Root Knot Nematode Comparative Genomics

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ROOT-KNOT NEMATODE COMPARATIVE GENOMICS UNDERSTANDING EVOLUTION, DIVERSITY AND THREAT EVOHULL: EVOLUTIONARY AND ENVIRONMENTAL GENOMICS UNIVERSITY OF HULL, UK @davelunt davelunt@gmail.com davelunt.net Dr Dave Lunt

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EVOHULL: EVOLUTIONARY AND ENVIRONMENTAL GENOMICS UNIVERSITY OF HULL, UK @davelunt davelunt@gmail.com davelunt.net Dr Dave Lunt invasive species ecological networks in forestry and agriculture biomonitoring bioinformatics metabarcoding genomics phylogenetics apomixis and breeding systems environmental DNA

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NERC, AFRICA GOMEZ, CHARLES OPPERMAN, DAVID BIRD, ETIENNE DANCHIN, PHILIPPE CASTAGNONE- SERENO, BOB ROBBINS, PABLO CASTILLO & MANY MANY OTHERS @davelunt davelunt@gmail.com data accession: PRJNA340324 Amir Szitenberg & Laura Salazar have carried out much of this work AMIR SZITENBERG, LAURA SALAZAR, VIVIAN BLOK, SOUMI JOSEPH, DOMINIK LAETSCH, VALERIE WILLIAMSON, MARK BLAXTER, DAVE LUNT THANKS images: Wikipedia, JD Eisenback, et al

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5 WHAT’S IN A GENOME & WHY? RESEARCH QUESTION 1 HeLa cell nuclei

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Marbled lungﬁsh ~133 Gbp Pratylenchus coffeae, Plant-parasitic nematode ~20Mb 1/6,650

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Bear garlic Allium ursinum 31.5 GB genome Chives, Allium schoenoprasum 7 GB genome Ricroch et al. Evolution of genome size across some cultivated Allium species. Genome. 2005;48: 511–520. doi:10.1139/g05-017 1/4.5

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onion, Allium cepa 15 GB genome human, Homo sapiens 3 GB genome

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JD Eisenback onion, Allium cepa 15 GB genome nematode, Meloidogyne sp 150 MB genome infected uninfected

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MOSTLY TRANSPOSONS, REPEATS, AND SEQUENCES OF UNKNOWN ORIGIN WHAT’S IN A GENOME? BUT WHY? onion, Allium cepa 15 GB genome human, Homo sapiens 3 GB genome

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RECOMBINATION GENE FLOW GENETIC DRIFT MUTATION SELECTION EVOLUTIONARY FORCES WHY IS GENOME CONTENT AS IT IS?

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TRANSPOSABLE ELEMENTS IN THE NEMATODA 42 GENOMES 500 MILLION YEARS OF EVOLUTION Szitenberg et al Genome Biology & Evolution 2016 doi:10.1093/gbe/evw208

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Szitenberg et al Genome Biology & Evolution 2016 doi:10.1093/gbe/evw208 DNA TEs LTRs transposons 500 million years of evolution 42 genomes 13 NEMATODE TRANSPOSABLE ELEMENTS FOLLOW PHYLOGENY THE GENOMIC TRANSPOSABLE ELEMENT LOAD IS EXPLAINED BY PHYLOGENY NOT BREEDING SYSTEM

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HOW CAN WE APPLY GENOMICS & BIOINFORMATICS FOR SUSTAINABLE AGRICULTURE? RESEARCH QUESTION 2

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GLOBALLY IMPORTANT CROP PESTS ROOT-KNOT NEMATODES genus Meloidogyne ENORMOUS PLANT HOST RANGE ~5% WORLD AGRICULTURE? ALL MAJOR CROPS SPECIES 15

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WIDE VARIETY OF REPRODUCTIVE MODES IN SINGLE GENUS ▸ Asexual (mitotic) ▸ mitotic parthenogenesis- apomixis ▸ no meiosis or exchange ▸ Sexual (meiotic) ▸ meiotic parthenogenesis- automixis ▸ outbreeding sexuality- amphimixis ▸ meiosis and genetic exchange 17 RKN juveniles

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18 RKN female SEM overlaid with juveniles WIDE VARIETY OF REPRODUCTIVE MODES IN SINGLE GENUS 18S structural alignment ML tree Reproductive mode changes frequently within the genus

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19 RKN female SEM overlaid with juveniles WIDE VARIETY OF REPRODUCTIVE MODES IN SINGLE GENUS Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190

