H Angulo

C Benhabiles

R Borcea

P Borcea

Bourgault

J C Dalouzy

L Achouri

M Aliotta

C Angulo

H Benhabiles

C Borcea

P Bourgault

A Buta

A Coc

A Damman

T Davinson

F De Grancey

F De Oliveira

Santos

N De Séréville

J Kiener

M G Pellegriti

F Negoita

A M Sánchez-Benítez

O Sorlin

M Stanoiu

I Stefan

P J Woods

Discovery of a new

Keywords:

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Novae are nuclear explosions caused by the accretion of hydrogen onto the surface of a white dwarf star. In these events, temperatures can reach 3.5 × 10 8 K, allowing fast synthesis of stable and radioactive nuclei [1]. The observation of γ rays from nova ejecta should provide a rather direct way to investigate the nucleosynthesis and matter ejection mechanism [2]. The most powerful γ-ray emission coming from novae is predicted to be at energies of 511 keV and below, originating from positron annihilations. When the nova envelope becomes transparent enough for γ rays to escape, the main contribution to positron production is the long-lived 18 F radioactive nucleus (half-life 109.77 min). Therefore, the amount of radiation emitted scales with the 18 F content of the nova ejecta, which in turn depends strongly on its production and destruction rates. The 18 F (p, α) 15 O reaction is believed to be the most important destructive reaction [3]. Despite a lot of experimental efforts [4][5][6][7][8][9][START_REF] De Séréville | International Symposium on Nuclear Astrophysics, Nuclei in the Cosmos-IX[END_REF], uncertainties remain in the determination of the rate of this reaction at novae temperatures. One potentially important source of uncertainty comes from interference between three 3 2 + resonances [11,12].

More recently, M. Dufour and P. Descouvemont [13] predicted the existence of two not yet observed 1 2 + states, one at 0.41 MeV below the proton emission threshold S p , and a second broad resonance at about 1.49 MeV above the threshold. If the existence and properties of these 1 2 + states were confirmed, the reaction rate at typical novae temperatures would be dominated by reactions through these states. It would follow that the importance of the interference contribution between the + resonances would be much reduced. In the present paper, we report the results of a new approach developed to study excited states in 19 N e through the reaction 1 H( 19 N e, p) 19 N e * (p) 18 F . Implications to the 18 F (p, α) 15 O rate in novae are subsequently discussed.

The experiment was performed at the Centre de Recherches du Cyclotron at Louvain-la-Neuve (Belgium) using a 19 N e 6+ radioactive beam. The 19 N e beam was produced with a mean intensity of ≈ 8 × 10 7 pps and accelerated to 9 A•MeV. The beam was incident on a 3.5 µm thick polypropylene target during 100 hours. During the experiment the beam intensity was monitored in a beam catcher in which it was stopped. The measured signal of the catcher was normalized to the one of a Faraday cup to provide an absolute determination of the integrated beam current. With an upper limit of 1%, no contamination of the beam by 19 F was observed. A dedicated setting used a 19 F beam under similar conditions to calibrate the target and beam catcher thicknesses as well as the energies of the detectors using the inelastic scattering reaction 1 H( 19 F, p) 19 F * (α) 15 N .

Excited states in 19 N e were populated by inelastic scattering reactions 1 H( 19 N e, p) 19 N e * occurring in the target. Scattered protons were detected at zero degree by a ∆E -E telescope of silicon detectors located 50 cm downstream of the target, composed of a 500 µm ∆E detector (18 keV FWHM resolution) and a 6 mm Si(Li) E detector (28 keV FWHM) cooled to -25 • C. The tele-FIG. 1: The measured differential cross section for the H( 19 N e, p) 19 N e * reaction, with the associated proton detected at zero degree, is presented as a function of the 19 N e * excitation energy (lower axis) and the measured energy in the laboratory (upper axis) corrected for energy losses in the beam catcher. This figure is conditioned with the detection of another proton in CD-PAD coming from the emission channel 19 N e * (p) 18 F . Gaussian-shaped peaks were used to fit five of the observed peaks. The broad peak E was fitted with a Breit-Wigner shape using an energy-dependent proton width.

scope covered a solid angle of 5 msr. We chose to detect scattered protons at 0 • for three reasons: First, the best energy resolution for the excited states in 19 N e * is obtained for this angle as the reaction is made in inverse kinematics. Second, due to the axial symmetry of this experimental configuration, the analysis of the proton-proton angular correlation is simplified. Third, a strong alignment of the populated states in 19 N e [START_REF] Ferguson | Angular correlation methods in gamma-ray spectroscopy[END_REF] is obtained, resulting in a pronounced spin-dependent angular distribution when studying their decay. A 250 µmthick aluminium foil, placed between the target and the telescope, was used as a beam catcher. The intense radioactive beam was stopped inside this catcher while the scattered protons lost less than 20% of their energy when passing through it.

The study of unbound states in 19 N e * , (S p = 6.411 MeV and S α = 3.529 MeV), was obtained by using a telescope (CD and PAD) of annular silicon detectors which measured the energy losses and residual energies of the emitted particles, respectively. Their characteristics are as follows : CD detector [30µm thick, 16 annular strips and 24 radial strips, resolution 28 keV FWHM] , PAD detector [1.5 mm thick, 130 keV FWHM] [START_REF] Ostrowski | [END_REF]16]. This telescope was positioned between the target and the beam catcher, 10 cm downstream of the target. It covered laboratory angles between 4.3 • and 21.6 • .

