Does a genome made of multiple divergent copies allow genomic plasticity in absence of sexual reproduction in a plant-parasitic animal ? – ASEXEVOL

- Detecting multi-copy genes in Meloidogyne genomes.
For this step, we have used McScanX and reciprocal BLASTs of predicted proteins and CDS in 3 genomes of asexually-reproducing Meloidogyne and one with sexual reproduction.

- Detecting duplicated genomic blocks
We used McScanX to detect blocks made of at least 3 collinear genes present on at least two copies in each of the 4 Meloidogyne genomes.

- Evaluating % nucleotide divergence.
We used nucMer and BLAST as well as informations present on GFF annotation files to evaluate average nucleaotide divergence between blocks in the coding, intergenic and intronic regions.

- Detecting gene copies under positive and diversifying selection.
We calculated ratios of rates of non-synonymous / synonymous substitutions (dN/dS) between gene copies coming from duplicate blocks. We detected ''episodic diversifying selection« (EDS) via phylogenetic methods.

- Determining gene expression patterns and gene copies with divergent expression.
We will use software such as Cufflinks and Trinity for the reconstruction of transcript sequences. We will use RPKM approaches as well as EdgeR and DEseq to identify differentially expressed gene copies.

- We have shown that more than 90% of genes in asexually-reproducing Meloidogyne are duplicated, whereas only 47% sont are duplicated in the sexual species. In asexually reproducing Meloidogyne, a proportion of this genes form thousands of duplicated blocks that span several megabases of their genomes. In comparison, only a dozen of blocks are found in the sexual species and they span a few kilobases of the genome.

- The average nucleotide divergence between duplicated blocks is ~8%, within each of the 3 asexual species. These duplicated blocks are sometimes present on a same scaffold and they generally form synteny breakpoints. These observations are consistent with an evolution in the absence of meiosis.

- We have performed a phylogenomic analysis of genes present on duplicated blocks. Our analyzes show that within a same species, the different blocks do have different evolutionary histories and origins. This suggests that multiple hybridization events have given rise to this peculiar duplicated genome structure.

- Using dN/dS approaches, we have detected signs of positive selection for ~20% of pairs of genes resulting from duplicated genomic blocks. Furthermore, using phylogenetic methods, we found between 100-200 cases of episodic diversifying selection (EDS) in gene copies for each asexually-reproducing species.

- We have shown that positive and diversifying selection events impact almost all functional categories in these species.

- We have generated all the raw material for Illumina transcriptome sequencing of the different developmental life stages of the root-knot nematodes. Sequencing material for nematode infestation on common vs. specific plant hosts is being produced.

- As soon as we obtain RNAseq data for the different developmental life-stages of the nematodes, we will test whether within pairs of genes originating from duplicated blocks; differential gene expression patterns exist. This will be the first experimental support for possible functional divergence between genes copies and thus for a functional consequence of the peculiar genome structure. Expression data will be combined with positive and diversifying selection data.

- RNAseq data on infestation tests on common vs. specific plant hosts will allow determining whether gene copies coming from duplicated blocks are differentially expressed as a function of the infested plant host. This would indicate a potential impact of duplicated genes on the host spectrum of asexually-reproducing Meloidogyne.

- This ensemble of generated data will contribute to the resolution of what appears like an evolutionary paradox: the higher parasitic success of asexually-reproducing Meloidogyne as compared to their sexual cousins. Indeed, our data suggest that presence of multiple genomic copies within a same species could provide functional plasticity and possibly an advantage in terms of host range. The phylogenomic analyses performed during this project suggest that genomic copies originate from hybridization events.

Oral presentations:
• 7 Feb. 2014: Seminar of Institut Sophia Agrobiotech. Romain Blanc-Mathieu «How does an obligate asexual plant-parasitic species adapts to its host? Looking for evidences in the multiple diverged copies genome of the root-knot nematode Meloidogyne incognita.« 45'

• 02 - 04 Jul. 2014: Workshop: Genome Evolution in Asexual Animals, Lausanne, Suisse. Romain Blanc-Mathieu «Genome singularities in the asexually-reproducing root-knot nematodes; Genome structure and functional divergence of gene duplicates«. 45', invited speaker (by Tanja Schwander prof. Université de Lausanne).