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20 RKN female SEM overlaid with juveniles WIDE VARIETY OF REPRODUCTIVE MODES IN SINGLE GENUS Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190 THE GENUS HAS >6 TRANSITIONS TO MITOTIC PARTHENOGENESIS LOSS OF SEX/MEIOSIS FACULTATIVE MEIOTIC PARTHENOGENESIS (AUTOMIXIS) AND SEXUALITY (AMPHIMIXIS)

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21 RKN female SEM overlaid with juveniles TAXONOMY AND CROP DAMAGE: THE TROPICAL APOMICTS Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190

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22 RKN female SEM overlaid with juveniles TAXONOMY AND CROP DAMAGE: THE TROPICAL APOMICTS Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190

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23 RKN female SEM overlaid with juveniles TAXONOMY AND CROP DAMAGE: THE TROPICAL APOMICTS Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190

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24 ALL APOMICTS EXCEPT M. FLORIDENSIS Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190 These are the tropical root-knot nematodes, causing major economic loss

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25 Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190 These are the tropical root-knot nematodes, causing major economic loss THE MELOIDOGYNE INCOGNITA GROUP THE MIG OUTGROUP

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26 Janssen et al 2017 Fig 7. Majority rule consensus tree based on 18S ribosomal rDNA sequences with karyology doi:10.1371/journal.pone.0172190 These are the tropical root-knot nematodes, causing major economic loss THE MELOIDOGYNE INCOGNITA GROUP THE MIG OUTGROUP We have genome sequenced 19 new genomes

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GENOMICS AND BIOINFORMATICS CAN REVEAL COMPLEX BIOLOGICAL STORIES, AND SUGGEST NOVEL APPROACHES TO AGRICULTURAL PROBLEMS

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GENOMICS AND BIOINFORMATICS ▸Phylogeny and diversity ▸Genome structure ▸Origins and speciation ▸Hybrids? ▸Polyploids? 28

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29 SMALL MTDNA DIVERGENCES Edinburgh Genomics mtDNA genome phylogeny mtDNA is a poor diagnostic tool

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30 SMALL MTDNA DIVERGENCES mtDNA genome phylogeny Edinburgh Genomics

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MIG GENOMES ARE HYBRID, COMPLEX, AND CONTAIN TWO GENOMIC COPIES UNDERSTANDING THE COMPLEXITY WILL INFORM BIOLOGY AND CONTROL Lunt et al. 2014 The complex hybrid origins of the root knot nematodes revealed through comparative genomics. doi:10.7717/peerj.356

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Apomicts M. ﬂoridensis M. hapla M. chitwoodi M. incognita M. javanica M. arenaria INTRA-GENOMIC BLAST ANALYSIS 32

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MOST MIG GENOMES HAVE A MIX OF DIVERGENT AND HOMOZYGOUS REGIONS Number of Orthology Groups M. incognita M. javanica M. arenaria M. ﬂoridensis Copies per genome has mostly lost the divergent second genome copy 33

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PHYLOGENOMICS OF 2 DIVERGENT GENOME COPIES 533 Ortholog CDS supermatrix ML tree RAxML Single origin of 2 genome copies predating speciation Phylogeny in each genome copy A/B is identical M. incognita M. javanica M. arenaria M. ﬂoridensis colours are different species not reproductive system B A 34

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35 NUCLEAR PHYLOGENOMICS RESOLVES THE RELATIONSHIPS M. ﬂoridensis M. incognita M. arenaria M. javanica

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36 VERY LITTLE GENETIC DIVERGENCE GLOBALLY M. ﬂoridensis M. incognita M. arenaria M. javanica Libya USA French West Indies Ivory Coast USA Morocco French West Indies USA ROOT-KNOT NEMATODES LIKELY SPREAD WITH MODERN AGRICULTURE

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Eisenback and Triantaphyllou 1991 37

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ADAPTATION TO AGRICULTURAL ENVIRONMENT ONCE THOUGHT THAT HYBRID SPECIATION WAS RARE AND INCONSEQUENTIAL IN ANIMALS Heliconius Lake Malawi Polar and brown GENOME BIOLOGY IS REVEALING A VERY DIFFERENT VIEW HYBRID SPECIATION IN MELOIDOGYNE?

