THE TETRAPLOID CITRUS HYBRID CHALLENGE: GREAT POTENTIAL IN SALT-TOLERANT ROOTSTOCK SELECTION AND A COMPLEX MATERIAL TO WORK WITH

THE TETRAPLOID CITRUS HYBRID CHALLENGE: GREAT POTENTIAL IN SALT-TOLERANT ROOTSTOCK SELECTION AND A COMPLEX MATERIAL TO WORK WITH | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 May 2025 V1 Latest version Share on THE TETRAPLOID CITRUS HYBRID CHALLENGE: GREAT POTENTIAL IN SALT-TOLERANT ROOTSTOCK SELECTION AND A COMPLEX MATERIAL TO WORK WITH Authors : Marie Bonnin , Alexandre Soriano , Radia Lourkisti , Julie Oustric , Lenny Calvez , Maëva Miranda , Nathalie Leonhardt , Patrick Ollitrault , Liliane Berti , Jeremie Santini , Raphael Morillon , and Bénédicte Favreau 0000-0003-2786-9374 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174677060.00112058/v1 280 views 165 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Water limitation associated with climate changes leads to increased use of poor-quality water for irrigation, in turn enhancing soil salinization. Soil salinity has become a major threat in many regions of the world, limiting plant growth and productivity. The development of alternative salt-tolerant rootstocks that can also withstand biotic stresses is a priority for the citrus industry. Polyploidy is one of the major forces driving plant evolution and provides great advantages in coping with environmental constraints. Tetraploidy has been shown to enhance plant adaptation to various abiotic stresses, including salt stress. The evaluation of allotetraploid citrus hybrids, which can combine desired traits from both parents, appears to be an effective way to address the rootstock challenge. Cleopatra mandarin ( Citrus reshni Hort. Ex Tan. ) (CL), a well-known citrus rootstock, displays good tolerance to cold, drought, chlorides, and limestone. However, it is susceptible to tristeza, exocortis, and phytophtora. On the contrary, Trifoliate orange ( Poncirus trifoliata (L.) Raf. ) (PO) is a salt-sensitive rootstock but is resistant to phytophtora and tristeza. The present study is the first to evaluate the salt stress response of a Citrandarin hybrid generated from a cross between Cleopatra and Poncirus at two ploidy levels: diploid (2x) and tetraploid (4x). To dissect the hybrid response, photosynthetic activity, antioxidant metabolism, mineral uptake, and transcriptomic regulation were studied in two tissues: leaves and roots. Using various multivariate approaches, the study successfully deciphered the complexity of the multilevel response of each 2x and 4x hybrid. Although ploidy level accounted for very limited differences between 2x and 4x, they both showed a high and common tolerance to stress. However, the 4x exhibited a higher level of tolerance than the 2x thanks to its specific leaf photosynthesis and root antioxidant metabolism. Finally, this study provides insights into the finely tuned mechanisms underlying the respective salt stress adaptation of the 2x and 4x Citrandarins thanks to the identification of key genes selected using covariance analysis. Plant, Cell and Environment (2025) XX, XXX–XXX doi: XX.XXXX/X.XXXX-XXXX.2025.XXXXX.x© 2025 Blackwell Publishing Ltd THE TETRAPLOID CITRUS HYBRID CHALLENGE: GREAT POTENTIAL IN SALT-TOLERANT ROOTSTOCK SELECTION AND A COMPLEX MATERIAL TO WORK WITH Marie Bonnin 1,2* and Alexandre Soriano 2 , Radia Lourkisti 1 , Julie Oustric 1 , Lenny Calvez 2 , Maëva Miranda 2 , Nathalie Leonhardt 3 , Patrick Ollitrault 2 , Liliane Berti 1 , Jérémie Santini 1 , Raphaël Morillon 2 , Bénédicte Favreau 2* 1 CNRS, Equipe d’Adaptation des végétaux aux changements globaux, Projet Ressources Naturelles, UMR 6134 SPE, Université́ de Corse, Corsica, France. 2 Unité Mixte de Recherche Amélioration Génétique et Adaptation des Plantes méditerranéennes et tropicales (UMR AGAP) Institut, Univ. Montpellier, Centre de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Agro, Montpellier, France. 3 CEA, CNRS, BIAM, UMR7265, Aix Marseille Université, 13108 Saint Paul-Lez-Durance, France * Correspondence: [email protected] , [email protected] ABSTRACT Water limitation associated with climate changes leads to increased use of poor-quality water for irrigation, in turn enhancing soil salinization. Soil salinity has become a major threat in many regions of the world, limiting plant growth and productivity. The development of alternative salt-tolerant rootstocks that can also withstand biotic stresses is a priority for the citrus industry. Polyploidy is one of the major forces driving plant evolution and provides great advantages in coping with environmental constraints. Tetraploidy has been shown to enhance plant adaptation to various abiotic stresses, including salt stress. The evaluation of allotetraploid citrus hybrids, which can combine desired traits from both parents, appears to be an effective way to address the rootstock challenge. Cleopatra mandarin ( Citrus reshni Hort. Ex Tan. ) (CL), a