• 16 - 19 Sept. 2014: «18th Evolutionary Biology Meeting at Marseilles« (EBM). Romain Blanc-Mathieu «How does an obligate asexual plant parasitic species adapts to its host? Looking for evidences in the multiple diverged copies genome of the root-knot nematode Meloidogyne incognita.« 20'

• 21 - 25 Jun. 2015: Workshop «Mathematical and Computational Evolutionary Biology«, Porquerolles. Etienne G.J. Danchin «Evolutionary successful and asexual: the paradox of the root-knot nematodes?« 20'

• 26 - 28 Aug. 2015: COST SUSTAIN Congress «Evolutionary Genomics of Plant Pathogens«, Kiel, Germany. Invited speaker: Etienne G.J. Danchin «Comparative genomics of root-knot nematodes provides clues to their parasitic success despite asexual reproduction.« 30'

Article in preparation for submission to Genome Research :
• Blanc-Mathieu R, Perfus-Babeoch L, Da Rocha M, Gouzy M, Sallet E, Martin-Jimenez C, Castagnone-Sereno P, Kozlowski D, Aury JM, Couloux A, Flot JF, Da Silva C, Schiex T, Abad P, Danchin EGJ. Evolutionary successful and asexual: the paradox of the root-knot nematodes?

Sexual reproduction allows elimination of deleterious mutations and genomic plasticity in animals through meiotic recombination and exchange of alleles. Hence, absence of meiosis and sexual reproduction is viewed as an evolutionary dead end in animals. Challenging these views, a few animals are considered as ancient strict asexuals. Their genomes may hold the secrets of survival and adaptation in the absence of sexual reproduction.
The genome sequence of the root-knot nematode Meloidogyne incognita is the only one currently available for an animal that reproduces exclusively asexually. This notorious pest, causing billions of € damage to agriculture annually reproduces without meiosis via parthenogenesis. Annotation and analysis of its genome, coordinated by our laboratory, revealed a singular architecture, mainly constituted of pairs of similar yet divergent and re-arranged regions that might represent former allelic haplotypes or result from past hybridization. In Bdelloid rotifers, another animal with strict asexual reproduction, a similar genome structure in multiple re-arranged and divergent copies has been observed. Such a structure in multiple copies could favor functional divergence between corresponding gene copies through neo- or sub-functionalization, exactly as for paralogous genes. This kind of phenomenon could represent a mechanism of genomic plasticity despite absence of sex. However, it has never been tested at the whole genome scale in an animal. Surprisingly, root-knot nematodes with obligate asexual reproduction have a wider host range and geographical distribution than their “sexual” cousins. Whether these features, contradictory with the supposed benefits of sexual reproduction, are correlated to the observed peculiar genomic structure is unknown.
In this project, we propose to combine whole genome and transcriptome data in Meloidogyne to assess whether the observed sequence divergence between pairs has functional consequences on corresponding gene copies and provides adaptability in an asexually-reproducing animal. We will first establish an inventory of genes present in multiple copies due to the peculiar genomic structure of M. incognita. We will then assess whether some of these diverged copies potentially present divergent functions, by analyzing their rate of non-synonymous versus synonymous mutations. Using RNA-seq expression data obtained from different developmental stages of M. incognita, we will assess whether divergent copies at the nucleotide level present different expression patterns. Identification of gene copies with different expression patterns will provide the first evidence at a whole genome scale for a functional consequence of the peculiar genome structures observed in asexually-reproducing animals. To determine whether the genome architecture of M. incognita can be linked to its wider host plant range compared to its sexual cousin, M. hapla, we will infest different host plants with M. incognita and generate the corresponding parasitic transcriptomes using RNA-seq technique. The selected host plants are either hosts compatible both with M. hapla and M. incognita (Tomato, Pepper) or hosts compatible only with M. incognita (Watermelon, Rice). If, through comparison of RNA-seq transcriptomes, we identify structure-specific M. incognita gene copies expressed differentially on host plants incompatible with M. hapla, our analysis will show that the genome structure in pairs, observed in M. incognita, can provide adaptability to a strictly parthenogenetic animal.
With the unique genomics and transcriptomics resources we have generated, our project represents an unparalleled opportunity to provide the first models and highlight the possible mechanisms that would allow asexually-reproducing species to evolve and adapt in the absence of meiotic recombination, an important evolutionary question unresolved so far.