Astrid Cruaud
email: astrid.cruaud@inra.fr

Sabine Nidelet

Pierre Arnal

Audrey Weber

Lucian Fusu

Alex Gumovsky

John Huber

Andrew Polaszek

Jean-Yves Yves Rasplus

Optimized DNA extraction and library preparation for minute arthropods: Application to target enrichment in chalcid wasps used for biocontrol

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

through the inference of statistically robust phylogenies. Limitations to routine target enrichment by research laboratories are both the complexity of current protocols and low input DNA quantity. Thus, working with tiny organisms such as micro-arthropods can be challenging. Here, we propose easy to set up optimisations for DNA extraction and library preparation prior to target enrichment. Prepared libraries were used to capture 1432 Ultra-Conserved Elements (UCEs) from microhymenoptera (Chalcidoidea), which are among the tiniest insects on Earth and the most commercialized worldwide for biological control purposes. Results show no correlation between input DNA quantities (1.8-250ng, 0.4 ng with an extra whole genome amplification step) and the number of sequenced UCEs. Phylogenetic inferences highlight the potential of UCEs to solve relationships within the families of chalcid wasps, which has not been achieved so far. The protocol (library preparation + target enrichment), allows processing 96 specimens in five working days, by a single person, without requiring the use of expensive robotic molecular biology platforms, which could help to generalize the use of target enrichment for minute specimens.

Key words : Chalcidoidea, library construction, low DNA quantity, micro-arthropods, target enrichment, UCEs.

INTRODUCTION

Enriching subsets of the genome prior to sequencing (target enrichment, [START_REF] Mamanova | Target-enrichment strategies for nextgeneration sequencing[END_REF]) allows effort to be concentrated on genomic regions that are relevant to answer specific research questions. Using this approach also contributes to reducing cost, as experimental design can be adapted to sequence many samples simultaneously. In the fields of ecology and evolution, target enrichment has been used for genotyping or phylogenomics of plant and animal species [START_REF] Gasc | Sequence capture by hybridization to explore modern and ancient genomic diversity in model and nonmodel organisms[END_REF][START_REF] Lemmon | High-throughput genomic data in systematics and phylogenetics[END_REF], to characterize phenotypic traits (e.g., [START_REF] Muraya | Targeted sequencing reveals large-scale sequence polymorphism in maize candidate genes for biomass production and composition[END_REF] or to explore microbial ecosystems [START_REF] Gasc | Hybridization capture reveals microbial diversity missed using current profiling methods[END_REF].

However, routine target enrichment by research laboratories is limited both by the complexity of current protocols, and by input DNA quantity that may be very low for some minute species (e.g. micro-arthropods < 1mm) and /or old /rare (museum) specimens. Indeed, current protocols (DNA extraction, library preparation, target-enrichment) are time consuming and require handling expertise. They have been initially developed to work with large amounts of input DNA (e.g., vertebrates or large/medium size insects; Faircloth et al. 2015;[START_REF] Mccormack | A Phylogeny of Birds Based on Over 1,500 Loci Collected by Target Enrichment and High-Throughput Sequencing[END_REF]) and include many purification steps that increase DNA loss. Working on hyperdiverse groups of microarthropods is challenging, as it requires one to perform the extraction on i) a large number of specimens/species to be representative of the overall diversity of the group, without the possibility of using pipetting robots that increase DNA loss, ii) single individuals because species complexes are frequent (Al [START_REF] Khatib | An integrative approach for species discrimination in the Eupelmus urozonus complex (Hymenoptera, Eupelmidae), with the descriptions of eleven new species from the West Palaearctic[END_REF][START_REF] Kenyon | Uncovering cryptic parasitoid diversity in Horismenus (Chalcidoidea, Eulophidae)[END_REF][START_REF] Mottern | Revision of the Cales noacki species complex (Hymenoptera, Chalcidoidea, Aphelinidae)[END_REF], iii) the whole insect without destruction for vouchering and often prior to species identification, iv) rare species that have been collected once and may be represented in collection by a few specimens or only one specimen and, sometimes, v) old and dry museum specimens used for species description (types).

In this study, we propose optimised protocols for DNA extraction and library preparation for target enrichment purposes, as well as a custom pipeline to analyse the sequence data obtained. We used these protocols and customised pipeline to capture and analyse Ultra Conserved Elements (UCEs) in minute wasps, the chalcids (Insecta: Hymenoptera: Chalcidoidea, [START_REF] Heraty | A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera)[END_REF][START_REF] Noyes | Universal Chalcidoidea Database[END_REF], that are key components of terrestrial ecosystems. Chalcids are key models for basic and applied research. With an estimated diversity of more than 500,000 species these microhymenoptera have colonised almost all extant terrestrial habitats. Many of them develop as parasitoids of arthropod eggs, larvae or pupae. As such, they are both key regulators of the populations of many other arthropod species in natural ecosystems and are increasingly used worldwide as biocontrol agents (e.g., Consoli et al. 2010;[START_REF] Heraty | Parasitoid biodiversity and insect pest management[END_REF]. A few of them, especially Nasonia (Pteromalidae) or Trichogramma (Trichogrammatidae) species are also used as model systems to answer challenging questions about sex determination, genetics of speciation, host-symbiont interactions or behavioural ecology (e.g., [START_REF] Pinto | Variation in reproductive compatability in Trichogramma and its taxonomic significance (Hymenoptera: Trichogrammatidae)[END_REF][START_REF] Stouthamer | Antibiotics cause parthenogenetic Trichogramma (Hymenoptera/ Trichogrammatidae) to revert to sex[END_REF][START_REF] Werren | The parasitoid wasp Nasonia: an emerging model system with haploid male genetics[END_REF]. Chalcidoidea has undergone a spectacular radiation resulting in a huge diversity of morphologies and sizes [START_REF] Gibson | Phylogenetics and classification of Chalcidoidea and Mymarommatoidea -a review of current concepts (Hymenoptera, Apocripta)[END_REF][START_REF] Heraty | A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera)[END_REF], but are generally small insects (< 2 mm long). Among them, Kikiki huna Huber (Mymaridae) at 158 µm long is the smallest winged insect currently known and the wingless male of Dicopomorpha echmepterygis Mockford at 139 µm is the smallest insect currently known [START_REF] Huber | A new genus and species of fairyfly, Tinkerbella nana (Hymenoptera, Mymaridae), with comments on its sister genus Kikiki, and discussion on small size limits in arthropods[END_REF]. Notably, most species used for biological control, belonging mainly to five families (Aphelinidae, Encyrtidae, Eulophidae, Mymaridae and Trichogrammatidae) are among the tiniest wasps on earth (< 1mm).

Their small size, huge diversity and widespread morphological convergence make chalcid wasps difficult to identify to species by non-expert taxonomists, which limits their use in biological control. Attempts have been made to resolve the phylogeny of the whole superfamily [START_REF] Heraty | A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera)[END_REF][START_REF] Munro | A molecular phylogeny of the Chalcidoidea (Hymenoptera)[END_REF] or a few families [START_REF] Burks | Combined molecular and morphological phylogeny of Eulophidae (Hymenoptera: Chalcidoidea), with focus on the subfamily Entedoninae[END_REF][START_REF] Chen | A molecular phylogeny of eurytomid wasps inferred from DNA sequence data of 28S, 18S, 16S, and COI genes[END_REF][START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and fig-pollinating wasps[END_REF][START_REF] Desjardins | Phylogeny of pteromalid parasitic wasps (Hymenoptera : Pteromalidae): Initial evidence from four protein-coding nuclear genes[END_REF][START_REF] Janšta | Torymidae (Hymenoptera, Chalcidoidea) revised: molecular phylogeny, circumscription and reclassification of the family with discussion of its biogeography and evolution of life-history traits[END_REF][START_REF] Owen | A molecular phylogeny of the Trichogrammatidae (Hymenoptera : Chalcidoidea), with an evaluation of the utility of their male genitalia for higher level classification[END_REF]) but none has succeeded. Indeed, the few markers that could be targeted with Sanger sequencing were not informative enough to solve deeper relationships. A study based on transcriptome data (3,239 single-copy genes) obtained from 37 species of chalcids and 11 outgroups also failed to solve relationships within the superfamily [START_REF] Peters | Transcriptome sequence-based phylogeny of chalcidoid wasps (Hymenoptera: Chalcidoidea) reveals a history of rapid radiations, convergence, and evolutionary success[END_REF]. As only a representative sampling in both markers and taxa will allow one to draw accurate conclusions on the history of this hyperdiverse group, target enrichment approaches appear relevant. More specifically, targeting UCEs and their flanking regions that have been proven useful to solve ancient and recent divergences [START_REF] Faircloth | Ultraconserved Elements Anchor Thousands of Genetic Markers Spanning Multiple Evolutionary Timescales[END_REF][START_REF] Smith | Target capture and massively parallel sequencing of ultraconserved elements for comparative studies at shallow evolutionary time scales[END_REF]) seems pertinent. Indeed, a set of probes has been developed to target UCEs in Hymenoptera (Faircloth et al. 2015). This set and an enriched one (Branstetter et al. 2017c) were successfully used to solve the phylogeny of a few groups of ants, wasps and bees for which the amount of DNA was not limiting [START_REF] Blaimer | Phylogenomic methods outperform traditional multi-locus approaches in resolving deep evolutionary history: a case study of formicine ants[END_REF]Blaimer et al. 2016a;Blaimer et al. 2016b;[START_REF] Bossert | The impact of GC bias on phylogenetic accuracy using targeted enrichment phylogenomic data[END_REF]Branstetter et al. 2017a;Branstetter et al. 2017b;[START_REF] Jesovnik | Phylogenomic species delimitation and host-symbiont coevolution in the fungus-farming ant genus Sericomyrmex Mayr (Hymenoptera: Formicidae): ultraconserved elements (UCEs) resolve a recent radiation[END_REF][START_REF] Prebus | Insights into the evolution, biogeography and natural history of the acorn ants, genus Temnothorax Mayr (hymenoptera: Formicidae)[END_REF]Ward & Branstetter 2017). Thus, contributing to the global effort to solve the Hymenoptera tree of life while addressing the challenge of the phylogeny of chalcid wasps seemed sound.

Here, we provide a detailed description of the optimised protocol for DNA extraction and library preparation, followed by a description of the phylogenetic trees obtained through target enrichment of UCEs from 96 species belonging to seven families and one subfamily of chalcids used for biological control (Aphelinidae, Azotidae, Encyrtidae, Eulophidae, Mymaridae, Pteromalidae: Eunotinae, Signiphoridae, Trichogrammatidae) as well as three outgroups in Mymmaromatidae, the putative sister group to Chalcidoidea [START_REF] Gibson | Diversity, Classification and higher relationships of Mymarommatoidea (Hymenoptera)[END_REF][START_REF] Heraty | A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera)[END_REF].

MATERIALS AND METHODS

Sampling

Samples were taken from the personal collections of the co-authors of this paper, or borrowed from the Queensland Museum (Australia) or the Australian National Insect Collection, Canberra. Details of the samples included in the analysis are presented in Table S1. Most specimens sampled in the field were placed directly into ethanol for storage. On average, specimens spent 3.5 years in ethanol before being processed (maximum storage time in alcohol = 34 years). Two specimens were critical point dried 25 or 34 years ago. UCE data for three specimens were retrieved from a previous study (Branstetter et al. 2017a

DNA extraction

DNA was extracted using the Qiagen DNeasy Blood and Tissue kit. All extractions were conducted without destruction of the specimens' external (and certain internal) structures, with digestion and lysis of just the soft tissues. In this way, actual or potential type specimens are preserved. An often-essential feedback to the morphology is also preserved which is critical in this difficult group. The following modifications were made to manufacturer's protocol. Samples were incubated overnight in an Eppendorf thermomixer (temperature = 56°C, mixing frequency = 300 rpm). To increase DNA yield, two successive elutions (50 µL each) were performed with heated buffer AE (56°C) and an incubation step of 15 minutes followed by centrifugation (8000 rpm for 1 minutes at room temperature). Eppendorf microtubes LoBind 1,5ml were used for elution and to store DNA at -20°C until library preparation. DNA was quantified with a Qubit® 2.0 Fluorometer (Invitrogen). The final version of the DNA extraction protocol is available as an additional file 1. Vouchers were deposited at CBGP, Montferrier-sur-Lez, France or returned to their owner.

Whole genome amplification

DNA extracted from two specimens was subjected to ethanol precipitation and whole genome amplification (WGA) using the GenomiPhi™ V2 DNA Amplification kit (GE Healthcare) as described in Cruaud et al. (in press). 1µl of concentrated DNA was used as input (i.e. 4 ng or 0.4 ng, Table S1).

Library preparation.

Our starting point was the protocol described in http://ultraconserved.org and Faircloth et al. (2015). The final goal was to obtain a standardized protocol that could be implemented by one person and which, in 5 working days, would made possible the manual preparation of libraries and the capture of UCEs from 96 samples in parallel. Steps by steps optimisations were made specially to remove time-consuming purification steps and different reagents were tested. The final version of the protocol is available as additional file 2. Briefly, input DNA was sheared to a size of ca 400 bp using the Bioruptor® Pico (Diagenode). End repair, 3'-end adenylation, adapters ligation and PCR enrichment were then performed with the NEBNext Ultra II DNA Library prep kit for Illumina (NEB). We used barcoded adapters that contained amplification and Illumina sequencing primer sites, as well as a nucleotide barcode of 5 or 6 bp long for sample identification (additional file 3). Pools of 16 samples were made at equimolar ratio. We enriched each pool using the 2749 probes designed by Faircloth et al. (2015) using MYbaits kits (MYcroarray, Inc.). We followed manufacturer's protocol (MYbaits, user manual version 3, http://www.mycroarray.com/pdf/MYbaits-manual-v3.pdf).

The hybridization reaction was run for 24h at 65°C. Post enrichment amplification was performed on beads with the KAPA Hifi HotStart ReadyMix. The enriched libraries were quantified with Qubit, an Agilent Bioanalizer and qPCR with the Library Quantification Kit -Illumina/Universal from KAPA (KK4824). They were then pooled at equimolar ratio. Pairedend sequencing (2*300bp) was performed on an Illumina Miseq platform at UMR AGAP (Montpellier, France) to get longer flanking regions and, as a consequence, more information to differentiate closely related species.

Raw data cleaning

The analytical workflow is summarized in figure S1. In the next paragraph, chosen parameter values (different from default value) are provided between parentheses. Quality control checks were performed on raw sequence data with FastQC v.0.11.2 [START_REF] Andrews | FastQC -A Quality Control application for FastQ files[END_REF]. Quality filtering and adapter trimming were performed with Trimmomatic-0.36 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] (LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:180, with PrefixPE/1= AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGA TCT and PrefixPE/2 = CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTC CGATCT). Overlapping reads were merged using FLASH-1.2.11 (-M 300) [START_REF] Magoc | FLASH: fast length adjustment of short reads to improve genome assemblies[END_REF]. Demultiplexing was performed using a bash custom script (no mismatch in barcode sequences was allowed, additional file 4). Assembly of cleaned reads was performed using CAP3 (-i 25 -o 25 -s 400) [START_REF] Huang | CAP3: A DNA sequence assembly program[END_REF]. The 2749 probes designed by Faircloth et al. (2015) were assembled into non-overlapping UCEs (hereafter called reference UCEs, n=1432, additional files 5 and 6) using Geneious 8.1.8 [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF]) and contigs were aligned to this set of reference UCEs using LASTZ Release 1.02.00 [START_REF] Harris | Improved pairwise alignment of genomic DNA[END_REF].