The excitation energies in 19 N e produced by inelastic scattering were derived from the proton energies detected at zero degree. Above the proton emission threshold the requirement of another proton detected in CD-PAD (from 19 N e * (p) 18 F ) was used to suppress the background induced by reactions of the beam in the catcher. The corresponding differential cross section for the H( 19 N e, p) 19 N e * reaction is presented as a function of the 19 N e * excitation energy in Fig. 1. The energy resolution in excitation energy of 19 N e was measured to be 30 keV FWHM. This spectrum was analyzed with a multiple peak-fitting program. A minimum of 6 states is required to fit the proton spectrum within the excitation energy range ≈ 6.9 -8.4 MeV. They are reported in Table I. These peaks correspond to excited states in 19 N e produced by the inelastic scattering reaction, which have a large branching ratio (> 10%) for proton emission. Gaussian shapes were used for all peaks, except for the broad resonance (labeled E in Fig. 1) at ≈ 7.9 MeV for which an energy dependent Breit-Wigner shape [19] was used instead.

Angular distributions of the unbound protons were derived from the annular strips of the CD detector. As the scattered protons were detected at 0 • , the excited states in 19 N e * were expected to be nearly totally aligned [START_REF] Ferguson | Angular correlation methods in gamma-ray spectroscopy[END_REF]. Consequently, pronounced angular distributions (see Fig. 2) are obtained for the 6 peaks labeled in Fig. 1. These distributions were generated using energy regions where the overlap with neighboring peaks was minimized. The angular coverage in the center-ofmass was higher than 80% for most of the states but, due to the kinematics of the reaction, it was lower for states having excitation energies above 7.8 MeV. The analysis of the angular-correlation was used to assign the spin of the emitting states in 19 N e by following the general method outlined by Pronko and Lindgren [20]. Note that it is analogous to the collinear γ-ray angular correlation method of Ferguson [START_REF] Ferguson | Angular correlation methods in gamma-ray spectroscopy[END_REF]. As described in [START_REF] Ferguson | Angular correlation methods in gamma-ray spectroscopy[END_REF]20,21] the angular distribution should be of the form dσ(θ CM )/dΩ = k=even A k P k (cos(θ CM )), where P k are Legendre polynomials. Then, in the present case it would be isotropic for the spin J = 1 2 state, parabolic for J = 3 2 , biquadratic for J = 5 2 and so on. As stated in Ref. [20] the angular distributions are parity independent, meaning that the parity of the observed states could not be inferred from this sole information. However, the most likely parity value can be derived from the following property. The proton-unbound states observed here have large proton widths. These states therefore emit protons with the lowest possible angular momentum value, for which the centrifugal barrier is the smallest. Based on this fact and on the ground state spin value 1 + of 18 F, a parity value (+) is derived for the 19 N e * spins J = 1 2 , J = 3 2 , J = 7 2 ... and (-) for spins J = 5 2 , J = 9 2 etc. The measured spins and favored parity values are presented in Table I.

Energies, widths and spin values of the known states A and D in 19 Ne are remarkably reproduced by the present work, which demonstrates the reliability of the experimental method. For example, the first state labeled A is found at an excitation energy of E x = 7.079 (5) MeV and a width Γ = 32(8) keV, to be compared to E x = 7075(1.6) keV and Γ = 39(2.2) keV [7,12]. The angular distribution of the corresponding protons can be fitted with a polynomial of order 2 (see Fig. 2), leading to J = 3 2 and a favored (+) parity, which is in accordance with J π = 3 2 + obtained in Refs. [7,12]. In addition to a good energy and width matching for the states B, C, and F, their spin assignments are deduced for the first time. They were so far only based on comparison with the mirror nucleus 19 F. The 7.5 to 8.4 MeV region in Fig. 1 cannot be fitted without introducing at least one broad state. The analysis of the spectrum using a Breit-Wigner shaped peak gives an energy E x =7.863 (39) MeV and a total width Γ = 292 (107) keV. The angular distribution corresponding to this peak (labeled as E in Fig. 2) is flat, meaning that its spin value is J = 1 2 (+) . This state is observed for the first time. Excellent agreement is found with the predictions of Ref. [13] which proposed the existence of a broad 1/2 + state at E x =7.901 MeV with Γ = 296 keV. In the range of energies considered in this paper, the alpha emission channel H( 19 N e, p) 19 N e * (α) 15 O could not be analyzed since it could not be distinguished from the competing reaction channel H( 19 N e, α) 16 F * (p) 15 O. Therefore the alpha branching ratio, or alpha width Γ α , could not be determined. Following the remarkable agreement between theory [13] and the present experiment for E x and Γ, the theoretical value of Γ α = 139 keV will be retained in the following to estimate the impact of this broad resonance on the 18 F (p, α) 15 O reaction cross section.

