
10.1016/j.xplc.2023.100646
PMC
PMC10811346
OpenAlex
W4383197277
Cited by
103
References
102
Citations by year
2026: 25 · 2025: 26 · 2024: 42
FWCI
16.23
Type
journal article
Language
en
Open access
Licence
Locations
  • Plant Communications (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc-nd)
Funders
  • Genome Canada
  • United States-Israel Binational Science Foundation (2019654)
  • Israel Science Foundation (1366/18, 2289/16, 2342/18)
  • National Science Foundation (1937855)
  • Council for Higher Education
  • European Regional Development Fund (CZ.02.1.01/0.0/0.0/16_019/0000827)
  • United States - Israel Binational Science Foundation
  • U.S. Department of Agriculture (2020-67013-32577)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Wheat and Barley Genetics and Pathology
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Powdery Mildew Fungal Diseases
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Disease Resistance and Genetics
Keywords
powdery mildew resistance, disease resistance genes, NLR, nucleotide-binding leucine-rich repeat, wild emmer wheat, tetraploid wheat, long-read genome sequencing, Oxford Nanopore, physical map, suppressed recombination, structural variants, virus-induced gene silencing, candidate gene identification
MeSH terms
Chromosome Mapping, Cloning, Molecular, Multigene Family, Triticum, Genes, Plant

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