Contigs that aligned with more than one reference UCE and different contigs that aligned with the same reference UCE were filtered out using Geneious 8.1.8.

Data analysis

UCEs for which sequences were available for more than 25% of the taxa were kept in the next steps of the analysis. Alignments were performed with MAFFT v7.245 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]) (-linsi option). Ambiguously aligned blocks were removed using Gblock_0.91b with relaxed constrains (-t=d -b2=b1 -b3=10 -b4=2 -b5=h) [START_REF] Talavera | Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments[END_REF]. The final data set was analysed using supermatrix approaches and coalescent-based summary methods. Two gene tree reconciliation approaches were used: ASTRAL-III v5.6.1 [START_REF] Zhang | ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees[END_REF], which computes the phylogeny that agrees with the largest number of quartet trees induced by the set of input gene trees and ASTRID [START_REF] Vachaspati | ASTRID: Accurate Species TRees from Internode Distances[END_REF] which takes a set of gene trees, computes a distance matrix (ca sum of number of edges in the path between two samples divided by the number of gene trees in which the two samples are represented) and infers a phylogeny from this distance matrix. Following recommendations for incomplete distance matrices, BioNJ was used to compute the phylogeny. Individual trees were inferred from each UCE using raxmlHPC-PTHREADS-AVX [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] (version 8.2.4; -f a -x 12345 -p 12345 -# 100 -m GTRGAMMA). ASTRAL and ASTRID analyses were performed with 100 multi-locus bootstrapping (MLBS, site-only resampling [START_REF] Seo | Calculating bootstrap probabilities of phylogeny using multilocus sequence data[END_REF]). Phylogenetic trees were estimated from the concatenate, unpartitioned data set using Maximum Likelihood (ML) approaches as implemented in RAxML and IQTREE v1.6.4 (Nguyen et al. 2015). For the RAxML analysis, a rapid bootstrap search (100 replicates) followed by a thorough ML search (-m GTRGAMMA) was performed. For the IQTREE analysis, a ML search with the best-fit substitution model automatically selected was performed with branch supports assessed with ultrafast bootstrap [START_REF] Minh | Ultrafast approximation for phylogenetic bootstrap[END_REF]) and SH-aLRT test [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF]) (1000 replicates).

Summary statistics for all data sets (alignment length, number of samples, number of variable sites, number of parsimony informative sites etc.) were calculated using AMAS [START_REF] Borowiec | AMAS: a fast tool for alignment manipulation and computing of summary statistics[END_REF]. Tree annotation was performed with TreeGraph 2.13 [START_REF] Stöver | TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses[END_REF].

Linear correlation between the number of UCEs and the quantity of DNA used to build the library was tested with the Pearson's correlation coefficient in R (R Core Team 2015).

Analyses were performed on a Dell PowerEdge T630 with 10 Intel Xeon E5-2687 dual-core CPUs (3.1 GHz, 9.60 GT/s), 125 Go RAM and 13 To hard drive and on the Genotoul Cluster (INRA, Toulouse).

RESULTS

Optimisations made for DNA extraction are detailed in the additional file 1. The final version of the library preparation protocol is available as additional files 2 and 3. Hereafter, the range of values provided between parentheses refers to the range of data that fall between the 2.5th percentile (LB=lower bound) and 97.5th percentile (UP = upper bound). Table S1 contains sequencing information for all samples. The median amount of input DNA was 25ng (LB=1.8ng; UP=250ng). An average of 76,330 reads (cleaned and merged) per sample was obtained (LB=3,359;UP=348,326). The average number of contigs was 3,454 (LB=546; UP=14,012) and the average sequencing depth was 18X (LB=3X; UP=44.0X). The average number of UCEs obtained per sample after filtering of problematic contigs was 687 (LB=193-UP=1,082) with a length comprised between LB=315 and UP=816bp (mean=603bp). The final dataset (25%-complete matrix; i.e. at least 25 taxa on the 99 should have a sequence to keep the locus in the analysis) comprised 1,139 UCEs and 340,286bp (missing data = 47.0 %; parsimony informative sites = 72.3 %; GC-content = 42.6%).

Specimens retrieved from a previous study (Branstetter et al. 2017a) that were either represented by empirical data (Euplectrus sp.) or UCEs extracted from published genomes (Copidosoma floridanum and Trichogramma pretiosum) displayed a number of UCEs comparable to what was obtained for other specimens. Their placement in the trees (Figures 2; S3-S5) was in accordance with their morphology. Whatever the method used (supermatrix approaches versus coalescent-based summary methods), all families, except for Aphelinidae, were recovered as monophyletic with high support. Aphelinids were split into three groups: 1) a monophyletic Aphelininae + Eretmocerinae; 2) Coccophaginae; 3) Cales sp. (Calesinae).

The position of Cales was ambiguous. Cales was either recovered as sister to Trichogrammatidae (RAxML, low support) or as a lineage distinct from all other chalcidoids (all other analyses). Except for Mymaridae that was strongly placed as sister to all other Chalcidoidea in all analyses, the tree backbone remained poorly resolved. Statistical support was much higher within families. In all analyses Azotidae clustered with Signiphoridae, with strong support.

DISCUSSION

To our knowledge this study is the second after Sproul and Maddison (2017) to demonstrate success in library preparation from such low input using commercial kits, and the first to report successful sequencing of >1000 low copy genes in 96 specimens in parallel, from such low input and processing time. Our optimisations differ from what was proposed by Sproul and Maddison (2017). First, we tried to optimize DNA extraction itself by using overnight lysis with gentle mixing to preserve fragile specimens, heated elution buffer and increased incubation time before elution. Second, instead of increasing the number of time-consuming purification steps we decreased them. It is noteworthy that in this library only two historical specimens were included. This may have masked challenges posed by adapter dimers [START_REF] Burrell | The use of museum specimens with highthroughput DNA sequencers[END_REF]Sproul & Maddison 2017;[START_REF] Tin | Sequencing degraded DNA from nondestructively sampled museum specimens for RADtagging and low-coverage shotgun phylogenetics[END_REF]) that led Sproul and Maddison (2017) to add a second bead clean-up prior to library amplification. However, we have already used this protocol on hundreds of chalcid and moth species, including historical specimens that were processed the same way as fresh ones and we never had such an issue. Finally, instead of increasing the number of amplification cycles prior to target enrichment, we used a new generation mastermix including a hot start, processive and high-fidelity polymerase (NEBNext Ultra II Q5 Master Mix). Our protocol also allows one to back-up DNA at several steps that allows for multiple attempts without delay in case the first attempt fails. Finally, sequencing was performed on a MiSeq to get longer flanking regions and, as a consequence, more information to differentiate closely related species.

The protocol was successfully used on minute chalcid wasps widely used for biological control purposes. Up to 1165 valid UCEs were captured from 25ng DNA (median amount of DNA used for this study). No correlation was observed between the quantity of DNA used for library preparation and the number of captured UCEs. The average number of captured UCEs validated by our quality control workflow was 687, and 685 valid UCEs were captured from a tiny aphelinid (1.8ng of input DNA). The number of UCEs obtained per individual appears to drop within the basal clades (i.e., Mymaridae and Trichogrammatidae, Figure S2), a result probably linked to the relatively long branches observed in these groups, and that could reduce the efficiency of the probes that were designed from the genome of Nasonia (Faircloth et al. 2015). Trees were well resolved at the family level, with high statistical support, showing the potential of UCEs to solve long-standing taxonomic issues. However, the tree backbone remained unresolved, a pattern that confirms the rapid diversification of the group [START_REF] Heraty | A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera)[END_REF][START_REF] Peters | Transcriptome sequence-based phylogeny of chalcidoid wasps (Hymenoptera: Chalcidoidea) reveals a history of rapid radiations, convergence, and evolutionary success[END_REF]. Understanding the evolutionary history of the group was not the purpose of this paper. Indeed, only a representative sampling in both markers and taxa as well as cutting-edge data analysis will allow drawing accurate conclusions. By providing suitable tools for a fast, easy and affordable acquisition of data, this paper is a first step.

Interestingly, as it has been shown previously on RADseq data (Cruaud et al. in press), WGA does not seem to bias the results even when input DNA used for the WGA is below the recommendation on the manufacturer kit (here 0.4 and 4 ng when the Genomiphi kit V2 requires 10 ng). This further increases the possibilities opened by our protocol as input DNA as low as 0.2-0.3 ng may be used when an extra WGA step is included after DNA extraction (Cruaud et al. in press). It is noteworthy that reducing the amount of DNA required for library preparation allows one to use extracted DNA for different approaches in parallel (RADseq, amplicon, Shotgun etc.). This also allows one to send DNA back to museums from which specimens were borrowed, for archival purposes. We definitely agree with Sproul and Maddison (2017) who emphasize how important it is not to waste DNA obtained from irreplaceable specimens (whether fresh or historical). It is even more important to capitalize on existing collections as collecting samples for large-scale studies may be more and more difficult, given that many countries have imposed restrictive access regulations, even to academic researchers, to reduce the risk of supposed biopiracy (Divakaran Prathapan et al.

2018).

All the elements discussed above indicate that this protocol may be of great help to reconstruct phylogenetic hypotheses in multiple groups of tiny arthropods, e.g., springtails, sandflies, lice, whiteflies, mites, etc. Coupled or not with WGA, some steps of our protocol may also contribute to simplify and hasten construction of other reduced-representation libraries (RAD-Seq, ddRAD-Seq, etc). These methods may then be used to analyse species relationships, hybridization or population genomics [START_REF] Cruaud | Empirical assessment of RAD sequencing for interspecific phylogeny[END_REF][START_REF] Eaton | Inferring phylogeny and introgression using RADseq data: An example from flowering plants (Pedicularis: Orobanchaceae)[END_REF][START_REF] Emerson | Resolving postglacial phylogeography using high-throughput sequencing[END_REF][START_REF] Gagnaire | Mapping phenotypic, expression and transmission ratio distortion QTL using RAD markers in the Lake Whitefish (Coregonus clupeaformis)[END_REF][START_REF] Hipp | A framework phylogeny of the american oak clade based on sequenced RAD data[END_REF]) on minute arthropods or on endangered species using non-invasive DNA sampling [START_REF] Vila | Effect of non-lethal sampling on life-history traits of the protected mothGraellsia isabelae(Lepidoptera: Saturniidae)[END_REF]. help of AC, JYR, LF; sequenced libraries: AW; analysed data: AC, JYR; drafted the manuscript: AC, JYR. All authors commented on the manuscript.

TABLES AND FIGURES

Supplementary material to:

Optimised DNA extraction and library preparation for minute arthropods: application to target enrichment in chalcid wasps used for biocontrol. Astrid Cruaud 1* , Sabine Nidelet 1 , Pierre Arnal 1, 2 , Audrey Weber 3 , Lucian Fusu 4 , Alex Gumovsky 5 , John Huber 6 , Andrew Polaszek The DNA quantity used to build the library as well as the number of UCEs analysed for each sample is given in brackets.

Figure S4

ASTRAL tree obtained from the analysis of the 25%-complete data set (page 139) Bootstrap supports (site-only resampling) are indicated at nodes. The DNA quantity used to build the library as well as the number of UCEs analysed for each sample is given in brackets.

Figure S5

ASTRID tree obtained from the analysis of the 25%-complete data set (page 140) Bootstrap supports (site-only resampling) are indicated at nodes. The DNA quantity used to build the library as well as the number of UCEs analysed for each sample is given in brackets.

Additional file 1: Optimized protocol for DNA extraction

Optimisations were done to the DNeasy Blood & Tissue Kit (250) protocol (Qiagen)

Consumables

Tweezers and fine brush/or forceps to manipulate specimens; 1 pot with bleach at a 1:10 dilution with osmosis cleaned water; 1 pot with osmosis cleaned water to wash tweezers/forceps, cup, paper towels, sterile gloves, 50ml Falcon™ tube, 1.5ml Safe-lock tubes (Eppendorf), 1,5ml Microtubes DNA LoBind (Eppendorf).

---------day 1 / afternoon---------

• Label 1.5ml safe-lock tubes with sample codes. Close caps. Put under UV light for 10 min.

• Equilibrate frozen specimens to room temperature before processing.

• Dry specimens on clean paper towel to remove EtOH. Place specimen in tube. Clean forceps to avoid contamination between 2 different specimens: bleach, then water, then dry with paper towel.

• Add 200µl buffer ATL (Qiagen) to each tube. (NB DNeasy® Blood & Tissue Handbook uses 180µl; we use 200µl to improve diffusion of the DNA from the specimen)

• Add 20µl proteinase K to each tube (or prepare a master mix of buffer ATL + proteinase K and add master mix to tubes.)

• Vortex + Pulse spin. (NB if the specimens are fragile, i.e. old and dry mounted collection specimens, dispense the master mix of buffer ATL + proteinase K to each tube and then add specimens; or dispense specimens to tubes and then add the master mix. Do not vortex at all to avoid damage to specimens and place the tubes directly in the thermomixer after ensuring that the specimens are in the liquid.)

• Place tubes in thermomixer (Eppendorf) at 56°C and 300 rpm overnight.

---------day 2 / morning---------

• Remove tubes from thermomixer.

• Vortex + pulse spin. (NB if working with fragile specimens do not vortex. Pulse spin, transfer all the liquid to a new labelled tube leaving the specimen behind and then proceed to the next step.) (DNeasy® Blood & Tissue Handbook, p. 15, recommends using carrier DNA or RNA for samples containing less than 5 µg of DNA. Though this was not used for this publication, poly (A) RNA (2 µl of 2 µg/µl) is added to the lysate before the next step by one of us (LF) for routine DNA purification from small or old specimens. This should not interfere with library preparation as it was used for example by Sproul & Maddison (2017) as recommended in the QIAamp DNA Micro Kit by Qiagen.)

• Add 200µl buffer AL to each tube (kit Qiagen). (DNeasy® Blood & Tissue Handbook,p. 19,29 recommends adding RNase A if RNA-free genomic DNA is required. We do not use it because the copurified RNA is not interfering with target enrichment anyway and because RNase A also degrades DNA; see [START_REF] Donà | Expect the Unexpected -RNase A may still catch you by surprise Labtimes 1[END_REF].)

A white precipitate may appear that will dissolve during incubation at 70°C. • Vortex + pulse spin.

• Incubate tubes in thermomixer at 70°C and 300 rpm for 10 minutes.

