Characterisation and mapping of a Globodera pallida resistance derived from the wild potato species Solanum spegazzinii.

KEY MESSAGE: A new resistance locus acting against the potato cyst nematode Globodera pallida was mapped to chromosome VI in the diploid wild potato species Solanum spegazzinii CPC 7195. The potato cyst nematodes (PCN) Globodera pallida and Globodera rostochiensis are economically important potato pests in almost all regions where potato is grown. One important management strategy involves deployment through introgression breeding into modern cultivars of new sources of naturally occurring resistance from wild potato species. We describe a new source of resistance to G. pallida from wild potato germplasm. The diploid species Solanum spegazzinii Bitter accession CPC 7195 shows resistance to G. pallida pathotypes Pa1 and Pa2/3. A cross and first backcross of S. spegazzinii with Solanum tuberosum Group Phureja cultivar Mayan Gold were performed, and the level of resistance to G. pallida Pa2/3 was determined in progeny clones. Bulk-segregant analysis (BSA) using generic mapping enrichment sequencing (GenSeq) and genotyping-by-sequencing were performed to identify single-nucleotide polymorphisms (SNPs) that are genetically linked to the resistance, using S. tuberosum Group Phureja clone DM1-3 516 R44 as a reference genome. These SNPs were converted into allele-specific PCR assays, and the resistance was mapped to an interval of roughly 118 kb on chromosome VI. This newly identified resistance, which we call Gpa VIlspg, can be used in future efforts to produce modern cultivars with enhanced and broad-spectrum resistances to the major pests and pathogens of potato.

A process-based, stage-structured model of potato cyst nematode population dynamics: Effects of temperature and resistance.

Potato cyst nematodes (PCN) are responsible for large losses in potato yields in many of the world's potato-growing regions. As soil temperatures increase due to climate change, there is potential for faster growth rates of PCN, allowing development of multiple generations in a growing season. We develop a process-based temperature-dependent model representing the life cycle of Globodera pallida, comprising juvenile, adult and cyst/diapause stages. To incorporate variability in the amount of time spent in each stage caused by genetic/environmental variation, the model is based on a mix of ordinary differential equations (ODEs) with sub-stages, and delay differential equations (DDEs). The effect of climate change is incorporated through the influence of soil temperature on the rate of development and survival in the hatching and juvenile stages. The level of the plant resistance to PCN is incorporated via the proportion of juveniles which become adults. After comparing the model with field data we run simulations to explore the effects of temperature and resistance on PCN populations. We find that with higher temperatures and longer growing seasons multiple generations of PCN can develop within a season, provided any required diapause period is short. Despite this, we show that growing resistant potatoes is a very effective control strategy and planting potatoes with even moderate levels of resistance can counter the effects of climate change.

Mapping the H2 resistance effective against Globodera pallida pathotype Pa1 in tetraploid potato.

KEY MESSAGE: The nematode resistance gene H2 was mapped to the distal end of chromosome 5 in tetraploid potato. The H2 resistance gene, introduced into cultivated potatoes from the wild diploid species Solanum multidissectum, confers a high level of resistance to the Pa1 pathotype of the potato cyst nematode Globodera pallida. A cross between tetraploid H2-containing breeding clone P55/7 and susceptible potato variety Picasso yielded an F1 population that segregated approximately 1:1 for the resistance phenotype, which is consistent with a single dominant gene in a simplex configuration. Using genome reduction methodologies RenSeq and GenSeq, the segregating F1 population enabled the genetic characterisation of the resistance through a bulked segregant analysis. A diagnostic RenSeq analysis of the parents confirmed that the resistance in P55/7 cannot be explained by previously characterised resistance genes. Only the variety Picasso contained functionally characterised disease resistance genes Rpi-R1, Rpi-R3a, Rpi-R3b variant, Gpa2 and Rx, which was independently confirmed through effector vacuum infiltration assays. RenSeq and GenSeq independently identified sequence polymorphisms linked to the H2 resistance on the top end of potato chromosome 5. Allele-specific KASP markers further defined the locus containing the H2 gene to a 4.7 Mb interval on the distal short arm of potato chromosome 5 and to positions that correspond to 1.4 MB and 6.1 MB in the potato reference genome.

Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages.

Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.

A Rapid Diagnostic for Detection of Aphelenchoides besseyi and A. fujianensis Based on Real-Time PCR.

Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.