well-known citrus rootstock, displays good tolerance to cold, drought, chlorides, and limestone. However, it is susceptible to tristeza, exocortis, and phytophtora. On the contrary, Trifoliate orange ( Poncirus trifoliata (L.) Raf. ) (PO) is a salt-sensitive rootstock but is resistant to phytophtora and tristeza. The present study is the first to evaluate the salt stress response of a Citrandarin hybrid generated from a cross between Cleopatra and Poncirus at two ploidy levels: diploid (2x) and tetraploid (4x). To dissect the hybrid response, photosynthetic activity, antioxidant metabolism, mineral uptake, and transcriptomic regulation were studied in two tissues: leaves and roots. Using various multivariate approaches, the study successfully deciphered the complexity of the multilevel response of each 2x and 4x hybrid. Although ploidy level accounted for very limited differences between 2x and 4x, they both showed a high and common tolerance to stress. However, the 4x exhibited a higher level of tolerance than the 2x thanks to its specific leaf photosynthesis and root antioxidant metabolism. Finally, this study provides insights into the finely tuned mechanisms underlying the respective salt stress adaptation of the 2x and 4x Citrandarins thanks to the identification of key genes selected using covariance analysis. Keywords: Citrus, tetraploidy, salt stress, oxidative stress, transcriptomic. Graphical abstract: 1. INTRODUCTION Since the beginning of the 21st century, climate change (Wheeler & Watts 2018) has affected temperatures and the pattern of precipitations. Thus, water limitation will lead to plant evapotranspiration decrease, as well as reduced growth and productivity. Rising sea levels trigger salinity increase in both surface and ground water through saltwater intrusion (Ullah, Bano & Khan 2021) in coastal agriculture lands, such as around the Mediterranean ring. In these areas, where citrus crops are widely cultivated, salinity is becoming an increasing concern (Ullah et al. 2021). Salinity reduces flowering intensity, the number of fruits produced, and their quality (Storey & Walker 1998; García-Sánchez, Carvajal, Porras, Botı́a & Martínez 2003) because of the decrease in juice quantity, total soluble solids, and titratable acidity (García-Sánchez et al. 2003). Finally, salinity reduces the thickness of the fruit skin. However, salinity has no significant effect on other fruit characteristics such as size, length–weight ratio, or colour (Maas 1993). Osmotic stress induced by increased salinity at the root level leads to stomatal closure and reduction in CO 2 supply, in turn limiting the photosynthetic processes (Loreto, Centritto & Chartzoulakis 2003; Zahra et al. 2022). Salt stress affects photosynthesis by induction of high oxygen burst via highly energized electrons, as well as production of over-oxidized moieties, also known as reactive oxygen species (ROS). ROS produces rapid cell damages by triggering a chain of reactions, which make the plant susceptible. Previous studies have investigated intergeneric hybridizations between the Poncirus and Citrus genera with the goal to combine traits of interest (Cabasson, Luro, Ollitrault & Grosser 2001; Albrecht & Bowman 2012; Ruiz et al. 2018) for the production of rootstock genotypes responding to the new challenges of the citrus industry (Dambier et al. 2022). Intergeneric citrus hybrids, such as Swingle citrumelo ( C. paradisi Macf. × P. trifoliata (L.) Raf. ), Citrandarin ( C. reticulata × P. trifoliata ), Carrizo, Troyer, and C35 citrange ( C. sinensis (L.) Osb. × P. trifoliata ), are already being used all around the world. Tetraploidy was shown to provide better adaptation to various abiotic stresses including salt stress (Ruiz et al. 2016; Oustric et al. 2019; Wei, Wang & Liu 2020; Bonnin et al. 2023, 2024a). Consequently, the use of 4x hybrids with enhanced tolerance to salt stress appears to be of a high agronomic interest. Trifoliate orange ( P. trifoliata (L.) Raf ) is an important rootstock for citrus because it confers Citrus tristeza virus tolerance, but it is unfortunately salt sensitive (Bonnin et al. 2023). The high salt stress tolerance of Cleopatra mandarin ( C. reshni hort. ex ) makes it a crucial genetic resource in rootstock-improvement programmes. In a previous work, we studied the physiological and biochemical determinants of the salt stress tolerance in 2x and 4x Trifoliate orange and Cleopatra mandarin (Bonnin et al. 2023). The transcriptomic responses of these two genotypes were also investigated (Bonnin et al. 2024a). Overall, these studies highlighted the higher physiological and biochemical salt tolerance of the 4x genotypes compared with the respective 2x (Bonnin et al. 2023). We found that, in 4x Cleopatra mandarin, genes involved in carbohydrate biosynthesis and cell wall remodelling were regulated under salt stress. In Trifoliate orange genotypes, oxidative