• During that time, prepare DNeasy mini spin Qiagen columns and 1.5 ml DNA LoBind Eppendorf tubes (label tubes with sample codes). (NB LoBind tubes minimize DNA loss during storage by reducing sample-to-surface binding.)

• Aliquot buffer AE in Eppendorf tubes and heat at 56°C in thermomixer (heated buffer will improve the release of DNA from the resin.)

• Add 200µl absolute ethanol (not provided with the Qiagen kit) to each tube. (NB only high purity ethanol should be used; cheap ethanol may contain traces of other chemicals that will interfere with the solubilisation of the DNA from the membrane in the last step.)

• Vortex + Pulse spin.

• Pipette the liquid (including precipitate) from each tube and transfer into a DNeasy mini spin Qiagen column placed in a 2 ml collection tube (Qiagen kit).

• Centrifuge columns + collection tubes at 6000 x g (8000 rpm) for 1 minute; discard the flow-through and collection tubes. Keep the columns.

• Place spin columns in new collection tubes.

• Add 500µl buffer AW1 (kit Qiagen).

• Centrifuge at 6000 x g (8000rpm) for 1 minute; discard the flow-through and collection tubes. Keep the columns.

• Place spin columns in new collection tubes.

• Add 500µl buffer AW2 (kit Qiagen).

• Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the columns.

• Rotate columns to 180 degrees in their collection tubes and centrifuge again at 20000 xg (14000rpm) for 3 minutes (this will make sure the column is well dried).

• Place dried spin columns in 1.5ml LoBind tubes that you previously labelled.

• Add 50µl of heated AE buffer. Deposit buffer right in the middle of the resin.

• Incubate 15 min at room temperature (meanwhile, reheat buffer AE.) • Centrifuge at 6000 x g (8000 rpm) for 1 minute.

• Rotate columns to 180 degrees and centrifuge again at 6000 x g (8000 rpm) for 1 minute.

• Remove columns + tubes from the centrifuge.

• Add again 50µl of heated buffer right in the middle of the resin.

• Incubate 15 min at room temperature.

• Centrifuge at 6000 x g (8000 rpm) for 1 minute at room temperature.

• Rotate columns to 180 degrees and centrifuge again at 6000 x g (8000 rpm) for 1 minute.

• DNA (ca 2 x 50µl) is ready for use.

• Add distilled water to the extracted specimens and incubate at room temperature for 30 min.

(NB water is used to eliminate the residual salts left by the buffers. Otherwise they usually crystallise on the specimen upon storage in ethanol.)

• Remove water and replace with ethanol. Keep specimens in the dark in a freezer until mounting. (NB if the specimens are stored at room temperature under ambient light they will become sun-bleached very quickly, much quicker than unextracted specimens.)

• Use a critical point dryer, hexamethyldisilazane, acetone or amyl acetate to dry the specimens before mounting. Do not air dry as they will collapse.

Additional file 2: Optimized protocol for library preparation 1. DNA quantification

Qubit® 2.0 Fluorometer (Invitrogen); dsDNA HS Assay Kit.

DNA normalization and shearing

Bioruptor ® Pico, sonication bath-based rotor 12 samples in parallel.

Transfer 100µl of input DNA in tubes adapted to the Bioruptor ® Pico that will be used for DNA shearing (Diagenode tubes 0.5ml). [now we shear 30ng DNA in routine but you can use as little as 1.8 ng, see main text] Set up the following program for DNA shearing to obtain a mean fragment size of ca 400pb: 15sec ON / 90sec OFF for 8 cycles.

You can STOP here for up to 1 week (store tubes in a freezer at -20°C).

The NEBNext Ultra II DNA Library prep kit for Illumina by NEB will be used for End repair, A tailing, Adapter ligation and PCR enrichment of adaptor-ligated DNA.

End repair + A tailing

To keep a backup, only 50 µl of the sheared DNA is used [you may want to re-concentrate DNA on beads in 50 µl when you have a small amount i.e. < 5 ng] Vortex master mix gently + pulse spin.

-Add 10µl of master mix to tubes containing 50µl of sheared DNA.

-Vortex (mix well.) -Pulse spin.

-Place in a thermocycler. Enable heated lid and run the following program: 30 min at 20°C 30 min at 65°C Hold at 4°C.

PROCEED WITHOUT DELAY TO ADAPTER LIGATION

Adapter ligation and cleanup

See additional file 3 for adapter sequences and hybridization.

Thaw adapters (1.5µM). Centrifuge to avoid cross contamination. Open carefully.

For 96 samples use the following tagging scheme:

P1-1 P1-9 P1-1 P1-9 P1-1 P1-9 P1-1 P1-9 P1-1 P1-9 P1-1 P1-9 P1-2 P1-10 P1-2 P1-10 P1-2 P1-10 P1-2 P1-10 P1-2 P1-10 P1-2 P1-10 P1-3 P1-11 P1-3 P1-11 P1-3 P1-11 P1-3 P1-11 P1-3 P1-11 P1-3 P1-11 P1-4 P1-12 P1-4 P1-12 P1-4 P1-12 P1-4 P1-12 P1-4 P1-12 P1-4 P1-12 P1-5
P1-13 P1-5 P1-13 P1-5 P1-13 P1-5 P1-13 P1-5 P1-13 P1-5 P1-13 P1-6 P1-14 P1 (NB samples can be stored overnight at -20°C.)

Clean-up adaptor ligated DNA (remove dimers of adapters):

Purification was conducted with the Agencourt AMPure XP Purification system (Beckman Coulter) and a ALPAQUA magnet plate.

Pull Ampure beads out of fridge. Make sure they are at room temperature (RT) before use.

-Vortex Ampure beads. -Move strip back to magnetic rack and wait for 2 to 5min.

-Transfer the 15µl of DNA to a new strip.

PCR enrichment of adaptor-ligated DNA

Quantification, equimolar pooling and re-concentration

Qubit -quantify 2 µl of each library.

In our experience, a clean-up step of the librairies is not necessary.

Prepare 6 pools of 16 samples in 1.5ml LoBind tubes.

Pool 100 ng of each library (you want a final quantity of 100-500ng DNA per pool after concentration to fit with the requirements of the MYbaits® protocol.)

On beads Concentration of DNA (Agencourt AMPure XP Purification system (Beckman Coulter)). Concentration of DNA is required, as MyBaits require 7 µl of starting material.

Remove Ampure beads from fridge. Make sure they are at room temperature (RT) before use.

-Vortex Ampure beads.

-Add 0.8 vol. of Ampure beads for 1 vol. of DNA.

-Vortex + pulse spin.

-Incubate for 5min at RT.

-Move tubes to magnetic rack and wait for 2 to 5 min.

-Remove and discard supernatant without disturbing beads.

- Qubit quantify 1 µl of each library (dilution 1:2).

Check library profile on an Agilent Bioanalyzer with a High Sensitivity DNA Analysis Kit (load 1µl of library dilution 1:2).

Capture of UCEs

UCEs were captured on each pool using the probes designed by Faircloth et al. (2015) and synthetisized by MYbaits®. Capture was performed with the MYbaits® kit following manufacturer's instruction (http://www.mycroarray.com/pdf/MYbaits-manual-v3.pdf).

PCR Enrichment of captured libraries and final clean-up

At this step of the process we have obtained libraries and Streptavidin beads mixed in 30 µl of 10mM Tris-Cl, 0.05% TWEEN®-20 solution (pH 8.0 -8.5).

Here we want to release the bead-bound UCE-enriched library from the baits and amplif the resulting fragments.

PCR enrichment of the captured fragment is performed on beads with the following mix. We performed 2 PCR reactions for each pool of samples.

Remove Streptavidin beads:

Pool the 2 PCR products obtained for each pool of samples in DNA LoBind tubes (1.5ml.) Move the 6 tubes to a magnetic rack and wait for 2 to 5min.

Transfer supernatant to new tubes.

Final Cleanup (Agencourt AMPure XP Purification system (Beckman Coulter)

Remove Ampure beads from fridge. Make sure they are at room temperature (RT) before use.

-Vortex Ampure beads.

-Add 1V of Ampure beads for 1V of DNA (ie 100 µl of beads for 100 µl of DNA.) -Vortex + pulse spin.

-Incubate for 5min at RT.

-Move tubes to magnetic rack and wait for 2 to 5min.

-Remove and discard supernatant without disturbing beads. -Move strip back to magnetic rack and wait for 2 to 5min.

-Transfer the 25µl of DNA to new LoBind tubes.

Final quantification and equimolar pooling prior to sequencing

-Qubit Quantify 2µl of each.

-Check library profile on an Agilent Bioanalyzer with a High Sensitivity DNA Analysis Kit (1µl undiluted library). -Perform KAPA quantification.

-Equimolar pooling of the 6 librairies.

Additional file 3

Adapters (Ultramer Oligos, TruGrade synthesis, Standard Desalting) were synthesized by IDT (Integrated DNA Technologies, Inc.) /5Phos/ = 5' Phosphorylation; * = Phosphorothioate Bond, F=forward, R=reverse).

Adapter were hybridized as follows:

25µM oligo preparation:

-Thaw 100µM oligos. Vortex and spin well.

-Pool each pair of oligos (F/R) in DNA LoBind tubes (1.5ml) as follows: -Vortex and spin well the oligo's pools and split each pool in tow PCR tubes (0.2ml)

Adapters hybridization:

-Place the PCR tubes in a thermocycler:

Program: 15 cycles (with -1.7°C/cycle) 40 cycles (with -1°C/cycle) Time 2min 1min 1min ∞ Temperature 95°C 95°C 69°C 10°C
-Vortex and spin well the tubes.

-Qubit quantify 2µl of diluted adapters (1/20e.) -Adjust adapters concentration to 1.5µM if necessary and make aliquots.

-Store the aliquots at -20°C.

Adapters (1.5µM) were stored in tube strips for usage comfort. #use the barcodes.txt file to generate grep commands to find occurrences of barcodes at the beginning and end of each reads #and redirect output to fastq files named after your sample codes awk 'BEGIN{FS="\t";RS="\n"} {if (NR>1){print "grep -B 1 -i \047^"$2"\.*"$4"\$\047 input.extendedFrags.fastq > "$1"_fq"}}' /AbsolutePathTo/barcodes.txt |bash # remove barcodes from beginning / end of reads awk 'BEGIN{FS="\t";RS="\n"} {if (NR>1){print "sed -i \047s/^"$2"//g\;s/"$4"\$//g\047 "$1"_fq"}}' /AbsolutePathTo/barcodes.txt |bash

#id barcode sequence P1-1_F AAGTG AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGTG*T P1-2_F ACAAT AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACAAT*T P1-3_F CTAGA AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTAGA*T P1-4_F GATAG AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGATAG*T P1-5_F GCCTC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTC*T P1-6_F TGTGT AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTGT*T P1-7_F GTATT AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTATT*T P1-8_F CGGAC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGGAC*T P1-9_F AAACAA AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAACAA*T P1-10_F ACGTCA AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACGTCA*T P1-11_F CGTCTC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTCTC*T P1-12_F CCCAAA AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCCAAA*T P1-13_F GTTCGG AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTCGG*T P1-14_F TGCCCA AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCCCA*T P1-15_F TTGGGC AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGGGC*T P1-16_F

  ): Euplectrus sp. (empirical data); Copidosoma floridanum and Trichogramma pretiosum (in silico extraction of UCEs from genomes).

Figure 1

 1 Figure 1 and S2 show the variation of the number of UCEs obtained with regard to the

Figure 1 .

 1 Variation of the number of UCEs with regard to the amount of input DNA (ng).

Figure 2 .

 2 Figure 2. RAxML tree obtained from the analysis of the 25%-complete data set. Black squares indicate node supported with RAxML BP = 100, IQTREE aLRT = 100 / BP = 100 and ASTRAL/ASTRID BP > 75. Grey square indicates node with RAxML BP = 100, IQTREE aLRT = 100 / BP = 100 and ASTRAL BP > 75. White squares indicate nodes with RAxML BP > 95 and IQTREE aLRT > 80 / BP > 95. The DNA quantity used to build the library as well as the number of UCEs analysed for each sample is given in brackets. Photos ©J.-Y. Rasplus. Scale bars = 500 µm. IQTREE, ASTRAL and ASTRID trees are available in figures S3-S5.

  Figure S1: Workflow used for raw data cleaning (page 136)

Figure S3 :

 S3 Figure S3: IQTREE tree obtained from the analysis of the 25%-complete data set (page 138) SH-aLRT / UFboot values are indicated at nodes. The DNA quantity used to build the library as well as the number of UCEs analysed for each sample is given in brackets.

-

  Add 31 µl of master mix to tubes containing the 60 µl end prep reaction mixture obtained in the previous step. -Add 1.25 µl of P1 adapters (1.5µM) and 1.25 µl of P2 adapters (1.5 µM) to each tube. -Vortex (mix well.) -Pulse spin. -Place in a thermocycler. Disable heated lid and run the following program: 15 min at 20°C.

  Do not remove tubes from magnetic rack and add 200 µl of 70 % ethanol without disturbing beads (use freshly made 70% EtOH to ensure accurate concentration.) -Wait for 30s (leave cap open.) -Remove and discard supernatant without disturbing beads. -Add again 200 µl of 70 % ethanol. -Wait for 30s (leave cap open.) -Remove and discard supernatant without disturbing beads. -Make sure to remove all traces of Ethanol (use a 10µl micropipette if necessary.) -Dry for 5 min (leave cap open.) -Do not remove tubes from magnetic rack and add 9µl of buffer EB. -Close cap. -Remove tubes from magnetic rack. -Vortex + spin pulse. -Wait for 2min. -Move strip back to magnetic rack and wait for 2 to 5min. -Transfer the 9µl of DNA to a new 1.5ml LoBind tube.

  Transfer 50 µl of sheared DNA into a strip / plate. Thaw NEBNext Ultra II End Prep Reaction Buffer. Vortex + pulse spin. ATTENTION: Pull out NEBNext Ultra II End Prep Enzyme Mix out of freezer just before use. LIGHT vortex + pulse spin.

		Master Mix: x1 reaction x 96 reactions
	NEBNext Ultra II End Prep Enzyme Mix	3 µl	288 µl
	NEBNext Ultra II End Prep Reaction Buffer	7 µl	672 µl
	Master mix to add to each tube	10 µl

  Thaw NEBNext Ultra II Q5 Master Mix. Homogenize by pipetting + Pulse spin. DO NOT VORTEX. Leave on Ice.