To ensure that this broad peak (E) does not originate from parasitic reaction-induced background, several checks have been made: (i) Proton energies and relative angles measured for this broad peak follow the expected kinematics relationship of the H( 19 N e, p) 19 N e * (p) 18 F reaction, as well as the other peaks. Such correlations do not hold true for other possible reactions as H( 19 N e, p) 19 N e * (p) 18 F * reaction or carbon-induced fusion-evaporation reactions from the (CH 2 ) n target. (ii) Uncorrelated proton correlations were generated by taking the proton parameters of two distinct events. The resulting background on the differential cross section is flat over the studied energy range, and amounts to less than ≈ 10% of the peak height. (iii) A combination of more than one resonance, instead of a broad one, is unlikely as they should all exhibit the same angular distribution. Moreover when using several peaks to fit the data, the agreement is not improved. (iv) Finally no state was so + states is shown [13]. The contribution of the new s wave state is superimposed in this figure. In the range of interest this state could become the major contribution to the S-factor. far observed in this energy domain [12]. The presence of one broad resonance is therefore the only plausible interpretation to explain the excitation energy spectrum around 7.8 MeV.

The astrophysical factor S(E), that is the 18 F (p, α) 15 O reaction cross section with the Coulomb barrier penetrability function removed, is presented in Fig. 3 as a function of the center-of-mass energy. The first three 3 2 + states above the proton emission threshold lead to four distinct interference possibilities which are indicated by different continuous lines. In the range of interest corresponding to novae explosions, the S factor varies by more than three orders of magnitude, from < 10 2 to 10 5 KeV•b. The contribution of the new broad s state is superposed in this figure. Even if located far away from the proton emission threshold, the low energy tail of this resonance contributes significantly to the S(E) factor at novae temperatures. It becomes the dominant contribution in the case of destructive interferences between the 3/2 + states. It also brings so far the stringent lower limit of S(E).

To

summarize, the scattering reaction 1 H( 19 N e, p) 19 N e * (p) 18 F was used in inverse kinematics to study the excited states in 19 N e which can contribute to the 18 F (p, α) 15 O reaction cross section. Proton-proton coincidences between protons arising from the inelastic reaction and emitted from unbound 19 N e * states were used for determining energies, widths and for the first time spin values of the resonant states. Besides the remarkable agreement with the insofar known resonances, a new broad 1/2 + resonance was found at about 1.4 MeV above the reaction threshold. The low energy tail of this resonance contributes to a significant enhancement of the 18 F destruction rate at nova temperature. This reduces significantly the chance to observe γ-ray emission of 18 F from a nearby nova explosion with existing telescopes. More generally, this newly used experimental method opens perspectives to study resonant states of astrophysical interest in other nuclei.

FIG. 2 :

 2 FIG. 2: Center-of-mass angular distributions measured for each excited state in 19 N e * . Lines correspond to the best fits obtained in the present analysis. Part of the angular distribution of levels E and F was beyond the angular coverage of the detectors.

FIG. 3 :

 3 FIG. 3: The astrophysical S-factor of the 18 F (p, α) 15 O reaction is plotted as a function of the center-of-mass energy. The corresponding novae temperatures are indicated by upper marks in the top left corner of the figure. Interference pattern of the three known 3 2

TABLE I :

 I Properties of the19 N e excited states measured in the present experiment are compared to results from previous measurements.

			This experiment					Previous measurements
	Label	Er(keV)	Ex(MeV)	Γ (keV)	J π	Ex(MeV)	Γ (keV)	J π	ref
	A B	669(5) 793(31)	7.079(5) 7.203(31)	32(8) 35(12)	3 2 3 2	(+) (+)	7.075(1.6) 7.173(5) 7.238(6)	39(2.2) --	3 2 -+ -	[7] [5] [5]
	C	1092(30)	7.502(30)	17(7)	5 2	(-)	7.500(9) 7.531(11)	16(16) 31(16)	--	[5] [5]
	D	1206(5)	7.616(5)	21(10)	3 2	(+)	7.608(11) 7.644(12)	45(16) 43(16)	3 2	+	[17] [5]
	E F	1452(39) 1564(10)	7.863(39) 7.974(10)	292(107) 11(8)	1 2 ( 5 (+) -) 2	-7.944(15) 8.069(12)	---	---	[18] [18]

We thank the Centre de Recherches du Cyclotron at Louvain-la-Neuve crew for delivering the 19 Ne beam. We acknowledge the support of the European Commission within the Sixth Framework Programme through I3-EURONS (contract no. RII3-CT-2004-506065), the support from the French-Romanian collaboration agreement IN2P3-IFIN-HH Bucharest n • 03-33 and the support from the UK STFC. One of us (A.B) acknowledges also the partial financial support from CNCSIS, Romania.