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ADAPTATION TO AGRICULTURAL ENVIRONMENT PREVIOUS WORK SUGGESTS INTERSPECIFIC HYBRIDISATION MAY BE INVOLVED WITH MELOIDOGYNE ASEXUAL SPECIES Heliconius butterﬂies Lake Malawi cichlids Root knot nematodes? HYBRID SPECIATION IN MELOIDOGYNE? Lunt et al. 2014 The complex hybrid origins of the root knot nematodes revealed through comparative genomics. doi:10.7717/peerj.356

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X parent 1 parent 2 hybrid offspring phenotypic variation parental phenotypic variation TRANSGRESSIVE SEGREGATION phenotype could be anything, including nematode host-range is greater than sum of parental variation Transgressive segregation is when the absolute values of traits in some hybrids exceed the trait variation shown by either parental lineage small big very small very big 40

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THE APOMICTS ARE HYPO-TRIPLOID SOME REGIONS OF THE APOMICTS ARE PRESENT IN 3 COPIES A1,A2,B hypo-triploid = not full triploid, some parts of genome are diploid

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42 TROPICAL APOMICTS ARE HYPO-TRIPLOIDS hypo-triploid = not full triploid, some parts of genome are diploid copy A1 copy B copy A2 copy number 1 2 A1-A2 are ~100% identical to each other copy A copy B copy number 1 2 3% divergence in protein coding regions illustration of diversity at each diploid locus Some loci are diploid A,B, some triploid A1,A2,B illustration of diversity at each triploid locus

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TROPICAL APOMICTS ARE HYPO-TRIPLOIDS read depth analysis demonstrates hypo-triploidy illustration of diversity at each triploid locus not all loci are triploid, diploid loci add to read depth 100 peak many loci have read depth 100 some loci have read depth 200 A1 + A2 from high quality PacBio genome copy A1 copy B copy A2 copy number 1 2 sequence read depth A + B

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GENE CONVERSION IS AN IMPORTANT FORCE SHAPING THE APOMICT GENOMES THERE IS EVIDENCE OF GENE CONVERSION BUT NOT HOMOLOGOUS-EXCHANGE RECOMBINATION

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45 NO EVIDENCE OF RECIPROCAL EXCHANGE AMONG APOMICTS GENE CONVERSION IS EVIDENT RECIPROCAL EXCHANGE GENE CONVERSION A1 B A2 A1 B A2 Identical & no recombination detectable B A1 A2 or

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SUMMARY 46 GENOMES CONTAIN DIVERGENT A AND B COPIES MIG APOMICTS ARE HYPOTRIPLOID A AND B DIVERGED BEFORE MIG SPECIES GENE CONVERSION IS A POWERFUL FORCE MIG ARE DIVERGENT DUE TO MUCH MORE THAN SIMPLE MUTATION NUCLEAR GENOME RESOLVES PHYLOGENY

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FUTURE QUESTIONS 47 CAN WE LOCATE FUNCTIONAL LOCI FOR VIRULENCE AVIRULENCE? WHAT IS THE NATURE OF THE ADAPTIVE VARIATION? HAVE MIG OUTRUN THEIR PATHOGENS? DO GENOME COPIES FULFIL THE SAME ROLES?

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GENOMICS HAS ONLY SCRATCHED THE SURFACE OF ROOT-KNOT DIVERSITY 48

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GENOMICS HAS ONLY SCRATCHED THE SURFACE OF ROOT-KNOT DIVERSITY 49

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FUTURE ACTIONS BROADEN THE NUMBER OF SPECIES SAMPLED METADATA COLLECTION WITH GENOMES INDUSTRIAL AND ACADEMIC COLLABORATION CHARACTERISE MUCH MORE GENOMIC INTRASPECIFIC VARIATION 50 WHAT IF….

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WHAT IF BOTH CROP ISOLATE AND NEMATODE RACE GENOMES WERE KNOWN? By Mason Masteka - originally posted to Flickr as End of Summer Tomatoes, CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=11444911

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WHAT IF WE COULD MOVE FROM SINGLE INDIVIDUAL RKN TO GENOME- BASED DIAGNOSTIC OF LIKELY HOST RANGE AND CROP THREAT? JD Eisenback

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ROOT-KNOT NEMATODE COMPARATIVE GENOMICS UNDERSTANDING EVOLUTION, DIVERSITY AND THREAT Dr Dave Lunt EVOHULL: EVOLUTIONARY AND ENVIRONMENTAL GENOMICS UNIVERSITY OF HULL, UK @davelunt davelunt@gmail.com davelunt.net By Mason Masteka - originally posted to Flickr as End of Summer Tomatoes, CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=11444911

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Root-Knot Nematodes Meloidogyne spp