Genomes of the wild beets Beta patula and Beta vulgaris ssp. maritima

We present draft genome assemblies of Beta patula, a critically endangered wild beet endemic to the Madeira archipelago, and of the closely related Beta vulgaris ssp. maritima (sea beet). Evidence‐based reference gene sets for B. patula and sea beet were generated, consisting of 25 127 and 27 662 genes, respectively. The genomes and gene sets of the two wild beets were compared with their cultivated sister taxon B. vulgaris ssp. vulgaris (sugar beet). Large syntenic regions were identified, and a display tool for automatic genome‐wide synteny image generation was developed. Phylogenetic analysis based on 9861 genes showing 1:1:1 orthology supported the close relationship of B. patula to sea beet and sugar beet. A comparative analysis of the Rz2 locus, responsible for rhizomania resistance, suggested that the sequenced B. patula accession was rhizomania susceptible. Reference karyotypes for the two wild beets were established, and genomic rearrangements were detected. We consider our data as highly valuable and comprehensive resources for wild beet studies, B. patula conservation management, and sugar beet breeding research.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

The NLRomes of Zea mays NAM founder lines and Zea luxurians display presence–absence variation,integrated domain diversity,and mobility

Plant pathogens cause significant crop loss worldwide, and new resistance genes deployed to combat diseases can be overcome quickly. Understanding the existing resistance gene diversity within the germplasm of major crops, such as maize, is crucial for the development of new disease-resistant varieties. We analysed the nucleotide-binding leucine-rich repeat receptors (NLRs) of 26 recently sequenced diverse founder lines from the maize nested association mapping (NAM) population and compared them to the R gene complement present in a wild relative of maize, Zea luxurians. We found that NLRs in both species contain a large diversity of atypical integrated domains, including many domains that have not previously been found in the NLRs of other species. Additionally, the single Z. luxurians genome was found to have greater integrated atypical domain diversity than all 26 NAM founder lines combined, indicating that this species may represent a rich source of novel resistance genes. NLRs were also found to have very high sequence diversity and presence–absence variation among the NAM founder lines, with a large NLR cluster on Chr10 representing a diversity hotspot. Additionally, NLRs were shown to be mobile within maize genomes, with several putative interchromosomal translocations identified.     

DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.     

Genome‐wide association study identifies an NLR gene that confers partial resistance to Magnaporthe oryzae in rice

Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C‐RDP‐II), which contains 584 rice accessions and are genotyped with 700 000 single‐nucleotide polymorphism (SNP) markers. The C‐RDP‐II accessions were inoculated with three blast strains collected from different rice‐growing regions in China. Genome‐wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide‐binding site leucine‐rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up‐regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR‐Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non‐strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice.     

A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome‐arm identification,integration of genetic linkage groups and analysis of major repeat family distribution

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North–South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome‐specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S‐5.8S‐25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber‐FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.     

Cell death triggering and effector recognition by Sw‐5 SD‐CNL proteins from resistant and susceptible tomato isolines to Tomato spotted wilt virus

Only a limited number of dominant resistance genes acting against plant viruses have been cloned, and further functional studies of these have been almost entirely limited to the resistance genes Rx against Potato virus X (PVX) and N against Tobacco mosaic virus (TMV). Recently, the cell‐to‐cell movement protein (NS_M) of Tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant (Avr) of Sw‐5b‐mediated resistance, a dominant resistance gene which belongs to the class of SD‐CC‐NB‐LRR (Solanaceae domain‐coiled coil‐nucleotide‐binding‐leucine‐rich repeat, SD‐CNL) resistance genes. On transient expression of the NS_M protein in tomato and transgenic Nicotiana benthamiana harbouring the Sw‐5b gene, a hypersensitive cell death response (HR) is triggered. Here, it is shown that high accumulation of the Sw‐5b protein in N. benthamiana leaves, achieved by co‐expression of the Sw‐5b protein with RNA silencing suppressors (RSSs), leads to auto‐activity in the absence of NS_M. In a similar approach, Sw‐5a, the highest conserved paralogue of Sw‐5b from Solanum peruvianum, also triggered HR by auto‐activation, whereas the highest conserved orthologue from susceptible S. lycopersicum, named Sw‐5a^S, did not. However, neither of the last two homologues was able to trigger an NS_M‐dependent HR. Truncated and mutated versions of these Sw‐5 proteins revealed that the NB‐ARC [nucleotide‐binding adaptor shared by Apaf‐1 (from humans), R proteins and CED‐4 (from nematodes)] domain is sufficient for the triggering of HR and seems to be suppressed by the SD‐CC domain. Furthermore, a single mutation was sufficient to restore auto‐activity within the NB‐ARC domain of Sw‐5a^S. When the latter domain was fused to the Sw‐5b LRR domain, NS_M‐dependent HR triggering was regained, but not in the presence of its own Sw‐5a^S LRR domain. Expression analysis in planta revealed a nucleocytoplasmic localization pattern of Sw‐5b, in which the SD‐CC domain seems to be required for nuclear translocation. Although the Sw‐5 N‐terminal CC domain, in contrast with Rx, contains an additional SD, most findings from this study support a conserved role of domains within NB‐LRR (NLR) proteins against plant viruses.     