					Master Mix: x 1 reaction x 96 reactions
	NEBNext Ultra II Q5 Master Mix			25 µl	2400 µl
	Primer F (10µM)				2 µl	192 µl
	Primer R (10µM)				2 µl	192 µl
	Master mix to add to each tube			29 µl
	With
	Primer F = AATGATACGGCGACCACCG*A
	Primer R = CAAGCAGAAGACGGCATACG*A
	(synthesized by IDT (Integrated DNA Technologies, Inc.), HPLC Purification, * = Phosphorothioate
	Bond.)
	Vortex + pulse spin master mix
	-Add 29 µl of master mix to tubes containing the 15 µl of adaptor ligated DNA.
	-Vortex.
	-Pulse spin.
	-Place in a thermocycler. Enable heated lid and run the following program:
			15 cycles
	Time	30s	10s	75s	5min	∞
	Temperature	98°C	98°C	65°C	65°C	4°C

-

  Do not remove tubes from magnetic rack and add 200 µl of 70 % ethanol without disturbing beads (use freshly made 70% EtOH to ensure accurate concentration.) -Wait for 30s (leave cap open.) -Remove and discard supernatant without disturbing beads. -Add again 200

µl of 70 % ethanol.

	-Wait for 30s (leave cap open.)
	-Remove and discard supernatant without disturbing beads.
	-Make sure to remove all traces of ethanol (use a 10µl micropipette if necessary.)
	-Dry for 5 min (leave cap open.)
	-Do not remove tubes from magnetic rack and add 25µl of buffer EB.
	-Close cap.
	-Remove tubes from magnetic rack.
	-Vortex + pulse spin.
	-Wait for 2min.

ACKNOWLEDGEMENTS

We are grateful to Nicole Fisher (CSIRO, National Museum of Australia, Canberra), Chris Burwell, Christine Lambkin and Susan Wright (Queensland Museum, Brisbane, Australia) for facilitating access to samples and to the Genotoul bioinformatics platform Toulouse Midi-Pyrenees, France for providing computing resources. AC thanks Lisa Schlée, Philippe Cruaud and Maya for their patience in the field and Jean-Pierre Rossi for his help with R. AG thanks V. Fursov (Schmalhausen Institute of Zoology, Ukraine) for his help with specimen identification. This work is part of a large NSF project (Award#1555808) led by John Heraty (UC Riverside USA) that attempts to solve the phylogeny of Chalcidoidea with NGS approaches, and was partly funded by the ANR project TriPTIC (ANR-14-CE18-0002) led by JYR and the SPE department of the INRA (French National Agronomic Institute). LF travels to France were supported by the grants IntegPar (89BM/2017) and NEVPIT (PN-III-P4-ID-PCE-2016-0233) awarded by the Romanian National Authority for Scientific Research, CNCS -UEFISCDI. This paper is dedicated to the memory of our friend Dr John La Salle, whose kindness and sense of humor will be remembered for ever.