Discovery of a new broad resonance in 19 N e: Implications for the destruction of the cosmic γ-ray emitter 18 F


Benjamin Penaud

Benoît Laurent

Marine Milhes

Camille Noûs

François Ehrenmann

Cyril Dutech
email: cyril.dutech@inrae.fr

Benoit Laurent

Camille Noüs

A bio-informatic pipeline to isolate robust molecular markers for phylogeny, phylogeographical and population genetic studies of genetically orphan species

Keywords: Bursaphelenchus xylophilus Amplicon, biological invasion, forest diseases and pests, single-copy genes, wholegenome sequencing

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Introduction

Pest and disease emergences are an important threat to the biodiversity and functioning of world ecosystems (Fisher et al. 2012) and human well-being (Diagne et al. 2021). A dramatic increase in these events has been recorded for several decades [START_REF] Anderson | Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology[END_REF], Fisher et al. 2012, Lips 2016, Rohr et al. 2019, Santini et al. 2012). Specifically, in the context of invasions or recent expansions of pests and parasites, there is an urgent need to identify methods for avoiding, stopping, or, at least, reducing their spread and deleterious effects on ecosystems, species, and genetic diversity (Filipe et al. 2012, Gonthier et al. 2014, Hall et al. 2016 ). For many of the emerging pests and pathogens, however, taxonomic and biological knowledge is insufficient, with no or little information about their geographical origin, routes of colonisation, and ability to adapt to newly colonised environments and environmental change, for example, induced by human activities (Fraimout et al. 2017, Gross et al. 2021, Lavergne and Molofsky 2007) Evolutionary and demographic inferences may be obtained from population genetic analyses (Beichman et al. 2018, Estoup andGuillemaud 2010). For emerging species that are generally little studied before causing significant effects on ecosystems or native species, we need to develop new genetic markers because of no or few published genetic resources. This development is sometimes challenging because to obtain correct genetic estimates, markers should only amplify DNA of the species under investigation. Population genetic studies are often based on samples collected in the field. This sampling may cause, especially for micro-organisms, DNA contamination due to host or other associated micro-organisms in the samples (Ballenghien et al. 2017), or possible mistakes of identification due to the occurrence of cryptic species in the sampled area (i.e., species apparently identical morphologically but incapable of producing hybrids) (Gross et al. 2021, Mapondera et al. 2012, Queloz et al. 2011). In some cases, the situation is even more challenging when hybridization may occur among sympatric species or between invasive and native species (Sillo et al. 2015, Soghigian et al. 2020). However, the genotyping of multiple polymorphic markers may sometimes allow the identification of the different genetic lineages and their potential hybrids [START_REF] Altermann | How do you solve a problem like Letharia? A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPs from multilocus sequence data[END_REF], López-Vinyallonga et al. 2015). As late as the first decade of the 21 century, microsatellite markers (also called simple sequence repeats, SSR) were extensively used for population genetic analyses ( Selkoe and Toonen 2006). With the emergence of next-generation sequencing (NGS) technologies, single nucleotide polymorphism markers (SNP) have been increasingly used for population genetic studies.

Out of the methods used for SNP genotyping, genome-wide sequencing (GWS) allows to obtain thousands of markers by using high-throughput DNA sequencing on parts or whole genomes studied (Elshire et al. 2011). These methods can be powerful because they generate a huge quantity of genetic information. However, they may be costly in time and money, since they generally need good DNA quality for all the samples analysed, high sequencing coverage to identify duplicated genomic regions and several computational steps for removing putative false positive SNPs (Ribeiro et al. 2015). All these requirements can be difficult for some species having a genome with numerous repetitive elements, for species for which extraction of good quality DNA is not easy to obtain ( Dutech et al. 2020), or for research teams without bioinformatics expertise. In addition, the large genetic information generated by GWS methods may be beyond the need of only a few genetic markers to first estimate reproductive mode, gene flow, spatial structure, or origin of the expansion of emerging species (Brodde et al. 2019, Peccoud et al. 2008). Alternatively to GWS, several studies have shown that it is generally possible to isolate a few SNPs within introns of single-copy genes conserved in the genus or the family of targeted species (Feau et al. 2011, Ilves andLópez-Fernández 2014) or in ultraconserved elements (UCEs) (Blaimer et al. 2015, Bossert andDanforth 2018). The method developed by Feau et al. (2011) has been successfully applied to several fungal pathogens, and it also allowed to investigate population genetic structure, gene flow, and reproductive mode (Dutech et al. 2017, Dutech et al. 2020, Tsykun et al. 2017). Based on detecting a few single-copy genes in the genomes, this method has yielded less than fifty unlinked SNPs per study, and it has not been automated to reduce analysis time.

Therefore, the objective of the present software was to develop an automatic bioinformatics pipeline usable for a large number of species, especially focusing on emerging forest pest and pathogen species that are often orphan species (i.e., with no or poor published genomic resources). The pipeline hereafter called "SNP4OrphanSpecies" is designed to be easily installed and used by biologists, and based on limited genomic resources (i.e., one single shot-gun whole genome sequencing) in order to provide useful genetic markers for assignments to genetic lineages, identification of the origin of invading or expanding populations, and estimates of population diversity and structure.