The Absence of TIR-Type Resistance Gene Analogues in the Sugar Beet (Beta vulgaris L.) Genome

The majority of known plant resistance genes encode proteins with conserved nucleotide-binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 27 genomic and 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. The clones were classified into three subfamilies based on nucleotide sequence identity. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic analysis revealed an ancestral relationship among sugar beet nlRGAs and resistance genes from various angiosperm species. One group appeared to share the same common ancestor as Prf, Rx, RPP8, and Mi, whereas the second group originated from the ancestral gene from which 12C1, Xa1, and Cre3 arose. The predicted protein products of the nlRGAs isolated in this study are all members of the non-TIR-type resistance gene subfamily and share strong sequence and structural similarities with non-TIR-type resistance proteins. No representatives of the TIR-type RGAs were detected either by PCR amplification using TIR type-specific primers or by in silico screening of more than 16,000 sugar beet ESTs. These findings suggest that TIR type of RGAs is absent from the sugar beet genome. The possible evolutionary loss of TIR type RGAs in the sugar beet is discussed. These authors (Yanyan Tian, Longjiang Fan) contributed equally to this work.     

Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1

Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide‐binding leucine‐rich repeat (NLR) protein with a Toll‐like interleukin‐1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely‐related Nicotiana sylvestris and the distantly‐related beet plant (Beta vulgaris). Roq1 was found to co‐immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.     

Genetic localization of four genes for nematode (Heterodera schachtii Schm.) resistance in sugar beet (Beta vulgaris L.)

Sugar beet (Beta vulgaris L.) is highly susceptible to the beet cyst nematode (Heterodera schachtii Schm.). Three resistance genes originating from the wild beets B. procumbens (Hs1 ^pro-1) and B. webbiana (Hs1 ^web-1, Hs2 ^web-7) have been transferred to sugar beet via species hybridization. We describe the genetic localization of the nematode resistance genes in four different sugar beet lines using segregating F₂ populations and RFLP markers from our current sugar beet linkage map. The mapping studies yielded a surprising result. Although the four parental lines carrying the wild beet translocations were not related to each other, the four genes mapped to the same locus in sugar beet independent of the original translocation event. Close linkage (0–4.6 cM) was found with marker loci at one end of linkage group IV. In two populations, RFLP loci showed segregation distortion due to gametic selection. For the first time, the non-randomness of the translocation process promoting gene transfer from the wild beet to the sugar beet is demonstrated. The data suggest that the resistance genes were incorporated into the sugar beet chromosomes by non-allelic homologous recombination. The finding that the different resistance genes are allelic will have major implications on future attempts to breed sugar beet combining the different resistance genes.     

Evolution and selection of Rhg1, a copy‐number variant nematode‐resistance locus

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2‐kb unit contains four genes. One allele of Rhg1, Rhg1‐b, is responsible for protecting most US soybean production from SCN. Whole‐genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2‐kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high‐density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non‐neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1.     