uce-844_p1 uce-844_p2 locus0033 uce-841_p1 uce-841_p2 locus0034 uce-840_p1 uce-840_p2 locus0035 uce-832_p1 uce-832_p2 locus0036 uce-1343_p1 uce-1343_p2 locus0037 uce-822_p1 uce-822_p2 locus0038 uce-815_p1 uce-815_p2 locus0039 uce-805_p1 uce-805_p2 locus0040 uce-804_p1 uce-804_p2 locus0041 uce-801_p1 uce-801_p2 locus0042 uce-800_p1 uce-800_p2 locus0043 uce-785_p1 uce-785_p2 locus0044 uce-784_p1 uce-784_p2 locus0045 uce-781_p1 uce-781_p2 locus0046 uce-779_p1 uce-779_p2 locus0047 uce-1342_p1 uce-1342_p2 locus0048 uce-774_p1 uce-774_p2 locus0049 uce-773_p1 uce-773_p2 locus0050 uce-772_p1 uce-772_p2 locus0051 uce-770_p1 uce-770_p2 locus0052 uce-761_p1 uce-761_p2 locus0053 uce-759_p1 uce-759_p2 locus0054 uce-749_p1 uce-749_p2 locus0055 uce-744_p1 uce-744_p2 locus0056 uce-743_p1 uce-743_p2 locus0057 uce-735_p1 uce-735_p2 locus0058 uce-1337_p1 uce-1337_p2 locus0059 uce-723_p1 uce-723_p2 locus0060 uce-721_p1 uce-721_p2 locus0061 uce-715_p1 uce-715_p2 locus0062 uce-713_p1 uce-713_p2 locus0063 uce-711_p1 uce-711_p2 locus0064 uce-699_p1 uce-699_p2 locus0065 uce-691_p1 uce-691_p2 locus0066 uce-687_p1 uce-687_p2 locus0067 uce-684_p1 uce-684_p2 locus0068 uce-679_p1 uce-679_p2 locus0069 uce-1330_p1 uce-1330_p2 locus0070 uce-658_p1 uce-658_p2 locus0071 uce-656_p1 uce-656_p2 locus0072 uce-646_p1 uce-646_p2 locus0073 uce-642_p1 uce-642_p2 locus0074 uce-640_p1 uce-640_p2 locus0075 uce-635_p1 uce-635_p2 locus0076 uce-632_p1 uce-632_p2 locus0077 uce-631_p1 uce-631_p2 locus0078 uce-630_p1 uce-630_p2 locus0079 uce-621_p1 uce-621_p2 locus0080 uce-1325_p1 uce-1325_p2 locus0081 uce-620_p1 uce-620_p2 locus0082 uce-616_p1 uce-616_p2 locus0083 uce-610_p1 uce-610_p2 locus0084 uce-609_p1 uce-609_p2 locus0085 uce-606_p1 uce-606_p2 locus0086 uce-597_p1 uce-597_p2 locus0087 uce-584_p1 uce-584_p2 locus0088 uce-580_p1 uce-580_p2 locus0089 uce-571_p1 uce-571_p2 locus0090 uce-567_p1 uce-567_p2 locus0091 uce-1324_p1 uce-1324_p2 locus0092 uce-564_p1 uce-564_p2 locus0093 uce-561_p1 uce-561_p2 locus0094 uce-557_p1 uce-557_p2 locus0095 uce-556_p1 uce-556_p2 locus0096 uce-552_p1 uce-552_p2 locus0097 uce-551_p1 uce-551_p2 locus0098 uce-547_p1 uce-547_p2 locus0099 uce-544_p1 uce-544_p2 locus0100 uce-533_p1 uce-533_p2 locus0101 uce-531_p1 uce-531_p2 locus0102 uce-1287_p1 uce-1287_p2 locus0103 uce-528_p1 uce-528_p2 locus0104 uce-523_p1 uce-523_p2 locus0105 uce-518_p1 uce-518_p2 locus0106 uce-514_p1 uce-514_p2 locus0107 uce-506_p1 uce-506_p2 locus0108 uce-499_p1 uce-499_p2 locus0109 uce-498_p1 uce-498_p2 locus0110 uce-497_p1 uce-497_p2 locus0111 uce-492_p1 uce-492_p2 locus0112 uce-483_p1 uce-483_p2 locus0113 uce-825_p1 uce-825_p2 uce-826_p1 uce-826_p2 locus0114 uce-1278_p1 uce-1278_p2 locus0115 uce-481_p1 uce-481_p2 locus0116 uce-478_p1 uce-478_p2 locus0117 uce-471_p1 uce-471_p2 locus0118 uce-470_p1 uce-470_p2 locus0119 uce-468_p1 uce-468_p2 locus0120 uce-467_p1 uce-467_p2 locus0121 uce-466_p1 uce-466_p2 locus0122 uce-465_p1 uce-465_p2 locus0123 uce-461_p1 uce-461_p2 locus0124 uce-459_p1 uce-459_p2 locus0125 uce-1273_p1 uce-1273_p2 locus0126 uce-458_p1 uce-458_p2 locus0127 uce-453_p1 uce-453_p2 locus0128 uce-436_p1 uce-436_p2 locus0129 uce-431_p1 uce-431_p2 locus0130 uce-424_p1 uce-424_p2 locus0131 uce-416_p1 uce-416_p2 locus0132 uce-415_p1 uce-415_p2 locus0133 uce-413_p1 uce-413_p2 locus0134 uce-406_p1 uce-406_p2 locus0135 uce-401_p1 uce-401_p2 locus0136 uce-1261_p1 uce-1261_p2 locus0137 uce-397_p1 uce-397_p2 locus0138 uce-369_p1 uce-369_p2 locus0139 uce-349_p1 uce-349_p2 locus0140 uce-348_p1 uce-348_p2 locus0141 uce-345_p1 uce-345_p2 locus0142 uce-339_p1 uce-339_p2 locus0143 uce-337_p1 uce-337_p2 locus0144 uce-335_p1 uce-335_p2 locus0145 uce-333_p1 uce-333_p2 locus0146 uce-330_p1 uce-330_p2 locus0147 uce-1258_p1 uce-1258_p2 locus0148 uce-320_p1 uce-320_p2 locus0149 uce-319_p1 uce-319_p2 locus0150 uce-304_p1 uce-304_p2 locus0151 uce-296_p1 uce-296_p2 locus0152 uce-295_p1 uce-295_p2 locus0153 uce-275_p1 uce-275_p2 locus0154 uce-269_p1 uce-269_p2 locus0155 uce-242_p1 uce-242_p2 locus0156 uce-241_p1 uce-241_p2 locus0157 uce-240_p1 uce-240_p2 locus0158 uce-1257_p1 uce-1257_p2 locus0159 uce-237_p1 uce-237_p2 locus0160 uce-235_p1 uce-235_p2 locus0161 uce-233_p1 uce-233_p2 locus0162 uce-228_p1 uce-228_p2 locus0163 uce-217_p1 uce-217_p2 locus0164 uce-200_p1 uce-200_p2 locus0165 uce-199_p1 uce-199_p2 locus0166 uce-182_p1 uce-182_p2 locus0167 uce-180_p1 uce-180_p2 locus0168 uce-170_p1 uce-170_p2 locus0169 uce-1249_p1 uce-1249_p2 locus0170 uce-157_p1 uce-157_p2 locus0171 uce-148_p1 uce-148_p2 locus0172 uce-143_p1 uce-143_p2 locus0173 uce-139_p1 uce-139_p2 locus0174 uce-115_p1 uce-115_p2 locus0175 uce-110_p1 uce-110_p2 locus0176 uce-104_p1 uce-104_p2 locus0177 uce-98_p1 uce-98_p2 locus0178 uce-87_p1 uce-87_p2 locus0179 uce-86_p1 uce-86_p2 locus0180 uce-1248_p1 uce-1248_p2 locus0181 uce-79_p1 uce-79_p2 locus0182 uce-78_p1 uce-78_p2 locus0183 uce-77_p1 uce-77_p2 locus0184 uce-75_p1 uce-75_p2 locus0185 uce-74_p1 uce-74_p2 locus0186 uce-68_p1 uce-68_p2 locus0187 uce-67_p1 uce-67_p2 locus0188 uce-64_p1 uce-64_p2 locus0189 uce-59_p1 uce-59_p2 locus0190 uce-54_p1 uce-54_p2 locus0191 uce-1244_p1 uce-1244_p2 locus0192 uce-49_p1 uce-49_p2 locus0193 uce-44_p1 uce-44_p2 locus0194 uce-43_p1 uce-43_p2 locus0195 uce-41_p1 uce-41_p2 locus0196 uce-34_p1 uce-34_p2 locus0197 uce-29_p1 uce-29_p2 locus0198 uce-11_p1 uce-11_p2 locus0199 uce-9_p1 uce-9_p2 locus0200 uce-1387_p1 uce-1387_p2 locus0201 uce-666_p1 uce-666_p2 locus0202 uce-1243_p1 uce-1243_p2 locus0203 uce-1080_p1 uce-1080_p2 locus0204 locus0205 uce-1237_p1 uce-1237_p2 locus0206 uce-456_p1 uce-456_p2 uce-457_p1 uce-457_p2 locus0207 uce-1235_p1 uce-1235_p2 locus0208 uce-1233_p1 uce-1233_p2 locus0209 uce-1232_p1 uce-1232_p2 locus0210 uce-1225_p1 uce-1225_p2 locus0211 uce-1213_p1 uce-1213_p2 locus0212 uce-1212_p1 uce-1212_p2 locus0213 uce-1211_p1 uce-1211_p2 locus0214 uce-1200_p1 uce-1200_p2 locus0215 uce-1188_p1 uce-1188_p2 locus0216 uce-1182_p1 uce-1182_p2 locus0217 uce-1164_p1 uce-1164_p2 uce-1165_p1 uce-1165_p2 locus0218 uce-1181_p1 uce-1181_p2 locus0219 uce-1160_p1 uce-1160_p2 locus0220 uce-1151_p1 uce-1151_p2 locus0221 uce-1149_p1 uce-1149_p2 locus0222 uce-1147_p1 uce-1147_p2 locus0223 uce-1146_p1 uce-1146_p2 locus0224 uce-1134_p1 uce-1134_p2 locus0225 uce-1131_p1 uce-1131_p2 locus0226 uce-1128_p1 uce-1128_p2 locus0227 uce-1124_p1 uce-1124_p2 locus0228 uce-419_p1 uce-419_p2 uce-420_p1 uce-420_p2 locus0229 uce-1110_p1 uce-1110_p2 locus0230 uce-1099_p1 uce-1099_p2 locus0231 uce-1088_p1 uce-1088_p2 locus0232 uce-1074_p1 uce-1074_p2 locus0233 uce-1066_p1 uce-1066_p2 locus0234 uce-1065_p1 uce-1065_p2 locus0235 uce-1052_p1 uce-1052_p2 locus0236 uce-1051_p1 uce-1051_p2 locus0237 uce-1050_p1 uce-1050_p2 locus0238 uce-1049_p1 uce-1049_p2 locus0239 uce-1115_p1 uce-1115_p2 uce-1116_p1 uce-1116_p2 locus0240 uce-1036_p1 uce-1036_p2 locus0241 uce-1029_p1 uce-1029_p2 locus0242 uce-1025_p1 uce-1025_p2 locus0243 uce-996_p1 uce-996_p2 locus0244 uce-991_p1 uce-991_p2 locus0245 uce-990_p1 uce-990_p2 locus0246 uce-988_p1 uce-988_p2 locus0247 uce-987_p1 uce-987_p2 locus0248 uce-976_p1 uce-976_p2 locus0249 uce-975_p1 uce-975_p2 locus0250 uce-311_p1 uce-311_p2 uce-312_p1 uce-312_p2 locus0251 uce-962_p1 uce-962_p2 locus0252 uce-960_p1 uce-960_p2 locus0253 uce-958_p1 uce-958_p2 locus0254 uce-955_p1 uce-955_p2 locus0255 uce-948_p1 uce-948_p2 locus0256 uce-942_p1 uce-942_p2 locus0257 uce-937_p1 uce-937_p2 locus0258 uce-930_p1 uce-930_p2 locus0259 uce-920_p1 uce-920_p2 locus0260 uce-914_p1 uce-914_p2 locus0261 uce-1100_p1 uce-1100_p2 uce-1101_p1 uce-1101_p2 locus0262 uce-907_p1 uce-907_p2 locus0263 uce-906_p1 uce-906_p2 locus0264 uce-905_p1 uce-905_p2 locus0265 uce-901_p1 uce-901_p2 locus0266 uce-897_p1 uce-897_p2 locus0267 uce-892_p1 uce-892_p2 locus0268 uce-877_p1 uce-877_p2 locus0269 uce-866_p1 uce-866_p2 locus0270 uce-864_p1 uce-864_p2 locus0271 uce-858_p1 uce-858_p2 locus0272 uce-1310_p1 uce-1310_p2 uce-1311_p1 uce-1311_p2 locus0273 uce-852_p1 uce-852_p2 locus0274 uce-851_p1 uce-851_p2 locus0275 uce-848_p1 uce-848_p2 locus0276 uce-846_p1 uce-846_p2 locus0277 uce-836_p1 uce-836_p2 locus0278 uce-835_p1 uce-835_p2 locus0279 uce-831_p1 uce-831_p2 locus0280 uce-828_p1 uce-828_p2 locus0281 uce-823_p1 uce-823_p2 locus0282 uce-820_p1 uce-820_p2 locus0283 uce-83_p1 uce-83_p2 uce-84_p1 uce-84_p2 locus0284 uce-816_p1 uce-816_p2 locus0285 uce-812_p1 uce-812_p2 locus0286 uce-787_p1 uce-787_p2 locus0287 uce-778_p1 uce-778_p2 locus0288 uce-764_p1 uce-764_p2 locus0289 uce-751_p1 uce-751_p2 locus0290 uce-750_p1 uce-750_p2 locus0291 uce-745_p1 uce-745_p2 locus0292 uce-739_p1 uce-739_p2 locus0293 uce-720_p1 uce-720_p2 locus0294 uce-729_p1 uce-729_p2 uce-730_p1 uce-730_p2 uce-731_p1 uce-731_p2 locus0295 uce-970_p1 uce-970_p2 uce-971_p1 uce-971_p2 locus0296 uce-719_p1 uce-719_p2 locus0297 uce-710_p1 uce-710_p2 locus0298 uce-707_p1 uce-707_p2 locus0299 uce-702_p1 uce-702_p2 locus0300 uce-690_p1 uce-690_p2 locus0301 uce-661_p1 uce-661_p2 locus0302 uce-654_p1 uce-654_p2 locus0303 uce-650_p1 uce-650_p2 locus0304 uce-649_p1 uce-649_p2 locus0305 uce-644_p1 uce-644_p2 locus0306 uce-164_p1 uce-164_p2 uce-165_p1 uce-165_p2 locus0307 uce-643_p1 uce-643_p2 locus0308 uce-638_p1 uce-638_p2 locus0309 uce-615_p1 uce-615_p2 locus0310 uce-614_p1 uce-614_p2 locus0311 uce-607_p1 uce-607_p2 locus0312 uce-601_p1 uce-601_p2 locus0313 uce-594_p1 uce-594_p2 locus0314 uce-589_p1 uce-589_p2 locus0315 uce-582_p1 uce-582_p2 locus0316 uce-577_p1 uce-577_p2 locus0317 uce-890_p1 uce-890_p2 uce-891_p1 uce-891_p2 locus0318 uce-576_p1 uce-576_p2 locus0319 uce-554_p1 uce-554_p2 locus0320 uce-548_p1 uce-548_p2 locus0321 uce-542_p1 uce-542_p2 locus0322 uce-541_p1 uce-541_p2 locus0323 uce-537_p1 uce-537_p2 locus0324 uce-535_p1 uce-535_p2 locus0325 uce-522_p1 uce-522_p2 locus0326 uce-512_p1 uce-512_p2 locus0327 uce-507_p1 uce-507_p2 locus0328 uce-972_p1 uce-972_p2 uce-973_p1 uce-973_p2 locus0329 uce-504_p1 uce-504_p2 locus0330 uce-501_p1 uce-501_p2 locus0331 uce-496_p1 uce-496_p2 locus0332 uce-495_p1 uce-495_p2 locus0333 uce-488_p1 uce-488_p2 locus0334 uce-486_p1 uce-486_p2 locus0335 uce-476_p1 uce-476_p2 locus0336 uce-460_p1 uce-460_p2 locus0337 uce-455_p1 uce-455_p2 locus0338 uce-449_p1 uce-449_p2 locus0339 uce-283_p1 uce-283_p2 uce-284_p1 uce-284_p2 locus0340 uce-446_p1 uce-446_p2 locus0341 uce-437_p1 uce-437_p2 locus0342 uce-432_p1 uce-432_p2 locus0343 uce-427_p1 uce-427_p2 locus0344 uce-407_p1 uce-407_p2 locus0345 uce-395_p1 uce-395_p2 locus0346 uce-394_p1 uce-394_p2 locus0347 uce-393_p1 uce-393_p2 locus0348 uce-392_p1 uce-392_p2 locus0349 uce-390_p1 uce-390_p2 locus0350 uce-209_p1 uce-209_p2 uce-210_p1 uce-210_p2 locus0351 uce-379_p1 uce-379_p2 locus0352 uce-374_p1 uce-374_p2 locus0353 uce-367_p1 uce-367_p2 locus0354 uce-366_p1 uce-366_p2 locus0355 uce-360_p1 uce-360_p2 locus0356 uce-359_p1 uce-359_p2 locus0357 uce-342_p1 uce-342_p2 locus0358 uce-334_p1 uce-334_p2 locus0359 uce-329_p1 uce-329_p2 locus0360 uce-327_p1 uce-327_p2 locus0361 uce-1455_p1 uce-1455_p2 uce-1456_p1 uce-1456_p2 locus0362 uce-307_p1 uce-307_p2 locus0363 uce-303_p1 uce-303_p2 locus0364 uce-301_p1 uce-301_p2 locus0365 uce-299_p1 uce-299_p2 locus0366 uce-297_p1 uce-297_p2 locus0367 uce-291_p1 uce-291_p2 locus0368 