Project description

Title: SNP4OrphanSpecies

Design description: The method to isolate single-copy genes for orphan species is based on an automated de novo genome assembly without step of manual curation, using st sequence data generated by paired-end Illumina sequencing technology. The assembly quality is checked by looking at some summary statistics (i.e., genome size, degree of assembly fragmentation, completeness of the genome). For isolating hundreds of SNPs, we focused on the single-copy genes conserved in genomes at a given taxonomic level (i.e., genus, family, or order). The focus on these conserved genes allows 1) to control for the taxonomic assignment of the analysed genomic regions, 2) to remove duplicated genes in the genome which can produce possible false positive SNPs, 3) to isolate several SNPs generally present in the introns of these genes, and 4) to yield several pools of pairs of primers for amplification of around 400bp sequences which can be amplified together in one multiplex (100 sequences per pool). By automation of these steps, the method decreases the time of genomic analysis, while it selects DNA sequences specific to the studied species (discarding sequences due to, for example, laboratory or field DNA contamination; Ballenghien et al. 2017). Based on the first polymorphic sequences obtained from this method in Feau et al. (2011) andDutech et al. (2016), we expect that the isolated sequences with intron regions are polymorphic within species, and valuable candidates for future SNPs detection.

The basic pipeline steps are illustrated in Fig. 1. The first step of the pipeline is the wholegenome de novo assembly using paired-end short reads obtained from Illumina technology. Although not tested in this study, minimum coverage of 10X is recommended for correcting sequencing errors, and probably 20X minimum to obtain a correct de novo assembly (Jiang et al. 2019). This step starts with a quality analysis of the raw data using FastQC v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads are trimmed by using a sliding window, and filtered using a minimum length with the software trimmomatic v0.39 (Bolger et al. 2014). The parameters for trimming are defined in the parameter file of the pipeline (Snakemake_Config_SNP4OrphanSpecies.yaml). Then a de novo assembly is performed using IDBA-UD v1. 1.3 (Peng et al. 2010). A basic statistics report is then generated on the final assembly using Quast v5.0.2 (Gurevich et al. 2013). For Fungi, Bacteria, and Viruses only, detection for DNA contamination can be performed by an automatic assignation of the assembled contigs, using Kaiju v1. 7.4 (Menzel et al. 2016). Additionally, for fungal species only, isolation of the internal transcribed spacer (ITS) can be performed from the de novo genome assembly with ITSx v1.1b (Bengtsson-Palme et al. 2013).

The second step of this pipeline evaluates the completeness of the assembly and identify the genes which will be used to isolate short sequences (400 bp hereafter called "amplicon"). In this objective, BUSCO v4.1.4 (Seppey et al. 2019) runs on the de novo assembly obtained in the previous step to identify single-copy genes conserved at the taxonomic level defined in the the configuration file (Snakemake_Config_SNP4OrphanSpecies.yaml). The more narrowly the taxonomic information is defined, the more specific are the isolated markers. Then, only complete and not duplicate genes are kept for the definition of amplicons. Optionnally, a taxonomic assignment of the selected genes can be done using Kaiju. Genes assigned to another taxon than the taxon set in the configuration file are removed from the final selection. This verification step requires big disk spaces (ideally 125GB for the nr_euk database) and is possible only for Fungi, Bacteria, and Viruses.

The last step of the pipeline is the isolation of amplicons to be amplified in pools. For this step, the amplicons are chosen to encompass at least one intron in the sequence. For each amplicon, a pair of primers is designed using a home-made Perl script integrating the program Primer3 (v2.5 Koressaar and Remm 2007), with stringent parameters favouring the possibility to be amplified jointly in a single multiplex PCR. All the designed primers are subjected to Blastn (v2.10, Altschul et al. 1990) against the de novo genome assembly to test for the specificity of the targeted sequences. Each pair of primers for which one of the two primers was found in at least two copies in the genome, was removed. One pair of primers is finally randomly selected per BUSCO gene to amplify a maximum of physically unlinked sequences. The validated primers are dispatched in several pools for which the primer dimer formation compatibility during a multiplex DNA amplification is tested in silico, using Primer Pooler (v1.71, Brown et al. 2017).

Web location (URIs)

Homepage: https://doi.org/10.15454/GWKRKY

Technical specification

Programming language: snakemake v6 or later and singularity v3 or later

Implementation

Implements specification

Keeping in mind biologist users, we implemented this pipeline with Snakemake (Köster and Rahmann 2012) and Singularity (Kurtzer et al. 2017). These softwares allow organizing the whole bioinformatics workflow within a container, with all the software and libraries needed, and the automatic achievement of each step of the analysis from the initial input data. This pipeline is easy to install, easy to use and can be run on all Linux machines, including highperformance clusters. The pipeline, its associated notice, and parameter files can be downloaded from the Portail Data INRAE (https://doi.org/10.15454/GWKRKY).

For running the pipeline, users only must 1) produce a paired-end Illumina whole-genome sequencing of the species of interest, and 2) set parameters (i.e., taxonomy, filtering, number of amplicons), in the file "Snakemake_Config_SNP4OrphanSpecies.yaml". A README available on https://doi.org/10.15454/GWKRKY gives more details about these different steps and parameters.

