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1.  Improved structural annotation of protein-coding genes in the Meloidogyne hapla genome using RNA-Seq 
Worm  2014;3:e29158.
As high-throughput cDNA sequencing (RNA-Seq) is increasingly applied to hypothesis-driven biological studies, the prediction of protein coding genes based on these data are usurping strictly in silico approaches. Compared with computationally derived gene predictions, structural annotation is more accurate when based on biological evidence, particularly RNA-Seq data. Here, we refine the current genome annotation for the Meloidogyne hapla genome utilizing RNA-Seq data. Published structural annotation defines 14 420 protein-coding genes in the M. hapla genome. Of these, 25% (3751) were found to exhibit some incongruence with RNA-Seq data. Manual annotation enabled these discrepancies to be resolved. Our analysis revealed 544 new gene models that were missing from the prior annotation. Additionally, 1457 transcribed regions were newly identified on the ends of as-yet-unjoined contigs. We also searched for trans-spliced leaders, and based on RNA-Seq data, identified genes that appear to be trans-spliced. Four 22-bp trans-spliced leaders were identified using our pipeline, including the known trans-spliced leader, which is the M. hapla ortholog of SL1. In silico predictions of trans-splicing were validated by comparison with earlier results derived from an independent cDNA library constructed to capture trans-spliced transcripts. The new annotation, which we term HapPep5, is publically available at www.hapla.org.
doi:10.4161/worm.29158
PMCID: PMC4165543  PMID: 25254153
Meloidogyne hapla; annotation; NGS; RNA sequencing; trans-splicing
2.  Correction: A Novel Candidate Vaccine for Cytauxzoonosis Inferred from Comparative Apicomplexan Genomics 
PLoS ONE  2013;8(10):10.1371/annotation/943b121e-343b-4df1-a06b-7f8a205a057d.
doi:10.1371/annotation/943b121e-343b-4df1-a06b-7f8a205a057d
PMCID: PMC3806894  PMID: 24194819
3.  A Novel Candidate Vaccine for Cytauxzoonosis Inferred from Comparative Apicomplexan Genomics 
PLoS ONE  2013;8(8):e71233.
Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91–100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.
doi:10.1371/journal.pone.0071233
PMCID: PMC3748084  PMID: 23977000
4.  A Sequence-Anchored Linkage Map of the Plant–Parasitic Nematode Meloidogyne hapla Reveals Exceptionally High Genome-Wide Recombination 
G3: Genes|Genomes|Genetics  2012;2(7):815-824.
Root-knot nematodes (Meloidogyne spp.) cause major yield losses to many of the world’s crops, but efforts to understand how these pests recognize and interact with their hosts have been hampered by a lack of genetic resources. Starting with progeny of a cross between inbred strains (VW8 and VW9) of Meloidogyne hapla that differed in host range and behavioral traits, we exploited the novel, facultative meiotic parthenogenic reproductive mode of this species to produce a genetic linkage map. Molecular markers were derived from SNPs identified between the sequenced and annotated VW9 genome and de novo sequence of VW8. Genotypes were assessed in 183 F2 lines. The colinearity of the genetic and physical maps supported the veracity of both. Analysis of local crossover intervals revealed that the average recombination rate is exceptionally high compared with that in other metazoans. In addition, F2 lines are largely homozygous for markers flanking crossover points, and thus resemble recombinant inbred lines. We suggest that the unusually high recombination rate may be an adaptation to generate within-population genetic diversity in this organism. This work presents the most comprehensive linkage map of a parasitic nematode to date and, together with genomic and transcript sequence resources, empowers M. hapla as a tractable model. Alongside the molecular map, these progeny lines can be used for analyses of genome organization and the inheritance of phenotypic traits that have key functions in modulating parasitism, behavior, and survival and for the eventual identification of the responsible genes.
doi:10.1534/g3.112.002261
PMCID: PMC3385987  PMID: 22870404
root-knot nematode; SNP; integrated map
5.  RNAi Effector Diversity in Nematodes 
While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.