High-resolution mapping of YACs and the single-copy gene Hs1pro−1 on Beta vulgaris chromosomes by multi-colour fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful approach for physical mapping of DNA sequences along plant chromosomes. Nematode-resistant sugar beets (Beta vulgaris) carrying aBeta procumbens translocation were investigated by FISH with two differentially labelled YACs originating from the translocation. At mitotic metaphases, the translocation was identified with both YACs in the terminal region on a pair of chromosomes. Meiotic chromosomes, representing a far more extended hybridization target, were used to determine the orientation of YACs with respect to chromosomal domains in combination with chromosomal landmark probes for telomeres and centromeres. The in situ detection of plant single-copy sequences is technically difficult, and the wild beet translocation was used to explore the potential resolution of the FISH approach and to introduce the chromosomal mapping of single-copy genes into genome analysis of Beta species. An internal fragment of the nematode resistance gene Hs1 ^pro–1, 684 bp long, was detected on both chromatids of different Beta chromosomes and represents one of the shortest unique DNA sequences localized on mitotic plant chromosomes so far. Comparative chromosomal mapping of the 684 bp Hs1 ^pro–1 probe in the translocation line, a monosomic addition line and in B. procumbens revealed the origin of the wild beet translocation leading to nematode-resistant sugar beets.     

Characterization of disease resistance genes in the Brassica napus pangenome reveals significant structural variation

Methods based on single nucleotide polymorphism (SNP), copy number variation (CNV) and presence/absence variation (PAV) discovery provide a valuable resource to study gene structure and evolution. However, as a result of these structural variations, a single reference genome is unable to cover the entire gene content of a species. Therefore, pangenomics analysis is needed to ensure that the genomic diversity within a species is fully represented. Brassica napus is one of the most important oilseed crops in the world and exhibits variability in its resistance genes across different cultivars. Here, we characterized resistance gene distribution across 50 B. napus lines. We identified a total of 1749 resistance gene analogs (RGAs), of which 996 are core and 753 are variable, 368 of which are not present in the reference genome (cv. Darmor‐bzh). In addition, a total of 15 318 SNPs were predicted within 1030 of the RGAs. The results showed that core R‐genes harbour more SNPs than variable genes. More nucleotide binding site‐leucine‐rich repeat (NBS‐LRR) genes were located in clusters than as singletons, with variable genes more likely to be found in clusters. We identified 106 RGA candidates linked to blackleg resistance quantitative trait locus (QTL). This study provides a better understanding of resistance genes to target for genomics‐based improvement and improved disease resistance.     

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2–mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.     

Intragenic allele pyramiding combines different specificities of wheat Pm3 resistance alleles

Some plant resistance genes occur as allelic series, with each member conferring specific resistance against a subset of pathogen races. In wheat, there are 17 alleles of the Pm3 gene. They encode nucleotide‐binding (NB‐ARC) and leucine‐rich‐repeat (LRR) domain proteins, which mediate resistance to distinct race spectra of powdery mildew. It is not known if specificities from different alleles can be combined to create resistance genes with broader specificity. Here, we used an approach based on avirulence analysis of pathogen populations to characterize the molecular basis of Pm3 recognition spectra. A large survey of mildew races for avirulence on the Pm3 alleles revealed that Pm3a has a resistance spectrum that completely contains that of Pm3f, but also extends towards additional races. The same is true for the Pm3b and Pm3c gene pair. The molecular analysis of these allelic pairs revealed a role of the NB‐ARC protein domain in the efficiency of effector‐dependent resistance. Analysis of the wild‐type and chimeric Pm3 alleles identified single residues in the C‐terminal LRR motifs as the main determinant of allele specificity. Variable residues of the N‐terminal LRRs are necessary, but not sufficient, to confer resistance specificity. Based on these data, we constructed a chimeric Pm3 gene by intragenic allele pyramiding of Pm3d and Pm3e that showed the combined resistance specificity and, thus, a broader recognition spectrum compared with the parental alleles. Our findings support a model of stepwise evolution of Pm3 recognition specificities.     

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root‐knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest‐resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.     

Pm21 CC domain activity modulated by intramolecular interactions is implicated in cell death and disease resistance

Nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled-coil (CC) NLR that confers broad-spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1–159) domain has cell-death-inducing activity, whereas the CC-containing truncations, including CC-NB and CC-NB-LRR, do not induce cell-death responses. Coimmunoprecipitation (Co-IP) assay showed that the eCC domain self-associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V-mediated full-length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell-death induction. Two mutations (R73Q and E80K) interfered with D491V-mediated Pm21 autoactivation without affecting the cell-death-inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell-death-inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell-death signalling and disease-resistance signalling, which are potentially independent of each other.