Audience

We consider that the time of bioinformatics analyses to isolate and to develop new markers is seriously reduced thanks to this pipeline, easily installed on a personal computer, without the need to acess to internet after this setting. Then, this method is especially dedicated to research teams, governmental agencies, or organisations, which have limited human and financial resources. With the short sequences provided by this pipeline, the possibility of obtaining the first genetic information on recently emerging populations without the high cost of genome sequencing should help to identify the origin of emergence, and the risk of adaptation to new ecosystems and define the best practices to manage new disease or pest species.

was likely introduced to Europe at the beginning of the 20th century (Gross et al. 2021). Genomic resources have recently been published for this obligate biotroph species (i.e., non-culturable on axenic media), for which DNA contamination was detected in the genome assembly (Dutech et al. 2020). Armillaria ostoyae (Romagn.) Herink 1973 (Basidiomycota) is distributed throughout the northern hemisphere, infecting numerous conifer species, causing large loss of wood (Heinzelmann et al. 2019) and is associated with some expanding populations in planted forests, as assumed in south-western France ( Labbé et al. After filtering and trimming Illumina raw reads, new de novo genome assemblies were produced by the pipeline for each species (details of each assembly are given in Table 1). These genome assemblies were strongly fragmented with small L50 and large N50 values (Table 1). As expected for contaminated DNA extraction (Dutech et al. 2020), the E. alphitoides genome assembly was one of the most fragmented genomes together with T. pityocampa. It confirmed the originally published surprisingly large estimate of the genome size for a powdery mildew species (317 Mb Vs. less than 110 Mb for other published powdery mildew genome assemblies; Frantzeskakis et al. 2019). For fungal species, Identifying the ITS1 sequences using the Kaiju nr_euk database confirmed that at least a part of the genome assembly may be assigned to the expected genus for each sequenced species (Suppl. material 1). The software ITSx used for this identification detected several ITS1 haplotypes in the E. alphitoides genome, which is congruent with the detection of several contigs of the genome assigned to different phyla or fungal families (Suppl. material 2). Between 98.6 % (D. sapinea) and 42 % (T. pityocampa) of conserved singlecopy genes listed in the Busco database were isolated from the genome assemblies (Table 2). For the analysed fungal species and using Kaiju, a variable proportion of genes were actually identified as different from the targeted genus, leading to the removal of between 70% (E. alphitoides) and 0.6 % (A. ostoyae) of the initial list of single-copy genes. In the last steps of the analysis, the pipeline defined in each species between 20,991 (A. ostoyae ) and 1,829 (E. alphitoides) short 400bp sequences (i.e., amplicons), encompassing at least one intron region. The design of the primer pairs for DNA amplification for each amplicon (only one per gene) and the control for their potential duplication in the genomes yielded a final set of between 614 (E. alphitoides) and 3,426 (A. ostoyae) primer pairs (Table 2). All these primer pairs are pooled in five pools for multiple DNA amplification. Depending on the final number of designed primer pairs, the redundancy rates of primer pairs among the primer pools for each species varied between 19 % (A. ostoyae) and 57.4 % (E. alphitoides) (Table 2). This redundancy can be manually optimized among pools when rates are too high. Primer Pooler was not designed to build several pools of primers simultaneously, and it may be useful to sequentially remove the pairs of primers used in the first pools to build the next pools.

Perspectives for evolutionary genetic studies of non-model invasive and emergent insect and pathogen species

The analysis of genomes from different phyla suggested that the method can be used for a large number of invasive or emergent insect and pathogen species for which genetic markers are searched for. Some limitations may occur for large genomes (i.e., several hundreds Mb), since the assembly, even without any curation steps, requires a minimum of computation resources. For example, the analysis of the T. pityocampa genome for which the size was estimated to be more than 500Mb, generated 268 GB (only 134 GB for the trimmed fastaQ files) and took more than 18 hrs on a Linux cluster using 20 CPUs. For such a large genome, it could be interesting to assess the method with a reduced wholegenome sequencing (i.e., a lower sequencing coverage or randomly amplified genome). Based on our results obtained from the highly fragmented and contaminated E. alphitoides genome assembly, we speculate that several hundreds of the conserved single-copy genes can be generally isolated by the present method, even from a low-quality or partial genome assembly. Another limitation would be the use of contaminated genome assemblies which may be quite frequent in whole-genome sequencing (Ballenghien et al. 2017), especially for not easily cultivated micro-organisms. The smallest number of validated sequences was obtained for E. alphitoides for which such contamination was assumed (Dutech et al. 2020) and confirmed in this study. When it involves genetically related species (for example between fungal species), such contamination may be difficult to identify and remove from the genomic data, because of the genetic similarity among the sequences. Using the Kaiju nr_euk database to assign the identified genes is then useful to detect this DNA contamination and discard the sequences with the incorrect taxonomic assignment. For the identification of conserved single-copy genes, a taxonomic determination is a sensitive parameter. An incorrect taxonomic identification at the genus level or higher of the emergent species may lead to discard several BUSCO genes present in the genome assembly but too different from the chosen reference genome. Conversely, if BUSCO genes are searched at a taxonomical level that includes the emergent species and a contaminating related species, some BUSCO genes non-specific to the emergent species may be selected despite the control performed by Kaiju. To avoid the selection of these unspecific genes, the isolated BUSCO genes can be evaluated again before the step of primers design. This evaluation can be done using the fasta files of the isolated genes (available in the outputs of the pipeline) and a phylogenetic reconstruction for each gene by extracting BUSCO genes of the taxonomical group studied from a database (for example, Mycocosm;Grigoriev et al. 2013) and the use of one of the numerous published phylogenetic software (see for example Dereeper et al. 2008).