uce-278_p1 uce-278_p2 locus0369 uce-267_p1 uce-267_p2 locus0370 uce-265_p1 uce-265_p2 locus0371 uce-258_p1 uce-258_p2 locus0372 uce-1389_p1 uce-1389_p2 uce-1390_p1 uce-1390_p2 locus0373 uce-257_p1 uce-257_p2 locus0374 uce-253_p1 uce-253_p2 locus0375 uce-251_p1 uce-251_p2 locus0376 uce-250_p1 uce-250_p2 locus0377 uce-236_p1 uce-236_p2 locus0378 uce-219_p1 uce-219_p2 locus0379 uce-213_p1 uce-213_p2 locus0380 uce-212_p1 uce-212_p2 locus0381 uce-195_p1 uce-195_p2 locus0382 uce-189_p1 uce-189_p2 locus0383 uce-80_p1 uce-80_p2 uce-81_p1 uce-81_p2 locus0384 uce-185_p1 uce-185_p2 locus0385 uce-173_p1 uce-173_p2 locus0386 uce-171_p1 uce-171_p2 locus0387 uce-162_p1 uce-162_p2 locus0388 uce-155_p1 uce-155_p2 locus0389 uce-150_p1 uce-150_p2 locus0390 uce-144_p1 uce-144_p2 locus0391 uce-136_p1 uce-136_p2 locus0392 uce-132_p1 uce-132_p2 locus0393 uce-131_p1 uce-131_p2 locus0394 uce-1153_p1 uce-1153_p2 uce-1154_p1 uce-1154_p2 locus0395 uce-122_p1 uce-122_p2 locus0396 uce-121_p1 uce-121_p2 locus0397 uce-117_p1 uce-117_p2 locus0398 uce-116_p1 uce-116_p2 locus0399 uce-114_p1 uce-114_p2 locus0400 uce-108_p1 uce-108_p2 locus0401 uce-100_p1 uce-100_p2 locus0402 uce-99_p1 uce-99_p2 locus0403 uce-96_p1 uce-96_p2 locus0404 uce-60_p1 uce-60_p2 locus0405 uce-755_p1 uce-755_p2 uce-756_p1 uce-756_p2 uce-757_p1 uce-757_p2 locus0406 uce-247_p1 uce-247_p2 uce-248_p1 uce-248_p2 locus0407 uce-55_p1 uce-55_p2 locus0408 uce-53_p1 uce-53_p2 locus0409 uce-48_p1 uce-48_p2 locus0410 uce-46_p1 uce-46_p2 locus0411 uce-38_p1 uce-38_p2 locus0412 uce-35_p1 uce-35_p2 locus0413 uce-33_p1 uce-33_p2 locus0414 uce-27_p1 uce-27_p2 locus0415 uce-21_p1 uce-21_p2 locus0416 uce-20_p1 uce-20_p2 locus0417 uce-281_p1 uce-281_p2 uce-282_p1 uce-282_p2 locus0418 uce-18_p1 uce-18_p2 locus0419 uce-17_p1 uce-17_p2 locus0420 uce-10_p1 uce-10_p2 locus0421 uce-6_p1 uce-6_p2 locus0422 uce-5_p1 uce-5_p2 locus0423 uce-3_p1 uce-3_p2 locus0424 uce-225_p1 uce-225_p2 locus0425 uce-2_p1 uce-2_p2 locus0426 uce-1508_p1 uce-1508_p2 locus0427 uce-1500_p1 uce-1500_p2 locus0428 uce-1255_p1 uce-1255_p2 uce-1256_p1 uce-1256_p2 locus0429 uce-1492_p1 uce-1492_p2 locus0430 uce-1487_p1 uce-1487_p2 locus0431 uce-1485_p1 uce-1485_p2 locus0432 uce-1481_p1 uce-1481_p2 locus0433 uce-1475_p1 uce-1475_p2 locus0434 uce-1468_p1 uce-1468_p2 locus0435 uce-1466_p1 uce-1466_p2 locus0436 uce-1465_p1 uce-1465_p2 locus0437 uce-1462_p1 uce-1462_p2 locus0438 uce-1461_p1 uce-1461_p2 locus0439 uce-1270_p1 uce-1270_p2 uce-1271_p1 uce-1271_p2 locus0440 uce-1460_p1 uce-1460_p2 locus0441 uce-1446_p1 uce-1446_p2 locus0442 uce-1443_p1 uce-1443_p2 locus0443 uce-1429_p1 uce-1429_p2 locus0444 uce-1410_p1 uce-1410_p2 locus0445 uce-1409_p1 uce-1409_p2 locus0446 uce-1407_p1 uce-1407_p2 locus0447 uce-1378_p1 uce-1378_p2 locus0448 uce-1376_p1 uce-1376_p2 locus0449 uce-1372_p1 uce-1372_p2 locus0450 uce-667_p1 uce-667_p2 uce-668_p1 uce-668_p2 locus0451 uce-1352_p1 uce-1352_p2 locus0452 uce-1351_p1 uce-1351_p2 locus0453 uce-1350_p1 uce-1350_p2 locus0454 uce-1348_p1 uce-1348_p2 locus0455 uce-1346_p1 uce-1346_p2 locus0456 uce-1335_p1 uce-1335_p2 locus0457 uce-1333_p1 uce-1333_p2 locus0458 uce-1329_p1 uce-1329_p2 locus0459 uce-1326_p1 uce-1326_p2 locus0460 uce-1319_p1 uce-1319_p2 locus0461 uce-417_p1 uce-417_p2 uce-418_p1 uce-418_p2 locus0462 uce-1318_p1 uce-1318_p2 locus0463 uce-1315_p1 uce-1315_p2 locus0464 uce-1307_p1 uce-1307_p2 locus0465 uce-1296_p1 uce-1296_p2 locus0466 uce-1286_p1 uce-1286_p2 locus0467 uce-1284_p1 uce-1284_p2 locus0468 uce-1282_p1 uce-1282_p2 locus0469 uce-1281_p1 uce-1281_p2 locus0470 uce-1280_p1 uce-1280_p2 locus0471 uce-1279_p1 uce-1279_p2 locus0472 uce-61_p1 uce-61_p2 uce-62_p1 uce-62_p2 locus0473 uce-1253_p1 uce-1253_p2 locus0474 uce-1252_p1 uce-1252_p2 locus0475 uce-1247_p1 uce-1247_p2 locus0476 uce-1242_p1 uce-1242_p2 locus0477 uce-1240_p1 uce-1240_p2 locus0478 uce-1239_p1 uce-1239_p2 locus0479 uce-1231_p1 uce-1231_p2 locus0480 uce-1228_p1 uce-1228_p2 locus0481 uce-1220_p1 uce-1220_p2 locus0482 uce-1218_p1 uce-1218_p2 locus0483 uce-439_p1 uce-439_p2 uce-440_p1 uce-440_p2 locus0484 uce-1217_p1 uce-1217_p2 locus0485 uce-1215_p1 uce-1215_p2 locus0486 uce-1214_p1 uce-1214_p2 locus0487 uce-1203_p1 uce-1203_p2 locus0488 uce-1201_p1 uce-1201_p2 locus0489 uce-1199_p1 uce-1199_p2 locus0490 uce-1196_p1 uce-1196_p2 locus0491 uce-1193_p1 uce-1193_p2 locus0492 uce-1184_p1 uce-1184_p2 locus0493 uce-1175_p1 uce-1175_p2 locus0494 uce-444_p1 uce-444_p2 uce-445_p1 uce-445_p2 locus0495 uce-1169_p1 uce-1169_p2 locus0496 uce-1163_p1 uce-1163_p2 locus0497 uce-1162_p1 uce-1162_p2 locus0498 uce-1148_p1 uce-1148_p2 locus0499 uce-1144_p1 uce-1144_p2 locus0500 uce-1136_p1 uce-1136_p2 locus0501 uce-1133_p1 uce-1133_p2 locus0502 uce-1127_p1 uce-1127_p2 locus0503 uce-1121_p1 uce-1121_p2 locus0504 uce-1120_p1 uce-1120_p2 locus0505 uce-1470_p1 uce-1470_p2 uce-1471_p1 uce-1471_p2 locus0506 uce-1119_p1 uce-1119_p2 locus0507 uce-1111_p1 uce-1111_p2 locus0508 uce-1106_p1 uce-1106_p2 locus0509 uce-1104_p1 uce-1104_p2 locus0510 uce-1095_p1 uce-1095_p2 locus0511 uce-1094_p1 uce-1094_p2 locus0512 uce-1093_p1 uce-1093_p2 locus0513 uce-1083_p1 uce-1083_p2 locus0514 uce-1077_p1 uce-1077_p2 locus0515 uce-1076_p1 uce-1076_p2 locus0516 uce-1412_p1 uce-1412_p2 uce-1413_p1 uce-1413_p2 uce-1414_p1 uce-1414_p2 locus0517 uce-1000_p1 uce-1000_p2 uce-1001_p1 uce-1001_p2 locus0518 uce-1063_p1 uce-1063_p2 locus0519 uce-1061_p1 uce-1061_p2 locus0520 uce-1058_p1 uce-1058_p2 locus0521 uce-1053_p1 uce-1053_p2 locus0522 uce-1048_p1 uce-1048_p2 locus0523 uce-1037_p1 uce-1037_p2 locus0524 uce-1034_p1 uce-1034_p2 locus0525 uce-1031_p1 uce-1031_p2 locus0526 uce-1030_p1 uce-1030_p2 locus0527 uce-1026_p1 uce-1026_p2 locus0528 uce-1043_p1 uce-1043_p2 uce-1044_p1 uce-1044_p2 locus0529 uce-1024_p1 uce-1024_p2 locus0530 uce-1020_p1 uce-1020_p2 locus0531 uce-1016_p1 uce-1016_p2 locus0532 uce-1012_p1 uce-1012_p2 locus0533 uce-1010_p1 uce-1010_p2 locus0534 uce-998_p1 uce-998_p2 locus0535 uce-993_p1 uce-993_p2 locus0536 uce-989_p1 uce-989_p2 locus0537 uce-985_p1 uce-985_p2 locus0538 uce-984_p1 uce-984_p2 locus0539 uce-286_p1 uce-286_p2 uce-287_p1 uce-287_p2 locus0540 uce-978_p1 uce-978_p2 locus0541 uce-963_p1 uce-963_p2 locus0542 uce-956_p1 uce-956_p2 locus0543 uce-952_p1 uce-952_p2 locus0544 uce-950_p1 uce-950_p2 locus0545 uce-949_p1 uce-949_p2 locus0546 uce-941_p1 uce-941_p2 locus0547 uce-940_p1 uce-940_p2 locus0548 uce-936_p1 uce-936_p2 locus0549 uce-934_p1 uce-934_p2 locus0550 uce-703_p1 uce-703_p2 uce-704_p1 uce-704_p2 locus0551 uce-925_p1 uce-925_p2 locus0552 uce-923_p1 uce-923_p2 locus0553 uce-918_p1 uce-918_p2 locus0554 uce-915_p1 uce-915_p2 locus0555 uce-899_p1 uce-899_p2 locus0556 uce-898_p1 uce-898_p2 locus0557 uce-886_p1 uce-886_p2 locus0558 uce-875_p1 uce-875_p2 locus0559 uce-873_p1 uce-873_p2 locus0560 uce-870_p1 uce-870_p2 locus0561 uce-1039_p1 uce-1039_p2 uce-1040_p1 uce-1040_p2 locus0562 uce-856_p1 uce-856_p2 locus0563 uce-850_p1 uce-850_p2 locus0564 uce-849_p1 uce-849_p2 locus0565 uce-837_p1 uce-837_p2 locus0566 uce-833_p1 uce-833_p2 locus0567 uce-830_p1 uce-830_p2 locus0568 uce-824_p1 uce-824_p2 locus0569 uce-819_p1 uce-819_p2 locus0570 uce-811_p1 uce-811_p2 locus0571 uce-803_p1 uce-803_p2 locus0572 uce-622_p1 uce-622_p2 uce-623_p1 uce-623_p2 locus0573 uce-789_p1 uce-789_p2 locus0574 uce-786_p1 uce-786_p2 locus0575 uce-777_p1 uce-777_p2 locus0576 uce-767_p1 uce-767_p2 locus0577 uce-997_p1 uce-997_p2 locus0578 uce-754_p1 uce-754_p2 locus0579 uce-753_p1 uce-753_p2 locus0580 uce-752_p1 uce-752_p2 locus0581 uce-747_p1 uce-747_p2 locus0582 uce-746_p1 uce-746_p2 locus0583 uce-376_p1 uce-376_p2 uce-377_p1 uce-377_p2 locus0584 uce-738_p1 uce-738_p2 locus0585 uce-737_p1 uce-737_p2 locus0586 uce-736_p1 uce-736_p2 locus0587 uce-722_p1 uce-722_p2 locus0588 uce-718_p1 uce-718_p2 locus0589 uce-716_p1 uce-716_p2 locus0590 uce-714_p1 uce-714_p2 locus0591 uce-709_p1 uce-709_p2 locus0592 uce-708_p1 uce-708_p2 locus0593 uce-692_p1 uce-692_p2 locus0594 uce-1448_p1 uce-1448_p2 uce-1449_p1 uce-1449_p2 locus0595 uce-689_p1 uce-689_p2 locus0596 uce-688_p1 uce-688_p2 locus0597 uce-678_p1 uce-678_p2 locus0598 uce-675_p1 uce-675_p2 locus0599 uce-673_p1 uce-673_p2 locus0600 uce-672_p1 uce-672_p2 locus0601 uce-665_p1 uce-665_p2 locus0602 uce-663_p1 uce-663_p2 locus0603 uce-657_p1 uce-657_p2 locus0604 uce-655_p1 uce-655_p2 locus0605 uce-1488_p1 uce-1488_p2 uce-1489_p1 uce-1489_p2 locus0606 uce-645_p1 uce-645_p2 locus0607 uce-641_p1 uce-641_p2 locus0608 uce-634_p1 uce-634_p2 locus0609 uce-633_p1 uce-633_p2 locus0610 uce-629_p1 uce-629_p2 locus0611 uce-627_p1 uce-627_p2 locus0612 uce-625_p1 uce-625_p2 locus0613 uce-624_p1 uce-624_p2 locus0614 uce-611_p1 uce-611_p2 locus0615 uce-608_p1 uce-608_p2 locus0616 uce-1186_p1 uce-1186_p2 uce-1187_p1 uce-1187_p2 locus0617 uce-596_p1 uce-596_p2 locus0618 uce-595_p1 uce-595_p2 locus0619 uce-590_p1 uce-590_p2 locus0620 uce-588_p1 uce-588_p2 locus0621 uce-581_p1 uce-581_p2 locus0622 uce-574_p1 uce-574_p2 locus0623 uce-566_p1 uce-566_p2 locus0624 uce-560_p1 uce-560_p2 locus0625 uce-553_p1 uce-553_p2 locus0626 uce-550_p1 uce-550_p2 locus0627 uce-765_p1 uce-765_p2 uce-76_p1 uce-76_p2 locus0628 uce-724_p1 uce-724_p2 uce-725_p1 uce-725_p2 locus0629 uce-543_p1 uce-543_p2 locus0630 uce-540_p1 uce-540_p2 locus0631 uce-539_p1 uce-539_p2 locus0632 uce-536_p1 uce-536_p2 locus0633 uce-529_p1 uce-529_p2 locus0634 uce-526_p1 uce-526_p2 locus0635 uce-524_p1 uce-524_p2 locus0636 uce-521_p1 uce-521_p2 locus0637 uce-517_p1 uce-517_p2 locus0638 uce-515_p1 uce-515_p2 locus0639 uce-894_p1 uce-894_p2 uce-895_p1 uce-895_p2 locus0640 uce-513_p1 uce-513_p2 locus0641 uce-511_p1 uce-511_p2 locus0642 uce-509_p1 uce-509_p2 locus0643 uce-508_p1 uce-508_p2 locus0644 uce-505_p1 uce-505_p2 locus0645 uce-503_p1 uce-503_p2 locus0646 uce-500_p1 uce-500_p2 locus0647 uce-491_p1 uce-491_p2 locus0648 uce-480_p1 uce-480_p2 locus0649 uce-479_p1 uce-479_p2 locus0650 uce-25_p1 uce-25_p2 uce-26_p1 uce-26_p2 locus0651 uce-475_p1 uce-475_p2 locus0652 uce-472_p1 uce-472_p2 locus0653 uce-8_p1 uce-8_p2 locus0654 uce-441_p1 uce-441_p2 locus0655 uce-422_p1 uce-422_p2 locus0656 uce-409_p1 uce-409_p2 locus0657 uce-404_p1 uce-404_p2 locus0658 uce-403_p1 uce-403_p2 locus0659 uce-400_p1 uce-400_p2 locus0660 uce-391_p1 uce-391_p2 locus0661 uce-585_p1 uce-585_p2 uce-586_p1 uce-586_p2 locus0662 uce-389_p1 uce-389_p2 locus0663 uce-388_p1 uce-388_p2 locus0664 uce-386_p1 uce-386_p2 locus0665 uce-383_p1 uce-383_p2 locus0666 uce-380_p1 uce-380_p2 locus0667 uce-375_p1 uce-375_p2 locus0668 uce-373_p1 uce-373_p2 locus0669 uce-370_p1 uce-370_p2 locus0670 uce-364_p1 uce-364_p2 locus0671 uce-363_p1 uce-363_p2 locus0672 uce-1264_p1 uce-1264_p2 uce-1265_p1 uce-1265_p2 locus0673 uce-350_p1 uce-350_p2 locus0674 uce-347_p1 uce-347_p2 locus0675 uce-344_p1 uce-344_p2 locus0676 uce-343_p1 uce-343_p2 locus0677 uce-340_p1 uce-340_p2 locus0678 uce-338_p1 uce-338_p2 locus0679 uce-332_p1 uce-332_p2 locus0680 uce-328_p1 uce-328_p2 locus0681 uce-326_p1 uce-326_p2 locus0682 uce-325_p1 uce-325_p2 locus0683 uce-1370_p1 uce-1370_p2 uce-1371_p1 uce-1371_p2 locus0684 uce-321_p1 uce-321_p2 locus0685 uce-317_p1 uce-317_p2 locus0686 uce-316_p1 uce-316_p2 locus0687 uce-315_p1 uce-315_p2 locus0688 uce-313_p1 uce-313_p2 