# Development of a set of SNP markers for population genetics of the sea peach (Halocynthia papillosa)

Mariana Padrón, Marine Milhes, Adline Delcamp, Erwan Guichoux, Katell Guizien

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Mariana Padrón, Marine Milhes, Adline Delcamp, Erwan Guichoux, Katell Guizien. Development of a set of SNP markers for population genetics of the sea peach (Halocynthia papillosa). Conservation Genetics Resources, 2023, 10.1007/s12686-023-01313-7. hal-04004739

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Development of a set of SNP markers for population genetics of the sea peach

(Halocynthia papillosa)

Padrón, M¹., Milhes M.², Delcamp, A.³, Guichoux, E.³ & Guizien K.¹

¹ Laboratoire d'Ecogeochimie des Environnements Benthique, CNRS, Sorbonne Université,

UMR 8222, F-66650, Banyuls-sur-Mer, France

² INRA, GeT-PlaGe, Genotoul, 31326 Castanet- Tolosan, France

³ Univ. Bordeaux, INRAE, BIOGECO, F-33610 Cestas, France

Corresponding author: guizien@obs-banyuls.fr

Abstract

A set of single nucleotide polymorphism (SNP) markers was developed from the transcriptome of the solitary ascidian *Halocynthia papillosa*. 1399 putative SNPs with coverage larger than 100 reads were identified, and a selected set of 320 SNPs was tested using a MassARRAY System on 95 samples from the NW Mediterranean. A total of 175 SNPs were successfully genotyped, polymorphic and in Hardy-Weinberg equilibrium over all samples. The newly developed loci will be a valuable tool for population genetics studies of this endemic ascidian species of the Mediterranean Sea.

Keywords

Halocynthia papillosa, Single nucleotide polymorphism, MassARRAY

Introduction

The solitary ascidian *Halocynthia papillosa* is an endemic species of the Mediterranean sea (Pérès and Picard, 1964) and Eastern Atlantic from the Canary Islands to the Berlengas
26 Islands in Portugal (Ramos-Esplá, 1991). It is a common sessile mega-benthic species with
27 medium size of about 15 cm that distributes evenly with low population density (a few
28 individuals per meter square) on hard bottom between 10 to 100 m in the Mediterranean sea
29 (Antoniadou et al. 2006; Coppari et al. 2014). Tunicates have been extensively studied for
30 their tunique made of a unique animal cellulose (Helbert et al. 1998). In contrast, the ecology
31 of the species has driven less interest. The species is a filter-feeder with a broad natural diet
32 including detrital organic matter, heterotrophic bacteria, *Prochlorococcus* sp., *Synechococcus*
33 sp., protozoa and phytoplankton with a mean size range from 0.6 to 70 µm (Ribes et al.
34 1998). It is a simultaneous hermaphrodite with a spawning period extending over 2 to 3
35 months, between September and November (Becerro and Turon, 1992). Ecologically, it is
36 considered an indicator of good conditions and unperturbed rocky areas (Naranjo et al. 1996).
37 Low population density of *H. papillosa* has been correlated to high SCUBA diving frequency
38 (Luna-Pérez et al., 2010). The deleterious impact of SCUBA diving on *H. papillosa* was
39 further tested experimentally by applying treatments with various intensities of flapping, with
40 highest flapping intensity altering *H. papillosa* growth and abundance (Luna-Pérez et al.,
41 2011). Nevertheless, the species has been found on artificial reefs deployed over soft-bottom
42 substrates, showing the potential of the species to survive in turbid environments (Blouet et
43 al. 2022). In addition, the colonisation of artificial reefs located at a few kms from any rocky
44 substrate question the dispersal potential of this species, an essential mechanism for
45 population recovery in a sensitive species. The objective of the present study is to propose a
46 set of Single Nucleotide Polymorphism markers that will be useful to study *H. papillosa*
47 population genetics and connectivity.
48

48

49 **Material and Methods**

50 *Transcriptome sequencing*
The transcriptome of *H. papillosa* was sequenced from the total RNA of ten individuals collected by SCUBA diving between 10 and 20 meters depth in Banyuls sur Mer, France. Small fragments were immediately frozen in liquid nitrogen, and preserved at -80 °C. Total RNA extractions were performed with Maxwell 16 LEV simplyRNA purification kits. RNA concentrations and quality were analysed with an Agilent 2100 Bioanalyzer. RNA-seq libraries were prepared according to Illumina's protocols using the Illumina TruSeq Stranded mRNA sample prep kit, using 11 cycles of PCR to amplify libraries. Library quality was assessed using a Fragment Analyzer. Libraries were quantified by qPCR using the Kapa Library Quantification Kit. Sequencing was achieved on an Illumina HiSeq3000 using a paired-end read length of 2 × 150 pb.