Two strategies can then be developed after the isolation of these amplicons. The first one would be sequencing hundreds samples using one of the designed pools, and nextgeneration sequencers. The combination of SNPs identified in each amplicon can be treated as microhaplotypes (i.e., multi-SNP loci), potentially giving more power for population genetic analyses than analysing independent bi-allelic SNP loci (Baetscher et al. 2017). Microhaplotypes defined on short sequences, as those isolated by this pipeline, may be well adapted to identify fine population genetic structures, especially for field samples for which quality of DNA extraction is often poor (Morin et al. 2021), or for assignment of samples to populations and estimates of population admixture (McKinney et al. 2017). In a preliminary PCR test on 47 samples of D. sapinea, the majority of the 100 pairs of primers tested in multiplex allowed to obtain on average more than ten sequencing reads per sample and per amplicon. This last result is encouraging new tests on other pools of primers, and on other orphan species. A second option would be the genotyping of selected SNPs (one per locus), combined in different pools (i.e., plex), on several hundreds of samples, using for example, Mass-ARRAY technology by Sequenom (Chancerel et al. 2013, Dutech et al. 2017). Resequencing about ten genomes or partial genomes can first identified variations within each amplicon, and then, several SNP-plex can be designed for genotyping hundreds of samples (Dutech et al. 2016). Although the haplotypic information is missed in this case and this strategy of genotyping may introduce ascertainment bias due to choice of SNPs [START_REF] Albrechtsen | Ascertainment biases in SNP chips affect measures of population divergence[END_REF], it allows for genetically characterizing many samples from different geographical regions, a central objective when the genetic origin of emergent populations is investigated.

We are aware that SNPs isolated from conserved genes may be under selection. It may seriously affect the inferences of demographic dynamics of populations and should be carefully considered if historical scenarios are tested (Beichman et al. 2018). A first study having selected these conserved genes in Armillaria sp., detected 2 out of 20 tested ( Dutech et al. 2016). Notwithstanding this potential bias, we argue that for the first estimation of population genetic structures, or identification of the genetic origin of the emergent populations, the methodology presented here remains efficient. No significant difference in the estimates of genetic structures has been observed when comparing SSR and SNP loci isolated using this method in several European populations of A. cepistipes ( Tsykun et al. 2017). Basic statistics can also identify loci under selection (Vitalis 2003). Furthermore, because these loci are chosen in conserved genes at a given taxonomic level, the designed primers can theoretically amplify every species of this level. This robustness for DNA amplification is usefull for investigating genetic differentiation among closely related species and their hybrids [START_REF] Altermann | How do you solve a problem like Letharia? A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPs from multilocus sequence data[END_REF]. Some loci, such as SSR loci, are sometimes difficult to transfer even within the same genus (Dutech et al. 2007), and may produce many null alleles and missing data. By contrast, the short sequences obtained by this pipeline would be especially relevant for studying genetic diversity and differentiation among closely related species, because of the expected good repeatability of DNA amplification, the standardization of genotyping among species, experiments, or laboratories, as well as the assessment of the sequence orthology within genomes. They may be an efficient alternative to WGS methods for studying genetically related species or comparing, or combining, genetic studies produced by different studies or laboratories (see Harvey et al. 2016

  Operational system: Linux; Hardware requirements (Minimum): 32 GB of RAM, 1 CPU Interface language: Command line Repository Type: Dataverse Browse URI: https://data.inrae.fr/ Location: https://doi.org/10.15454/GWKRKY Usage licence Usage licence: Creative Commons Public Domain Waiver (CC-Zero)

  2017). The whole genome sequencing published by Sipos et al. (2017) was used for this study. Thaumetopoea pityocampa Denis & Schiffermüller 1775 (Lepidoptera) is expanding in Europe due to climatic changes, and causes significant defoliation in pine plantations and human health concerns (Battisti and Larsson 2015). The genome sequencing used to assess the pipeline has been published by Gschloessl et al. (2018). Bursaphelenchus xylophilus Steiner & Buhrer 1934, a pine wood nematode, infects several pine species, and was introduced from its native North America area to Asia and Europe, where it causes dramatic mortality in forests of the invaded areas (Vicente et al. 2011). The genome used for this study was published by Dayi et al. (2020).

Table 1 .

 1 for details).Description of the genome assemblies obtained for the five tested species. Data into brackets are from the original publication.