locus0689 uce-310_p1 uce-310_p2 locus0690 uce-309_p1 uce-309_p2 locus0691 uce-308_p1 uce-308_p2 locus0692 uce-305_p1 uce-305_p2 locus0693 uce-302_p1 uce-302_p2 locus0694 uce-1418_p1 uce-1418_p2 uce-292_p1 locus0695 uce-293_p1 uce-293_p2 locus0696 uce-288_p1 uce-288_p2 locus0697 uce-274_p1 uce-274_p2 locus0698 uce-271_p1 uce-271_p2 locus0699 uce-261_p1 uce-261_p2 locus0700 uce-260_p1 uce-260_p2 locus0701 uce-259_p1 uce-259_p2 locus0702 uce-256_p1 uce-256_p2 locus0703 uce-254_p1 uce-254_p2 locus0704 uce-243_p1 uce-243_p2 locus0705 uce-1363_p1 uce-1364_p1 uce-1364_p2 locus0706 uce-239_p1 uce-239_p2 locus0707 uce-234_p1 uce-234_p2 locus0708 uce-232_p1 uce-232_p2 locus0709 uce-230_p1 uce-230_p2 locus0710 uce-229_p1 uce-229_p2 locus0711 uce-226_p1 uce-226_p2 locus0712 uce-224_p1 uce-224_p2 locus0713 uce-206_p1 uce-206_p2 locus0714 uce-205_p1 uce-205_p2 locus0715 uce-201_p1 uce-201_p2 locus0716 uce-362_p1 uce-362_p2 uce-705_p2 locus0717 uce-191_p1 uce-191_p2 locus0718 uce-186_p1 uce-186_p2 locus0719 uce-181_p1 uce-181_p2 locus0720 uce-174_p1 uce-174_p2 locus0721 uce-172_p1 uce-172_p2 locus0722 uce-169_p1 uce-169_p2 locus0723 uce-167_p1 uce-167_p2 locus0724 uce-160_p1 uce-160_p2 locus0725 uce-159_p1 uce-159_p2 locus0726 uce-146_p1 uce-146_p2 locus0727 uce-15_p2 uce-16_p1 uce-16_p2 locus0728 uce-145_p1 uce-145_p2 locus0729 uce-130_p1 uce-130_p2 locus0730 uce-129_p1 uce-129_p2 locus0731 uce-127_p1 uce-127_p2 locus0732 uce-126_p1 uce-126_p2 locus0733 uce-125_p1 uce-125_p2 locus0734 uce-120_p1 uce-120_p2 locus0735 uce-118_p1 uce-118_p2 locus0736 uce-95_p1 uce-95_p2 locus0737 uce-88_p1 uce-88_p2 locus0738 uce-178_p1 uce-178_p2 uce-179_p1 uce-179_p2 locus0739 uce-943_p2 uce-944_p1 uce-944_p2 locus0740 uce-73_p1 uce-73_p2 locus0741 uce-72_p1 uce-72_p2 locus0742 uce-71_p1 uce-71_p2 locus0743 uce-70_p1 uce-70_p2 locus0744 uce-69_p1 uce-69_p2 locus0745 uce-65_p1 uce-65_p2 locus0746 uce-52_p1 uce-52_p2 locus0747 uce-47_p1 uce-47_p2 locus0748 uce-45_p1 uce-45_p2 locus0749 uce-42_p1 uce-42_p2 locus0750 uce-202_p1 uce-202_p2 uce-203_p1 locus0751 uce-39_p1 uce-39_p2 locus0752 uce-28_p1 uce-28_p2 locus0753 uce-23_p1 uce-23_p2 locus0754 uce-22_p1 uce-22_p2 locus0755 uce-13_p1 uce-13_p2 locus0756 uce-1223_p1 uce-1223_p2 locus0757 uce-734_p1 uce-734_p2 locus0758 uce-1510_p1 uce-1510_p2 locus0759 uce-1497_p1 uce-1497_p2 locus0760 uce-1495_p1 uce-1495_p2 locus0761 uce-1017_p1 uce-1017_p2 uce-1018_p1 locus0762 uce-1484_p1 uce-1484_p2 locus0763 uce-1478_p1 uce-1478_p2 locus0764 uce-1476_p1 uce-1476_p2 locus0765 uce-1472_p1 uce-1472_p2 locus0766 uce-1467_p1 uce-1467_p2 locus0767 uce-1464_p1 uce-1464_p2 locus0768 uce-1463_p1 uce-1463_p2 locus0769 uce-1457_p1 uce-1457_p2 locus0770 uce-1452_p1 uce-1452_p2 locus0771 uce-1450_p1 uce-1450_p2 locus0772 uce-1355_p2 uce-1356_p1 uce-1356_p2 locus0773 uce-1442_p1 uce-1442_p2 locus0774 uce-1439_p1 uce-1439_p2 locus0775 uce-1438_p1 uce-1438_p2 locus0776 uce-1433_p1 uce-1433_p2 locus0777 uce-1431_p1 uce-1431_p2 locus0778 uce-1428_p1 uce-1428_p2 locus0779 uce-1427_p1 uce-1427_p2 locus0780 uce-1423_p1 uce-1423_p2 locus0781 uce-1422_p1 uce-1422_p2 locus0782 uce-1421_p1 uce-1421_p2 locus0783 uce-1435_p2 uce-1436_p1 uce-1436_p2 locus0784 uce-1420_p1 uce-1420_p2 locus0785 uce-1417_p1 uce-1417_p2 locus0786 uce-1415_p1 uce-1415_p2 locus0787 uce-1408_p1 uce-1408_p2 locus0788 uce-1403_p1 uce-1403_p2 locus0789 uce-1399_p1 uce-1399_p2 locus0790 uce-1397_p1 uce-1397_p2 locus0791 uce-1392_p1 uce-1392_p2 locus0792 uce-1383_p1 uce-1383_p2 locus0793 uce-1382_p1 uce-1382_p2 locus0794 uce-578_p2 uce-579_p1 uce-579_p2 locus0795 uce-1381_p1 uce-1381_p2 locus0796 uce-1380_p1 uce-1380_p2 locus0797 uce-1379_p1 uce-1379_p2 locus0798 uce-1375_p1 uce-1375_p2 locus0799 uce-1361_p1 uce-1361_p2 locus0800 uce-1360_p1 uce-1360_p2 locus0801 uce-1359_p1 uce-1359_p2 locus0802 uce-1358_p1 uce-1358_p2 locus0803 uce-1349_p1 uce-1349_p2 locus0804 uce-1328_p1 uce-1328_p2 locus0805 uce-813_p1 uce-813_p2 uce-814_p1 locus0806 uce-1322_p1 uce-1322_p2 locus0807 uce-1314_p1 uce-1314_p2 locus0808 uce-1313_p1 uce-1313_p2 locus0809 uce-1308_p1 uce-1308_p2 locus0810 uce-1305_p1 uce-1305_p2 locus0811 uce-1298_p1 uce-1298_p2 locus0812 uce-1293_p1 uce-1293_p2 locus0813 uce-1275_p1 uce-1275_p2 locus0814 uce-1274_p1 uce-1274_p2 locus0815 uce-1266_p1 uce-1266_p2 locus0816 uce-1505_p1 uce-1505_p2 uce-1506_p1 locus0817 uce-1262_p1 uce-1262_p2 locus0818 uce-1260_p1 uce-1260_p2 locus0819 uce-1259_p1 uce-1259_p2 locus0820 uce-1245_p1 uce-1245_p2 locus0821 uce-1241_p1 uce-1241_p2 locus0822 uce-1236_p1 uce-1236_p2 locus0823 uce-1234_p1 uce-1234_p2 locus0824 uce-1222_p1 uce-1222_p2 locus0825 uce-1219_p1 uce-1219_p2 locus0826 uce-1208_p1 uce-1208_p2 locus0827 uce-1250_p2 uce-1251_p1 uce-1251_p2 locus0828 uce-1207_p1 uce-1207_p2 locus0829 uce-1194_p1 uce-1194_p2 locus0830 uce-1191_p1 uce-1191_p2 locus0831 uce-1178_p1 uce-1178_p2 locus0832 uce-1177_p1 uce-1177_p2 locus0833 uce-1174_p1 uce-1174_p2 locus0834 uce-1173_p1 uce-1173_p2 locus0835 uce-1161_p1 uce-1161_p2 locus0836 uce-1156_p1 uce-1156_p2 locus0837 uce-1155_p1 uce-1155_p2 locus0838 uce-994_p1 uce-994_p2 uce-995_p1 locus0839 uce-1142_p1 uce-1142_p2 locus0840 uce-1140_p1 uce-1140_p2 locus0841 uce-1139_p1 uce-1139_p2 locus0842 uce-1135_p1 uce-1135_p2 locus0843 uce-1130_p1 uce-1130_p2 locus0844 uce-1129_p1 uce-1129_p2 locus0845 uce-1125_p1 uce-1125_p2 locus0846 uce-1114_p1 uce-1114_p2 locus0847 uce-1113_p1 uce-1113_p2 locus0848 uce-1112_p1 uce-1112_p2 locus0849 uce-1299_p1 uce-1299_p2 uce-1300_p1 uce-1300_p2 locus0850 uce-1157_p2 uce-1158_p1 uce-1158_p2 locus0851 uce-1109_p1 uce-1109_p2 locus0852 uce-1108_p1 uce-1108_p2 locus0853 uce-1098_p1 uce-1098_p2 locus0854 uce-1097_p1 uce-1097_p2 locus0855 uce-1092_p1 uce-1092_p2 locus0856 uce-1091_p1 uce-1091_p2 locus0857 uce-1086_p1 uce-1086_p2 locus0858 uce-1078_p1 uce-1078_p2 locus0859 uce-1073_p1 uce-1073_p2 locus0860 uce-1072_p1 uce-1072_p2 locus0861 uce-592_p2 uce-593_p1 uce-593_p2 locus0862 uce-1062_p1 uce-1062_p2 locus0863 uce-1060_p1 uce-1060_p2 locus0864 uce-1055_p1 uce-1055_p2 locus0865 uce-1047_p1 uce-1047_p2 locus0866 uce-1046_p1 uce-1046_p2 locus0867 uce-1038_p1 uce-1038_p2 locus0868 uce-1014_p1 uce-1014_p2 locus0869 uce-1011_p1 uce-1011_p2 locus0870 uce-1006_p1 uce-1006_p2 locus0871 uce-992_p1 uce-992_p2 locus0872 uce-1056_p2 uce-1057_p1 uce-1057_p2 locus0873 uce-983_p1 uce-983_p2 locus0874 uce-982_p1 uce-982_p2 locus0875 uce-981_p1 uce-981_p2 locus0876 uce-969_p1 uce-969_p2 locus0877 uce-968_p1 uce-968_p2 locus0878 uce-965_p1 uce-965_p2 locus0879 uce-964_p1 uce-964_p2 locus0880 uce-961_p1 uce-961_p2 locus0881 uce-959_p1 uce-959_p2 locus0882 uce-954_p1 uce-954_p2 locus0883 uce-1002_p1 uce-1002_p2 uce-1003_p1 locus0884 uce-945_p1 uce-945_p2 locus0885 uce-939_p1 uce-939_p2 locus0886 uce-938_p1 uce-938_p2 locus0887 uce-935_p1 uce-935_p2 locus0888 uce-933_p1 uce-933_p2 locus0889 uce-929_p1 uce-929_p2 locus0890 uce-922_p1 uce-922_p2 locus0891 uce-911_p1 uce-911_p2 locus0892 uce-910_p1 uce-910_p2 locus0893 uce-902_p1 uce-902_p2 locus0894 uce-322_p1 uce-322_p2 uce-323_p1 locus0895 uce-900_p1 uce-900_p2 locus0896 uce-896_p1 uce-896_p2 locus0897 uce-881_p1 uce-881_p2 locus0898 uce-880_p1 uce-880_p2 locus0899 uce-879_p1 uce-879_p2 locus0900 uce-878_p1 uce-878_p2 locus0901 uce-876_p1 uce-876_p2 locus0902 uce-872_p1 uce-872_p2 locus0903 uce-867_p1 uce-867_p2 locus0904 uce-863_p1 uce-863_p2 locus0905 uce-726_p2 uce-727_p1 uce-727_p2 locus0906 uce-862_p1 uce-862_p2 locus0907 uce-860_p1 uce-860_p2 locus0908 uce-847_p1 uce-847_p2 locus0909 uce-845_p1 uce-845_p2 locus0910 uce-843_p1 uce-843_p2 locus0911 uce-842_p1 uce-842_p2 locus0912 uce-839_p1 uce-839_p2 locus0913 uce-838_p1 uce-838_p2 locus0914 uce-834_p1 uce-834_p2 locus0915 uce-827_p1 uce-827_p2 locus0916 uce-853_p2 uce-854_p1 uce-854_p2 locus0917 uce-821_p1 uce-821_p2 locus0918 uce-818_p1 uce-818_p2 locus0919 uce-809_p1 uce-809_p2 locus0920 uce-799_p1 uce-799_p2 locus0921 uce-797_p1 uce-797_p2 locus0922 uce-793_p1 uce-793_p2 locus0923 uce-792_p1 uce-792_p2 locus0924 uce-791_p1 uce-791_p2 locus0925 uce-790_p1 uce-790_p2 locus0926 uce-783_p1 uce-783_p2 locus0927 uce-451_p1 uce-451_p2 uce-452_p1 locus0928 uce-780_p1 uce-780_p2 locus0929 uce-776_p1 uce-776_p2 locus0930 uce-775_p1 uce-775_p2 locus0931 uce-771_p1 uce-771_p2 locus0932 uce-769_p1 uce-769_p2 locus0933 uce-766_p1 uce-766_p2 locus0934 uce-763_p1 uce-763_p2 locus0935 uce-758_p1 uce-758_p2 locus0936 uce-748_p1 uce-748_p2 locus0937 uce-741_p1 uce-741_p2 locus0938 uce-762_p1 uce-762_p2 locus0939 uce-728_p1 uce-728_p2 locus0940 uce-717_p1 uce-717_p2 locus0941 uce-706_p1 uce-706_p2 locus0942 uce-701_p1 uce-701_p2 locus0943 uce-696_p1 uce-696_p2 locus0944 uce-695_p1 uce-695_p2 locus0945 uce-694_p1 uce-694_p2 locus0946 uce-693_p1 uce-693_p2 locus0947 uce-686_p1 uce-686_p2 locus0948 uce-683_p1 uce-683_p2 locus0949 uce-1502_p1 uce-1502_p2 locus0950 uce-682_p1 uce-682_p2 locus0951 uce-677_p1 uce-677_p2 locus0952 uce-676_p1 uce-676_p2 locus0953 uce-670_p1 uce-670_p2 locus0954 uce-669_p1 uce-669_p2 locus0955 uce-664_p1 uce-664_p2 locus0956 uce-662_p1 uce-662_p2 locus0957 uce-659_p1 uce-659_p2 locus0958 uce-652_p1 uce-652_p2 locus0959 uce-651_p1 uce-651_p2 locus0960 uce-1453_p1 uce-1453_p2 uce-1454_p1 uce-1454_p2 locus0961 uce-1482_p1 uce-1482_p2 locus0962 uce-647_p1 uce-647_p2 locus0963 uce-637_p1 uce-637_p2 locus0964 uce-636_p1 uce-636_p2 locus0965 uce-618_p1 uce-618_p2 locus0966 uce-617_p1 uce-617_p2 locus0967 uce-613_p1 uce-613_p2 locus0968 uce-605_p1 uce-605_p2 locus0969 uce-604_p1 uce-604_p2 locus0970 uce-600_p1 uce-600_p2 locus0971 uce-598_p1 uce-598_p2 locus0972 uce-1474_p1 uce-1474_p2 locus0973 uce-591_p1 uce-591_p2 locus0974 uce-583_p1 uce-583_p2 locus0975 uce-573_p1 uce-573_p2 locus0976 uce-570_p1 uce-570_p2 locus0977 uce-569_p1 uce-569_p2 locus0978 uce-568_p1 uce-568_p2 locus0979 uce-565_p1 uce-565_p2 locus0980 uce-559_p1 uce-559_p2 locus0981 uce-546_p1 uce-546_p2 locus0982 uce-545_p1 uce-545_p2 locus0983 uce-1473_p1 uce-1473_p2 locus0984 uce-538_p1 uce-538_p2 locus0985 uce-534_p1 uce-534_p2 locus0986 uce-532_p1 uce-532_p2 locus0987 uce-530_p1 uce-530_p2 locus0988 uce-527_p1 uce-527_p2 locus0989 uce-525_p1 uce-525_p2 locus0990 uce-520_p1 uce-520_p2 locus0991 uce-519_p1 uce-519_p2 locus0992 uce-516_p1 uce-516_p2 locus0993 uce-510_p1 uce-510_p2 locus0994 uce-1469_p1 uce-1469_p2 locus0995 uce-502_p1 uce-502_p2 locus0996 uce-493_p1 uce-493_p2 locus0997 uce-489_p1 uce-489_p2 locus0998 uce-473_p1 uce-473_p2 locus0999 uce-450_p1 uce-450_p2 locus1000 uce-448_p1 uce-448_p2 locus1001 uce-443_p1 uce-443_p2 locus1002 uce-442_p1 uce-442_p2 locus1003 uce-438_p1 uce-438_p2 locus1004 uce-433_p1 uce-433_p2 locus1005 uce-4_p1 uce-4_p2 locus1006 uce-423_p1 uce-423_p2 locus1007 uce-421_p1 uce-421_p2 locus1008 uce-412_p1 uce-412_p2 locus1009 uce-411_p1 uce-411_p2 locus1010 uce-408_p1 uce-408_p2 locus1011 uce-402_p1 uce-402_p2 locus1012 uce-399_p1 uce-399_p2 locus1013 uce-396_p1 uce-396_p2 locus1014 uce-387_p1 uce-387_p2 locus1015 uce-368_p1 uce-368_p2 locus1016 uce-1447_p1 uce-1447_p2 locus1017 uce-365_p1 uce-365_p2 locus1018 uce-361_p1 uce-361_p2 locus1019 uce-355_p1 uce-355_p2 locus1020 uce-351_p1 uce-351_p2 locus1021 uce-341_p1 uce-341_p2 locus1022 uce-298_p1 