Author Summary
Many organisms regulate gene expression through an RNA interference (RNAi) pathway, first characterized in the nematode Caenorhabditis elegans. This pathway can be triggered experimentally using double-stranded (ds)RNA to selected gene targets, thereby allowing researchers to ‘silence’ individual genes and so investigate their function. It is hoped that this technology will facilitate gene silencing in important parasitic nematodes that impose a considerable health and economic burden on mankind. Unfortunately, differences in RNAi susceptibility have been observed between species. Here we investigated the possibility that differences in the complement of effector proteins involved in the RNAi pathway are responsible for these differences in susceptibility. Our data revealed that most facets of the RNAi pathway are well represented across parasitic nematodes, although there were fewer pathway proteins in other nematodes compared to C. elegans. In contrast, the proteins responsible for uptake and spread of dsRNA are not well represented in parasitic nematodes. However, the importance of these differences is undermined by our observation that the protein complements in all the parasites were qualitatively similar, regardless of RNAi-susceptibility. Clearly, differences in the RNAi pathway of parasitic nematodes do not explain the variations in susceptibility to experimental RNAi.
doi:10.1371/journal.pntd.0001176
PMCID: PMC3110158  PMID: 21666793
7.  Computational and phylogenetic validation of nematode horizontal gene transfer 
BMC Biology  2011;9:9.
Sequencing of expressed genes has shown that nematodes, particularly the plant-parasitic nematodes, have genes purportedly acquired from other kingdoms by horizontal gene transfer. The prevailing orthodoxy is that such transfer has been a driving force in the evolution of niche specificity, and a recent paper in BMC Evolutionary Biology that presents a detailed phylogenetic analysis of cellulase genes in the free-living nematode Pristionchus pacificus at the species, genus and family levels substantiates this hypothesis.
See research article: http://www.biomedcentral.com/1471-2148/11/13
doi:10.1186/1741-7007-9-9
PMCID: PMC3042989  PMID: 21342537
8.  The secret(ion) life of worms 
Genome Biology  2009;10(1):205.
The secretomes of plant and human parasitic nematodes reveal molecular similarities that reflect the shared need to counter host defenses.
Tandem mass spectrographic analysis of the secreted proteins of plant- and human-parasitic nematodes reveals molecular similarities that reflect the shared need to counter host defenses.
doi:10.1186/gb-2009-10-1-205
PMCID: PMC2687784  PMID: 19226434
9.  A Method for Generating Meloidogyne incognita Males 
Journal of nematology  2006;38(2):192-194.
A method for producing mass quantities of Meloidogyne incognita males free from other developmental stages was developed. Host plants were grown hydroponically to facilitate nematode harvest. Pruning stress was shown to cause a higher percentage of juveniles to develop as males vs. a no-stress control. Application of pruning stress in the first 48 hr post-inoculation was also shown to be more effective at driving male development than at later times.
PMCID: PMC2586453  PMID: 19259447
hydroponic; males; Meloidogyne incognita; method; pruning stress
10.  A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies 
Journal of nematology  2006;38(1):165-167.
A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.
PMCID: PMC2586442  PMID: 19259442
endospores; method; Pasteuria penetrans
11.  A White Paper on Nematode Comparative Genomics 
Journal of Nematology  2005;37(4):408-416.
In response to the new opportunities for genome sequencing and comparative genomics, the Society of Nematology (SON) formed a committee to develop a white paper in support of the broad scientific needs associated with this phylum and interests of SON members. Although genome sequencing is expensive, the data generated are unique in biological systems in that genomes have the potential to be complete (every base of the genome can be accounted for), accurate (the data are digital and not subject to stochastic variation), and permanent (once obtained, the genome of a species does not need to be experimentally re-sampled). The availability of complete, accurate, and permanent genome sequences from diverse nematode species will underpin future studies into the biology and evolution of this phylum and the ecological associations (particularly parasitic) nematodes have with other organisms. We anticipate that upwards of 100 nematode genomes will be solved to varying levels of completion in the coming decade and suggest biological and practical considerations to guide the selection of the most informative taxa for sequencing.
PMCID: PMC2620993  PMID: 19262884
Caenorhabditis elegans; comparative genomics; genome sequencing; systematics
13.  High society (of nematologists) 
Genome Biology  2004;5(11):353.
A report on the 43rd annual meeting of the Society of Nematologists (SON), Estes Park, USA, 7-11 August 2004.
