The Genome of Melampsora larici-populina, The Poplar Leaf Rust

Tree/Microbe Interactions, INRA/Nancy University

Genome Sequencing of Micro-organisms Associated to Poplar

- the ectomycorrhizal Laccaria bicolor (65 Mb) (released)

- the ectomycorrhizal Paxillus involutus (18 Mb) (sequencing agreed)

- the endomycorrhizal Glomus intraradices (x Mb) (annotation on-going)

- the rust fungus Melampsora larici-populina (100 Mb) (annotation on-going)

- the ectomycorrhizal Tuber melanosporum (120 Mb) (annotation on-going)

+ the mycorrhiza helper Pseudomonas fluorescens BBc6R8 (7 Mb) (annotation on-going)

Sequencing of the Populus Community GenomeF Martin, P Lammers & G Tuskan

Glomus

Melampsora

Laccaria

Populus

Tuber

Pseudomonas fluorescens

Martin et al, New Phytol 2004

The Melampsora Genome Consortium

DOE Joint Genome Institute (http://www.jgi.doe.gov/)Igor Grigoriev, Harris Shapiro, Erika Lindquist, Andrea Aerts, Jeremy Schmutz & Sequencing/Assembly staffs

INRA Nancy – UMR IaM (http://mycor.nancy.inra.fr/IMGC/MelampsoraGenome/index.php)Sébastien Duplessis, Francis Martin, Marie-Pierre Oudot-Le Secq, Emilie Tisserant, Benoît Hilselberger, Stéphane Hacquard, Pascal Frey, Fabien Halkett, Axelle Andrieu

Nancy Université – UMR IaMClaire Veneault-Fourrey, Nicolas Rouhier, Eric Gelhaye, Benjamin Selles

Ghent University, VIBYao-Cheng Lin, Pierre Rouzé, Yves Van de Peer

Canadian Forest ServiceNicolas Feau, David Joly, Philippe Tangay, Richard Hamelin

Marseille, Göttingen, Lausane UniversitiesPedro Coutinho, Bernard Henrissat – Ursula Kües, Hélène Niculita-Herzel

Melampsora larici-populina Genome

Comparative analysis of genomes from saprotrophic, pathogenic and symbiotic basidiomycetes, e.g. the symbiotic Laccaria, the white-rot Phanerochate, the coprophylic Coprinopsis

© Broad Institute

© INRA

saprotrophy

saprotrophy

symbiosissymbiosis

path

ogen

esis

path

ogen

esis

The complement of genes is very conserved in eukaryotic lineages. Evolutionary innovation in eukaryotes relies mostly on carrying different versions (alleles) of the same gene.

- genes (or gene networks) specific to each functional groups?

- acquisitions or loss of genes?

- common genes, but specific regulatory networks?

Comparative Genomics . between biotrophic fungi . among saprotrophic, symbiotic & pathogenic basidiomycetes

Melampsora larici-populinaPuccinia graminis

Aspergillus niger

Sporobolomyces roseusPhakopsora pachyrhizi

Melampsora larici-populinaDikariaBasidiomycotaUredinalesMelampsoraceae

UredosporesGenomicLibraries

&Sequencing

1st Assembly

200gHMW DNA

(S Duplessis)

Feb 2006

Dec 2007

Basidiomycetes, Uredinales, Melampsoracea, Melampsora larici-populina 98AG31

ab initio AnnotationJGI Web Portal

Melampsora larici-populina/Populus trichocarpa

© INRA

strain 98AG31dikaryotic (2 x N) (collected by P Frey)Mars 2005

CSP Proposal Submission–

May 2005CSP Proposal Approved

Jan 2007

Manual curationGenome analysis

June 2008

August 2008

1st GenomeAnnotationWorkshop

Sequencing

2nd Assembly

Coverage & Production Stats: LIB COV. READS INSERT ± STD

BTCO 2.71x 577,884 3,968 ± 346BZIT 3.15x 615,747 6,283 ± 467BTCP 0.03x 7,680 8,511 ± 766BTCS 0.89x 278,777 36,907 ± 4,627