uce-298_p2 locus1023 uce-290_p1 uce-290_p2 locus1024 uce-285_p1 uce-285_p2 locus1025 uce-279_p1 uce-279_p2 locus1026 uce-276_p1 uce-276_p2 locus1027 uce-1445_p1 uce-1445_p2 locus1028 uce-273_p1 uce-273_p2 locus1029 uce-270_p1 uce-270_p2 locus1030 uce-266_p1 uce-266_p2 locus1031 uce-263_p1 uce-263_p2 locus1032 uce-255_p1 uce-255_p2 locus1033 uce-252_p1 uce-252_p2 locus1034 uce-249_p1 uce-249_p2 locus1035 uce-245_p1 uce-245_p2 locus1036 uce-244_p1 uce-244_p2 locus1037 uce-238_p1 uce-238_p2 locus1038 uce-1444_p1 uce-1444_p2 locus1039 uce-222_p1 uce-222_p2 locus1040 uce-221_p1 uce-221_p2 locus1041 uce-208_p1 uce-208_p2 locus1042 uce-207_p1 uce-207_p2 locus1043 uce-204_p1 uce-204_p2 locus1044 uce-197_p1 uce-197_p2 locus1045 uce-194_p1 uce-194_p2 locus1046 uce-192_p1 uce-192_p2 locus1047 uce-188_p1 uce-188_p2 locus1048 uce-175_p1 uce-175_p2 locus1049 uce-1434_p1 uce-1434_p2 locus1050 uce-168_p1 uce-168_p2 locus1051 uce-163_p1 uce-163_p2 locus1052 uce-161_p1 uce-161_p2 locus1053 uce-156_p1 uce-156_p2 locus1054 uce-154_p1 uce-154_p2 locus1055 uce-153_p1 uce-153_p2 locus1056 uce-152_p1 uce-152_p2 locus1057 uce-142_p1 uce-142_p2 locus1058 uce-141_p1 uce-141_p2 locus1059 uce-128_p1 uce-128_p2 locus1060 uce-1432_p1 uce-1432_p2 locus1061 uce-124_p1 uce-124_p2 locus1062 uce-119_p1 uce-119_p2 locus1063 uce-113_p1 uce-113_p2 locus1064 uce-111_p1 uce-111_p2 locus1065 uce-109_p1 uce-109_p2 locus1066 uce-107_p1 uce-107_p2 locus1067 uce-106_p1 uce-106_p2 locus1068 uce-103_p1 uce-103_p2 locus1069 uce-102_p1 uce-102_p2 locus1070 uce-97_p1 uce-97_p2 locus1071 uce-1493_p1 uce-1493_p2 uce-1494_p1 uce-1494_p2 locus1072 uce-1424_p1 uce-1424_p2 locus1073 uce-93_p1 uce-93_p2 locus1074 uce-92_p1 uce-92_p2 locus1075 uce-91_p1 uce-91_p2 locus1076 uce-90_p1 uce-90_p2 locus1077 uce-82_p1 uce-82_p2 locus1078 uce-63_p1 uce-63_p2 locus1079 uce-58_p1 uce-58_p2 locus1080 uce-57_p1 uce-57_p2 locus1081 uce-51_p1 uce-51_p2 locus1082 uce-50_p1 uce-50_p2 locus1083 uce-1419_p1 uce-1419_p2 locus1084 uce-40_p1 uce-40_p2 locus1085 uce-37_p1 uce-37_p2 locus1086 uce-36_p1 uce-36_p2 locus1087 uce-32_p1 uce-32_p2 locus1088 uce-31_p1 uce-31_p2 locus1089 uce-24_p1 uce-24_p2 locus1090 uce-14_p1 uce-14_p2 locus1091 uce-12_p1 uce-12_p2 locus1092 uce-1504_p1 uce-1504_p2 locus1093 uce-1499_p1 uce-1499_p2 locus1094 uce-1416_p1 uce-1416_p2 locus1095 uce-1496_p1 uce-1496_p2 locus1096 uce-1486_p1 uce-1486_p2 locus1097 uce-1483_p1 uce-1483_p2 locus1098 uce-1479_p1 uce-1479_p2 locus1099 uce-1451_p1 uce-1451_p2 locus1100 uce-1440_p1 uce-1440_p2 locus1101 uce-1426_p1 uce-1426_p2 locus1102 uce-1425_p1 uce-1425_p2 locus1103 uce-1406_p1 uce-1406_p2 locus1104 uce-1398_p1 uce-1398_p2 locus1105 uce-1405_p1 uce-1405_p2 locus1106 uce-1394_p1 uce-1394_p2 locus1107 uce-1393_p1 uce-1393_p2 locus1108 uce-1377_p1 uce-1377_p2 locus1109 uce-1374_p1 uce-1374_p2 locus1110 uce-1373_p1 uce-1373_p2 locus1111 uce-1365_p1 uce-1365_p2 locus1112 uce-1362_p1 uce-1362_p2 locus1113 uce-1353_p1 uce-1353_p2 locus1114 uce-1340_p1 uce-1340_p2 locus1115 uce-1334_p1 uce-1334_p2 locus1116 uce-1401_p1 uce-1401_p2 locus1117 uce-1331_p1 uce-1331_p2 locus1118 uce-1327_p1 uce-1327_p2 locus1119 uce-1323_p1 uce-1323_p2 locus1120 uce-1321_p1 uce-1321_p2 locus1121 uce-1317_p1 uce-1317_p2 locus1122 uce-1316_p1 uce-1316_p2 locus1123 uce-1306_p1 uce-1306_p2 locus1124 uce-1304_p1 uce-1304_p2 locus1125 uce-1303_p1 uce-1303_p2 locus1126 uce-1301_p1 uce-1301_p2 locus1127 uce-1396_p1 uce-1396_p2 locus1128 uce-1297_p1 uce-1297_p2 locus1129 uce-1283_p1 uce-1283_p2 locus1130 uce-1277_p1 uce-1277_p2 locus1131 uce-1272_p1 uce-1272_p2 locus1132 uce-1269_p1 uce-1269_p2 locus1133 uce-1268_p1 uce-1268_p2 locus1134 uce-1267_p1 uce-1267_p2 locus1135 uce-1254_p1 uce-1254_p2 locus1136 uce-1238_p1 uce-1238_p2 locus1137 uce-1230_p1 uce-1230_p2 locus1138 uce-1391_p1 uce-1391_p2 locus1139 uce-1229_p1 uce-1229_p2 locus1140 uce-1227_p1 uce-1227_p2 locus1141 uce-1216_p1 uce-1216_p2 locus1142 uce-1209_p1 uce-1209_p2 locus1143 uce-1206_p1 uce-1206_p2 locus1144 uce-1205_p1 uce-1205_p2 locus1145 uce-1197_p1 uce-1197_p2 locus1146 uce-1192_p1 uce-1192_p2 locus1147 uce-1190_p1 uce-1190_p2 locus1148 uce-1189_p1 uce-1189_p2 locus1149 uce-1369_p1 uce-1369_p2 locus1150 uce-1183_p1 uce-1183_p2 locus1151 uce-1180_p1 uce-1180_p2 locus1152 uce-1176_p1 uce-1176_p2 locus1153 uce-1171_p1 uce-1171_p2 locus1154 uce-1167_p1 uce-1167_p2 locus1155 uce-1166_p1 uce-1166_p2 locus1156 uce-1159_p1 uce-1159_p2 locus1157 uce-1126_p1 uce-1126_p2 locus1158 uce-1117_p1 uce-1117_p2 locus1159 uce-1107_p1 uce-1107_p2 locus1160 uce-1368_p1 uce-1368_p2 locus1161 uce-1105_p1 uce-1105_p2 locus1162 uce-1102_p1 uce-1102_p2 locus1163 uce-1096_p1 uce-1096_p2 locus1164 uce-1085_p1 uce-1085_p2 locus1165 uce-1084_p1 uce-1084_p2 locus1166 uce-1082_p1 uce-1082_p2 locus1167 uce-1079_p1 uce-1079_p2 locus1168 uce-1075_p1 uce-1075_p2 locus1169 uce-1071_p1 uce-1071_p2 locus1170 uce-1069_p1 uce-1069_p2 locus1171 uce-1367_p1 uce-1367_p2 locus1172 uce-1067_p1 uce-1067_p2 locus1173 uce-1064_p1 uce-1064_p2 locus1174 uce-1059_p1 uce-1059_p2 locus1175 uce-1045_p1 uce-1045_p2 locus1176 uce-1035_p1 uce-1035_p2 locus1177 uce-1033_p1 uce-1033_p2 locus1178 uce-1032_p1 uce-1032_p2 locus1179 uce-1028_p1 uce-1028_p2 locus1180 uce-1027_p1 uce-1027_p2 locus1181 uce-1023_p1 uce-1023_p2 locus1287 uce-1_p1 locus1182 uce-1009_p1 locus1183 uce-101_p2 locus1184 uce-1021_p1 locus1186 uce-105_p2 locus1185 uce-1054_p2 locus1187 uce-1068_p2 locus1188 uce-1070_p2 locus1189 uce-1081_p1 locus1190 uce-1087_p1 locus1191 uce-1089_p2 locus1192 uce-1090_p1 locus1193 uce-1103_p1 locus1194 uce-1118_p2 locus1197 uce-112_p1 locus1195 uce-1122_p2 locus1196 uce-1123_p1 locus1198 uce-1132_p2 locus1199 uce-1137_p2 locus1200 uce-1138_p2 locus1201 uce-1141_p1 locus1202 uce-1143_p2 locus1203 uce-1145_p1 locus1204 uce-1150_p1 locus1205 uce-1152_p1 locus1206 uce-1168_p2 locus1207 uce-1170_p2 locus1208 uce-1172_p1 locus1209 uce-1179_p2 locus1210 uce-1185_p2 locus1211 uce-1195_p2 locus1212 uce-1198_p2 locus1213 uce-1202_p2 locus1214 uce-1204_p1 locus1215 uce-1210_p2 locus1216 uce-1221_p2 locus1217 uce-1224_p1 locus1218 uce-1226_p2 locus1219 uce-123_p1 locus1220 uce-1246_p2 locus1221 uce-1263_p1 locus1222 uce-1276_p2 locus1223 uce-1285_p2 locus1224 uce-1288_p1 locus1225 uce-1289_p2 locus1226 uce-1290_p2 locus1227 uce-1291_p1 locus1228 uce-1292_p1 locus1229 uce-1294_p1 locus1230 uce-1295_p1 locus1231 uce-1302_p1 locus1232 uce-1309_p1 locus1233 uce-1312_p1 locus1234 uce-1320_p1 locus1239 uce-133_p2 locus1235 uce-1332_p2 locus1236 uce-1336_p2 locus1237 uce-1338_p1 locus1238 uce-1339_p1 locus1242 uce-134_p2 locus1240 uce-1341_p1 locus1241 uce-1347_p2 locus1245 uce-135_p1 locus1243 uce-1354_p1 locus1244 uce-1357_p2 locus1246 uce-137_p1 locus1251 uce-138_p2 locus1247 uce-1384_p2 locus1248 uce-1385_p1 locus1249 uce-1386_p2 locus1250 uce-1388_p2 locus1252 uce-1395_p1 locus1256 uce-140_p2 locus1253 uce-1400_p1 locus1254 uce-1402_p1 locus1255 uce-1404_p2 locus1257 uce-1411_p2 locus1258 uce-1430_p2 locus1259 uce-1437_p1 locus1260 uce-1441_p2 locus1261 uce-1458_p1 locus1262 uce-1459_p2 locus1264 uce-147_p2 locus1263 uce-1477_p1 locus1265 uce-1480_p2 locus1269 uce-149_p1 locus1266 uce-1490_p1 locus1267 uce-1491_p2 locus1268 uce-1498_p1 locus1270 uce-1501_p1 locus1271 uce-1503_p2 locus1272 uce-1507_p2 locus1273 uce-1509_p2 locus1274 uce-151_p1 locus1275 uce-158_p2 locus1276 uce-166_p1 locus1277 uce-176_p2 locus1278 uce-177_p2 locus1279 uce-183_p1 locus1280 uce-184_p2 locus1281 uce-187_p2 locus1286 uce-19_p2 locus1282 uce-190_p2 locus1283 uce-193_p1 locus1284 uce-196_p2 locus1285 uce-198_p1 locus1288 uce-211_p1 locus1289 uce-214_p2 locus1290 uce-215_p1 locus1291 uce-216_p2 locus1292 uce-218_p1 locus1293 uce-220_p2 locus1294 uce-223_p1 locus1295 uce-227_p2 locus1296 uce-231_p2 locus1297 uce-246_p2 locus1298 uce-262_p1 locus1299 uce-264_p1 locus1300 uce-268_p1 locus1301 uce-272_p2 locus1302 uce-277_p2 locus1303 uce-280_p1 locus1304 uce-289_p2 locus1305 uce-292 locus1306 uce-294_p1 locus1309 uce-30_p1 locus1307 uce-300_p2 locus1308 uce-306_p1 locus1310 uce-314_p2 locus1311 uce-318_p1 locus1312 uce-324_p1 locus1313 uce-331_p2 locus1314 uce-336_p2 locus1315 uce-346_p1 locus1316 uce-352_p1 locus1317 uce-353_p2 locus1318 uce-354_p1 locus1319 uce-356_p2 locus1320 uce-357_p1 locus1321 uce-358_p1 locus1322 uce-371_p1 locus1323 uce-372_p2 locus1324 uce-378_p1 locus1325 uce-381_p1 locus1326 uce-382_p2 locus1327 uce-384_p2 locus1328 uce-385_p2 locus1329 uce-398_p1 locus1330 uce-405_p1 locus1331 uce-410_p1 locus1332 uce-414_p1 locus1333 uce-425_p1 locus1334 uce-426_p2 locus1335 uce-434_p2 locus1336 uce-435_p1 locus1337 uce-447_p2 locus1338 uce-454_p2 locus1339 uce-462_p2 locus1340 uce-463_p2 locus1341 uce-464_p1 locus1342 uce-469_p1 locus1343 uce-474_p1 locus1344 uce-477_p2 locus1345 uce-482_p1 locus1346 uce-484_p1 locus1347 uce-485_p2 locus1348 uce-487_p2 locus1349 uce-490_p1 locus1350 uce-494_p2 locus1351 uce-549_p2 locus1352 uce-555_p2 locus1353 uce-558_p1 locus1356 uce-56_p2 locus1354 uce-562_p2 locus1355 uce-563_p1 locus1357 uce-572_p1 locus1358 uce-575_p1 locus1359 uce-587_p2 locus1360 uce-599_p1 locus1361 uce-602_p1 locus1362 uce-603_p1 locus1363 uce-612_p2 locus1364 uce-619_p2 locus1365 uce-626_p1 locus1366 uce-628_p1 locus1367 uce-639_p2 locus1368 uce-648_p1 locus1369 uce-653_p2 locus1371 uce-66_p1 locus1370 uce-660_p2 locus1372 uce-671_p1 locus1373 uce-674_p2 locus1374 uce-680_p1 locus1375 uce-681_p1 locus1376 uce-685_p1 locus1377 uce-697_p1 locus1378 uce-698_p2 locus1394 uce-7_p1 locus1379 uce-700_p1 locus1380 uce-705 locus1381 uce-712_p1 locus1382 uce-732_p2 locus1383 uce-733_p1 locus1384 uce-740_p1 locus1385 uce-742_p1 locus1386 uce-760_p1 locus1387 uce-768_p2 locus1388 uce-782_p2 locus1389 uce-788_p1 locus1390 uce-794_p1 locus1391 uce-795_p2 locus1392 uce-796_p1 locus1393 uce-798_p1 locus1395 uce-802_p1 locus1396 uce-806_p2 locus1397 uce-807_p2 locus1398 uce-808_p2 locus1399 uce-810_p2 locus1400 uce-817_p2 locus1401 uce-829_p2 locus1404 uce-85_p1 locus1402 uce-855_p1 locus1403 uce-857_p1 locus1405 uce-865_p2 locus1406 uce-868_p2 locus1407 uce-869_p1 locus1408 uce-871_p1 locus1409 uce-874_p1 locus1410 uce-883_p1 locus1411 uce-884_p2 locus1412 uce-885_p1 locus1413 uce-887_p2 locus1414 uce-889_p1 locus1415 uce-89_p1 locus1416 uce-903_p2 locus1417 uce-904_p2 locus1418 uce-908_p1 locus1419 uce-917_p1 locus1420 uce-919_p1 locus1421 uce-921_p2 locus1422 uce-924_p1 locus1423 uce-926_p1 locus1424 uce-94_p1 locus1425 uce-951_p1 locus1426 uce-967_p2 locus1427 uce-974_p1 locus1428 uce-977_p1 locus1429 uce-979_p2 locus1430 uce-980_p2 locus1431 uce-986_p2 locus1432 uce-999_p2

AUTHOR CONTRIBUTIONS

Designed the study: AC, JYR; contributed samples and identifications: AC, LF, AG, JH, AP, JYR; optimized protocol: SN with the help of AC, JYR, PA; performed lab work: SN with the

Figure S1

Analytical workflow -Step 1. Pre-processing of the data: from raw reads to raw UCEs *The 2749 probes designed by Faircloth et al. (2015) were first assembled into a set of nonoverlapping loci using Geneious. This set of reference UCE loci (N=1432) was then used to align sample-specific contigs obtained with CAP3. The reference UCEs are available in additional file 4.