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		Nature 592 (7855): 571-576. https://doi.org/10.1038/s41586-021-03405-6
	•	Dutech C, Enjalbert J, Fournier E, Delmotte F, Barrès B, Carlier J, Tharreau D, Giraud T
		(2007) Challenges of microsatellite isolation in fungi. Fungal Genetics and Biology 44
		(10): 933-949. https://doi.org/10.1016/j.fgb.2007.05.003
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		identification of polymorphic sequences in non-model fungal species: the PHYLORPH
		method tested in Armillaria species. Forest Pathology 46 (4): 298-308. https://doi.org/
		10.1111/efp.12256
	•	Dutech C, Labbé F, Capdevielle X, Lung-Escarmant B (2017) Genetic analysis reveals
		efficient sexual spore dispersal at a fine spatial scale in Armillaria ostoyae, the causal
		agent of root-rot disease in conifers. Fungal Biology 121: 550-560. https://doi.org/
		10.1016/j.funbio.2017.03.001
	•	Dutech C, Feau N, Lesur I, Ehrenmann F, Letellier T, Li B, Mouden C, Guichoux E,
		Desprez-Loustau ML, Gross A (2020) An easy and robust method for isolation and
		validation of single-nucleotide polymorphic markers from a first Erysiphe alphitoides
		draft genome. Mycological Progress 19 (6): 615-628. https://doi.org/10.1007/
		s11557-020-01580-w
	•	Elshire R, Glaubitz J, Sun Q, Poland J, Kawamoto K, Buckler E, Mitchell S (2011) A
		robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species.
		PLOS One 6 (5). https://doi.org/10.1371/journal.pone.0019379
	•	Estoup A, Guillemaud T (2010) Reconstructing routes of invasion using genetic data:
		why, how and so what? Molecular Ecology 19 (19): 4113-4130. https://doi.org/10.1111/j.
		1365-294x.2010.04773.x
	•	Feau N, Decourcelle T, Husson C, Desprez-Loustau M, Dutech C (2011) Finding single
		copy genes out of sequenced genomes for multilocus phylogenetics in non-model fungi.
		PLOS One 6 (4). https://doi.org/10.1371/journal.pone.0018803
	•	Filipe JN, Cobb R, Meentemeyer R, Lee C, Valachovic Y, Cook A, Rizzo D, Gilligan C
		(2012) Landscape epidemiology and control of pathogens with cryptic and long-distance
		dispersal: Sudden oak death in northern Californian forests. PLOS Computational
		Biology 8 (1). https://doi.org/10.1371/journal.pcbi.1002328
	•	Fisher M, Henk DA, Briggs C, Brownstein J, Madoff L, McCraw S, Gurr S (2012)
		Emerging fungal threats to animal, plant and ecosystem health. Nature 484 (7393):
		186-194. https://doi.org/10.1038/nature10947
	•	Fraimout A, Debat V, Fellous S, Hufbauer R, Foucaud J, Pudlo P, Marin J, Price D,
		Cattel J, Chen X, Deprá M, François Duyck P, Guedot C, Kenis M, Kimura M, Loeb G,

Loiseau A, Martinez-Sañudo I, Pascual M, Polihronakis Richmond M, Shearer P, Singh N, Tamura K, Xuéreb A, Zhang J, Estoup A (2017) Deciphering the routes of invasion of Drosophila suzukii by means of ABC random forest. Molecular Biology and Evolution https://doi.org/10.1093/molbev/msx050 Figure 1.

Figure 1: steps of the SNP4OrphanSpecies pipeline

Table 2 .

 2 Summary of the genes and primers isolated by SNP4Orphanspecies pipeline for the five tested species. * one per gene and not duplicated in the genome.

Acknowledgements

We thank Pedro Crous (Westerdijk Fungal Biodiversity Institute, Netherlands) for providing us the Diplodia sapinea isolate and G. Sypos and C. Simang (University of Sopron, Hungary) for having sent us the raw sequencing data of the C17 Armillaria isolate genome. We also thank O. Lepais and the two reviewers for their comments on the previous versions of this manuscript that have greatly improved the quality of this manuscript. The genome sequencing of the Diplodia isolate was performed in collaboration with the GeT core facility, Toulouse, France (http://get.genotoul.fr). GeT was supported by France Génomique National infrastructure, funded as part of "Investissement d'avenir" program managed by Agence Nationale pour la Recherche (contract ANR-10-INBS-09). Preliminary tests were performed at the Genome Transcriptome Facility of Bordeaux (grants from the Conseil Regional d'Aquitaine n°20030304002FA and 20040305003FA, the European Union, FEDER n°2003227 and Investissements d'avenir, N°ANR-10-EQPX-16-01). Part of the computational resources and infrastructure used in the present publication were provided by the Bordeaux Bioinformatics Center (CbiB). This research has benefited from the European HOMED project and received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement N°771271.

Additional information

Pipeline assessment

We assessed the performance of the pipeline in a new de novo genome assembly of Diplodia sapinea isolate CBS117911. Diplodia sapinea or Sphaeropsis sapinea (fr.) Dicko & B. Sutton 1980 is a worldwide emergent fungal pathogen infecting many host trees, especially pine species (Brodde et al. 2019). A genomic library was constructed for this isolate using the Illumina TruSeq Nano DNA kit, following the company procedure. A total of 10,544,224 raw 150 bp paired-end reads were generated by an Illumina HiSeq3000 sequencer at the Get-Plage Genotoul facility (INRAE, France). In addition, we used this pipeline for analysing four other invasive species from different phyla, for which the genome assembly has been already published (Table 1). Erysiphe alphitoides (Griffon & Maubl.) U. Braun & S. Takam. 2000, (Ascomycota) infects many host plants worldwide and

Author contributions

BP designed the pipeline and wrote the informatic code and the manuscript, BL produced the Diplodia genome, helped in the development of the pipeline and wrote the manuscript, MM produced the Diplodia genome, CN supported all this collective work, FE helped to designed and developped the pipeline, CD designed the pipeline and wrote the manuscript.