Total 6.79x 1,480,088 

Melampsora larici-populina Genome

Melampsora larici-populina Assembly (Dec 2007)

Arachne assembly

- Main genome scaffold total: 462- Main genome contig total: 3254- Main genome scaffold sequence total: 101.1 MB- Main genome contig sequence total: 97.7 MB (3.4% gap) - Main genome scaffold N/L50: 27/1.1 MB- Main genome contig N/L50: 265/112.3 KB- Number of scaffolds > 50 KB: 155-% main genome in scaffolds > 50 KB: 96.5%- Main genome depth: 6.79X

2 x N Moderately polymorphic genome:

Variation within assembled reads of ~ 1 in 333 bps AltHaplotype: 102 scaffolds (650 Kb, 50% with > 98% homology with main genome scaffolds) Repetitive scaffolds: 22 scaffolds (165 Kb)Excluded scaffolds: 39 scaffolds (28 Kb)Mitochondrion: 4 scaffolds (~79 Kb)

2420 out of 2494 (97.03%) JGI EST clusters mapped (90% ID 90% coverage) to the assembly

J Schmutz et al.

JGI Melampsora portal will go public in November 2008 I Grigoriev & A Aerts

scaffold 212 scaffold 269

Althaplotypes

50% of Althaplotypes scaffolds present 98% homology with a larger scaffold in ‘main genome’

Althaplotype vs. main scaffolds (Blastn): Reconstruction of 10 scaffolds (75 kb)

Scaffold 5

Scaffold 6

Scaffold 212Scaffold 212

Haplotypes/Althaplotypes Synteny

WebACT: E Tisserand

29 largest Puccinia scaffolds vs 27 largest Melampsora scaffolds [Blastn](~ 50 % of each genome)

~ 15 syntenic blocks, inversions of 5 to 10 kb

Synteny: Melampsora vs. Puccinia

Supercontig 1 (Puccinia) vs Scaffold 10 (Melampsora)

WebACT: E Tisserand

tRNA

tRNAScan-SE (default parameters, eukaryotic model) on all scaffolds.

253 tRNAs (24 in mitochondrion scaffolds), 194 contain an intron49 out of 61 possible anti-codons tRNA are found as well as 2 tRNA for selenocysteine10 tRNA pseudogenes, No tRNA suppressor # of tRNA detected in other basidiomycetes:L. bicolor: 279C. neoformans: 141P. chrysosporium: 200

Ribosomal DNAMelampsora larici-populina 5.8S ITS sequence & Puccinia graminis 28S and 18S sequences from NCBI used as query against all M. larici-populina scaffolds (blastn)

rDNA tandem unit: 10.3 Kb

Complete rDNA sequence used against M. larici-populina scaffolds:

22 complete copies

There are 11 extra rDNA 18S and 20 extra rDNA 28S sequences elsewhere on other scaffolds

Althaplotype:

460 : 1

Repetitive:195 1476 1384 1337 1199 1

Scaffolds # repetitions

Main:34 177 172 220 236 14 351 2101 126 144 139 1

Melampsora Laccaria

snRNA U1 0 copie 0 copiesnRNA U2 7 copies 8 copiessnRNA U4 2 copies 2 copiessnRNA U5 0 copie 2 copiessnRNA U6 6 copies 6 copies

snRNA U11 0 copie 0 copie

Scaffold Position

U2 scaffold_1 3615920..3616100scaffold_9 517992..517803

scaffold_13 36050..36239scaffold_33 425878..425689scaffold_35 804437..804248scaffold_50 534638..534827scaffold_50 317326..317515

U4 scaffold_2 1492180..1492311scaffold_8 1969927 - 1970058

U6 scaffold_10 1876437 - 1876544scaffold_10 1370684 - 1370579scaffold_16 1076292 - 1076190scaffold_26 237438 - 237545scaffold_26 205763 - 205870scaffold_30 227436 - 227553

snRNA

cmsearch (INFERNAL): 6 covariance models with cutoff & window size (RFAM) parameters

Mitochondrial DNA

Four scaffolds:sc146: 47,057 bpsc218: 18,446 bpsc332: 8,679 bpsc545: 4,301 bp

Total 78,483 bp