The mitochondrial genome of Globodera ellingtonae is composed of two circles with segregated gene content and differential copy numbers.

BACKGROUND: The evolution of animal mitochondrial (mt) genomes has resulted in a highly conserved structure: a single compact circular chromosome approximately 14 to 20 kb long. Within the last two decades exceptions to this conserved structure, such as the division of the genome into multiple chromosomes, have been reported in a diverse set of metazoans. We report on the two circle multipartite mt genome of a newly described cyst nematode, Globodera ellingtonae. RESULTS: The G. ellingtonae mt genome was found to be comprised of two circles, each larger than any other multipartite circular mt chromosome yet reported, and both were larger than the single mt circle of the model nematode Caenorhabditis elegans. The genetic content of the genome was disproportionately divided between the two circles, although they shared a ~6.5 kb non-coding region. The 17.8 kb circle (mtDNA-I) contained ten protein-coding genes and two tRNA genes, whereas the 14.4 kb circle (mtDNA-II) contained two protein-coding genes, 20 tRNA genes and both rRNA genes. Perhaps correlated with this division of genetic content, the copy number of mtDNA-II was more than four-fold that of mtDNA-I in individual nematodes. The difference in copy number increased between second-stage and fourth-stage juveniles. CONCLUSIONS: The segregation of gene types to different mt circles in G. ellingtonae could provide benefit by localizing gene functional types to independent transcriptional units. This is the first report of both two-circle and several-circle mt genomes within a single genus. The differential copy number associated with this multipartite mt organization could provide a model system for deconstructing mechanisms regulating mtDNA copy number both in somatic cells and during germline development.

A metagenetic approach to determine the diversity and distribution of cyst nematodes at the level of the country, the field and the individual.

Distinct populations of the potato cyst nematode (PCN) Globodera pallida exist in the UK that differ in their ability to overcome various sources of resistance. An efficient method for distinguishing between populations would enable pathogen-informed cultivar choice in the field. Science and Advice for Scottish Agriculture (SASA) annually undertake national DNA diagnostic tests to determine the presence of PCN in potato seed and ware land by extracting DNA from soil floats. These DNA samples provide a unique resource for monitoring the distribution of PCN and further interrogation of the diversity within species. We identify a region of mitochondrial DNA descriptive of three main groups of G. pallida present in the UK and adopt a metagenetic approach to the sequencing and analysis of all SASA samples simultaneously. Using this approach, we describe the distribution of G. pallida mitotypes across Scotland with field-scale resolution. Most fields contain a single mitotype, one-fifth contain a mix of mitotypes, and less than 3% contain all three mitotypes. Within mixed fields, we were able to quantify the relative abundance of each mitotype across an order of magnitude. Local areas within mixed fields are dominated by certain mitotypes and indicate towards a complex underlying 'pathoscape'. Finally, we assess mitotype distribution at the level of the individual cyst and provide evidence of 'hybrids'. This study provides a method for accurate, quantitative and high-throughput typing of up to one thousand fields simultaneously, while revealing novel insights into the national genetic variability of an economically important plant parasite.

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida.

BACKGROUND: The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. RESULTS: The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. CONCLUSION: This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Activation of hatching in diapaused and quiescent Globodera pallida.

The potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis are major pests of potatoes. The G. pallida (and G. rostochiensis) life cycle includes both diapause and quiescent stages. Nematodes in dormancy (diapause or quiescent) are adapted for long-term survival and are more resistant to nematicides. This study analysed the mechanisms underlying diapause and quiescence. The effects of several compounds (8Br-cGMP, oxotremorine and atropine) on the activation of hatching were studied. The measurements of some morphometric parameters in diapaused and quiescent eggs after exposure to PRD revealed differences in dorsal gland length, subventral gland length and dorsal gland nucleolus. In addition, the expression of 2 effectors (IVg9 and cellulase) was not induced in diapaused eggs in water or PRD, while expression was slightly induced in quiescent eggs. Finally, we performed a comparative study to identify orthologues of C. elegans diapause related genes in plant-parasitic nematodes (G. pallida, Meloidogyne incognita, M. hapla and Bursaphelenchus xylophilus). This analysis suggested that it was not possible to identify G. pallida orthologues of the majority of C. elegans genes involved in the control of dauer formation. All these data suggest that G. pallida may use different mechanisms to C. elegans in regulating the survival stage.

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.