A report on the 43rd annual meeting of the Society of Nematologists (SON), Estes Park, USA, 7-11 August 2004.
doi:10.1186/gb-2004-5-11-353
PMCID: PMC545774  PMID: 15535876
17.  A Genetic Nomenclature for Parasitic Nematodes 
Journal of Nematology  1994;26(2):138-143.
A uniform system of genetic nomenclature for parasitic nematodes is presented. Conventions for naming strains, genes, alleles, and loci identified as DNA polymorphisms are established, and a standardized system for naming molecular clones is proposed.
PMCID: PMC2619496  PMID: 19279876
allele; DNA; DNA polymorphism; gene; genetic map; locus; molecular clone; nematode; nomenclature; RAPD; RFLP; strain; STS
18.  Horizontally transferred genes in plant-parasitic nematodes: a high-throughput genomic approach 
Genome Biology  2003;4(6):R39.
A method for a high-throughput genome screening for horizontally acquired genes is presented, and is illustrated using EST data from three species of root-knot nematode, Meloidogyne species.
Background
Published accounts of horizontally acquired genes in plant-parasitic nematodes have not been the result of a specific search for gene transfer per se, but rather have emerged from characterization of individual genes. We present a method for a high-throughput genome screen for horizontally acquired genes, illustrated using expressed sequence tag (EST) data from three species of root-knot nematode, Meloidogyne species.
Results
Our approach identified the previously postulated horizontally transferred genes and revealed six new candidates. Screening was partially dependent on sequence quality, with more candidates identified from clustered sequences than from raw EST data. Computational and experimental methods verified the horizontal gene transfer candidates as bona fide nematode genes. Phylogenetic analysis implicated rhizobial ancestors as donors of horizontally acquired genes in Meloidogyne.
Conclusions
High-throughput genomic screening is an effective way to identify horizontal gene transfer candidates. Transferred genes that have undergone amelioration of nucleotide composition and codon bias have been identified using this approach. Analysis of these horizontally transferred gene candidates suggests a link between horizontally transferred genes in Meloidogyne and parasitism.
doi:10.1186/gb-2003-4-6-r39
PMCID: PMC193618  PMID: 12801413
19.  Analysis and functional classification of transcripts from the nematode Meloidogyne incognita 
Genome Biology  2003;4(4):R26.
As an entrée to characterizing plant parasitic nematode genomes, 5,700 expressed sequence tags (ESTs) from the infective second-stage larvae (L2) of the root-knot nematode Meloidogyne incognita have been analyzed. In addition to identifying putative nematode-specific and Tylenchida-specific genes, sequencing revealed previously uncharacterized horizontal gene transfer candidates in Meloidogyne with high identity to rhizobacterial genes.
Background
Plant parasitic nematodes are major pathogens of most crops. Molecular characterization of these species as well as the development of new techniques for control can benefit from genomic approaches. As an entrée to characterizing plant parasitic nematode genomes, we analyzed 5,700 expressed sequence tags (ESTs) from second-stage larvae (L2) of the root-knot nematode Meloidogyne incognita.
Results
From these, 1,625 EST clusters were formed and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database. L2 larvae, which represent the infective stage of the life cycle before plant invasion, express a diverse array of ligand-binding proteins and abundant cytoskeletal proteins. L2 are structurally similar to Caenorhabditis elegans dauer larva and the presence of transcripts encoding glyoxylate pathway enzymes in the M. incognita clusters suggests that root-knot nematode larvae metabolize lipid stores while in search of a host. Homology to other species was observed in 79% of translated cluster sequences, with the C. elegans genome providing more information than any other source. In addition to identifying putative nematode-specific and Tylenchida-specific genes, sequencing revealed previously uncharacterized horizontal gene transfer candidates in Meloidogyne with high identity to rhizobacterial genes including homologs of nodL acetyltransferase and novel cellulases.
Conclusions
With sequencing from plant parasitic nematodes accelerating, the approaches to transcript characterization described here can be applied to more extensive datasets and also provide a foundation for more complex genome analyses.
doi:10.1186/gb-2003-4-4-r26
PMCID: PMC154577  PMID: 12702207

Results 1-19 (19)