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## Marker development and screening

Sequence read quality was assessed using FastQC v0.10.1 (Andrews 2010). Trimmomatic v0.32 was used to remove low quality reads with a Phred score below 20, as well as the Illumina adapters (Bolger et al. 2014). FastQC was performed again to verify the integrity of the remaining raw Illumina sequence reads. High-quality reads were then used for the de novo transcriptome assembly, using Trinity with its default k-mer value of 25 (Grabherr et al. 2011). DiscoSnp + + v2.2.10 (Uricaru et al. 2015) was used independently with kmer size of 61 and a minimal coverage per read 20 to call SNPs using the high-quality reads of the 10 individuals transcriptomes. A total of 28320 SNPs were found among which 1399 SNPs had good coverage (> 100 reads) in each of the 10 individual's transcriptomes. A set of 320 SNPs were randomly selected and submitted for assay design using the MassARRAY Assay Designer version 4.0.0.2 (Agena Biosciences). The assay was performed on the genomic DNA extracted from 95 samples of *H. papillosa* collected in 6 locations in the NW Mediterranean (Table 1, 5 locations in Agde and 1 location in Saint-Cyprien), using the
76 DNeasy Blood and Tissue kits from QIAGEN. Allele calling was carried out with Typer
77 Viewer v.4.0.24.71 (Agena Biosciences). Monomorphic SNPs, loci with weak or ambiguous
78 signal (i.e., displaying more than three clusters of genotypes or unclear cluster delimitation)
79 and loci with too much missing data were all discarded. The genetic parameters, including
80 observed heterozygosity, expected heterozygosity, and the Hardy-Weinberg equilibrium
81 (HWE), were calculated using Genepop 4.7.5 (Rousset 2008).
<table><caption>**Table 1.-** Location and number of individuals (N) of *Halocynthia papillosa* used for SNP validation using MassArray</caption><thead><tr><th>Location</th><th>Site</th><th>Latitude (decimal degrees)</th><th>Longitude (decimal degrees)</th><th>Depth (m)</th><th>N</th></tr></thead><tbody><tr><td rowspan="2">Cap d'Agde -<br>natural rocks</td><td>A1</td><td>43,252556</td><td>3,519840</td><td>13</td><td>20</td></tr><tr><td>A4</td><td>43,244578</td><td>3,519568</td><td>25</td><td>15</td></tr><tr><td rowspan="3">Cap d'Agde –<br>artificial reef</td><td>AGD26</td><td>43,2386</td><td>3,4992</td><td>20</td><td>10</td></tr><tr><td>AGD28</td><td>43,2521</td><td>3,5378</td><td>20</td><td>6</td></tr><tr><td>AGD30</td><td>43,2543</td><td>3,4756</td><td>10</td><td>20</td></tr><tr><td>Saint Cyprien –<br>artificial reef</td><td>CYP 54</td><td>42,6293</td><td>3,0720</td><td>30</td><td>20</td></tr></tbody></table>

## Results

A total of 220 SNPs were successfully amplified across more than half of the samples. A subset of 188 loci were found to be polymorphic, resulting in a detectable rate of 85.4%. The observed heterozygosity ranged from 0.011 to 0.989, while the expected heterozygosity varied from 0.011 to 0.503 (Supplementary Material). Among the 188 loci, 175 were found in HWE proportions over 95 samples ($p > 0.01$). These results provide a valuable resource for future population genetic studies of this endemic ascidian species in the Mediterranean Sea.

## Acknowledgements

This work was funded by the Agence de l'Eau Rhône-Méditerranée-Corse under project ICONE - Impacts actuels et potentiels de la CONnectivité Ecologique ajoutée par les récifs artificiels sur la biodiversité fixée des substrats durs du Golfe du Lion (PI, K. Guizien, AAP 2016). The authors gratefully acknowledge the helpful assistance during sampling of S. Blouet and the staff of the Aire Marine Protégée Agatoise. We also thank the scuba divers, JC Roca and B Hesse, for their technical assistance for field sampling in Banyuls sur Mer. We are grateful to the Bio2Mar core facility (http://bio2marobs-banyuls.fr) for providing access to instrumentation for RNA extractions. RNAseq was performed at the GeT-PlaGe core
facility, INRAE Toulouse. SNP genotyping was performed at the PGTB
(doi:10.15454/1.5572396583599417E12) with the help of Laure Dubois.

106

Author contributions: MP and KG designed the study. MM, AD, and EG, performed the sequencing and genotyping. MP analyzed the data. KG wrote the main manuscript text. All authors reviewed the manuscript.

110

Funding: This work was funded by the Agence de l'Eau Rhône-Méditerranée-Corse under project ICONE - Impacts actuels et potentiels de la CONnectivité Ecologique ajoutée par les récifs artificiels sur la biodiversité fixée des substrats durs du Golfe du Lion (PI, K. Guizien, AAP 2016, grant numbers : 2017 7267 / 2017 7268 / 2018 0697).

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Data availability: The RNA seq data will be released publicly through National Center for Biotechnology Information portal (https://submit.ncbi.nlm.nih.gov).

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