stress response and ion management capacity differed between 2x and 4x. In the present article, we aim to study the salt stress response of the 2x and 4x Citrandarin hybrid, which was obtained by sexual crossing between P. trifoliata and C. reshni. We initially selected the 4x hybrid among seedlings arising from chromosome set doubling of nucellar cells of the corresponding 2x intergeneric ( C. reshni × P. trifoliata ) hybrid. The 4x should therefore be considered an allotetraploid. We investigated hybrid responses at the physiological, biochemical, and transcriptomic levels, in both roots and leaves, and we analysed using multifactorial and multivariate analysis approaches. We first evaluated the variables that characterized each ploidy level, independently of the stress response, and those that were affected by both 2x and 4x under stress. We then highlighted the specific response of each ploidy level to salt stress and compared it with its 2x and 4x parents previously characterized by our team (Bonnin et al. 2023, 2024a). Overall, we showed that both the 2x and 4x allotetraploid Citrandarins were characterized by strong adaptive mechanisms to photosynthetic damage and enhanced antioxidant capability. However, the 4x hybrid was found to be more adapted than 2x due to the involvement of fine mechanisms to cope with salt stress. Finally, conclusions were drawn regarding the inheritance of this adaptation from either the Triofoliate orange or the Cleopatra mandarin parents and the 2x and 4x ploidy levels. 2. MATERIALS AND METHODS 2.1. Plant material Diploid and allotetraploid somatic hybrids, called Citrandarin, were generated from freestanding 2x Trifoliate orange ( Poncirus trifoliata ) (Poncirus Pomeroy ICVN-0110081) (PO2x) and 2x Cleopatra mandarin ( Citrus reshni ) (ICVN-0110274) (CL2x) and their two doubled diploid counterparts, 4x Trifoliate orange (ICVN-01011106) (PO4x) and 4x Cleopatra mandarin (ICVN-0101110) (CL4x) (Bonnin et al. 2023). All germplasm originated from the INRAE-CIRAD collection at the San Giuliano research station in Corsica (42.286, 9.520). The 4x genotypes result from spontaneous doubling of the chromosomal stock of nuclear tissues of somatic origin. They were selected through flow cytometry in seedlings of the corresponding 2x genotypes and then transferred into the collection (Aleza et al. 2011). Conformity of the genetic material was checked as described in (Bonnin et al. 2023). Here, freestanding 2x Citrandarin (ICVN-0110157) (CR2x) and 4x Citrandarin (ICVN 0101114) (CR4x) were investigated. 2.2. Salt stress experiment The experiment was conducted in a climate-controlled greenhouse from November 8th to December 17th 2021 as described in ((Bonnin et al. 2023)and article 2). Plants selected for experimentation were divided into two blocks comprising 17 stressed and 10 control plants and watered once a week. Stressed plants were watered with a nutrient solution supplemented with salt (NaCl) whereas control plants were irrigated with only a nutrient solution. The salt concentrations supplied were steadily increased from 30 mM to 90 mM at 20-mM increments per week and then stabilized at 90 mM. Gas exchanges were measured each time the salt concentration increased and were performed for each physiological parameter investigated. Samples for biochemistry and transcriptomic analyses were collected at week 4 (90 mM NaCl). Sampling for foliar and root chloride determinations was performed weekly. 2.3. Measurements of physiological and biochemical parameters All the photosynthetic parameters investigated were measured on the same fully developed leaves, with identical light exposure. Measurements were taken, as described in (Bonnin et al. 2023), between 8:00 and 11:30 am to avoid high temperatures. A portable infrared gas analyser LC-PRO-SD (ADC, BioScientific Ltd., Hoddeston, UK) was used to determine the net photosynthetic rate ( P net ) and stomatal conductance ( g s ), with photosynthetically active radiation (PAR) set at 1400 μmol.m -2 .s-1, leaf temperature was set at 28°C, and ambient carbon dioxide (CO 2 ) concentration set at 390 μmol.mol-1. An OS1p chlorophyll fluorometer (Opti-Sciences, Inc. Hudson, USA) was used to measure the maximum PSII quantum efficiency ( F v /F m ), effective PSII quantum yield (ΦPSII), and ETR, following the procedure described in (Bonnin et al. 2023).Determination of major cations and chloride in leaf and roots samples was performed following Bonnin et al., 2023 (Bonnin et al. 2023).Determination of MDA, hydrogen peroxide (H 2 O 2 ), Asa, TPC, and activity of enzymes involved in ROS detoxification (SOD, CAT, APX, and DHAR) was performed following Bonnin et al., 2023 (Bonnin et al. 2023). Three samples were collected for each genotype by pooling 20 leaves and/or 5 g of roots for each modality (control/stressed), freezing them in liquid nitrogen, grinding them to a fine powder, and storing them at -80 °C. 2.4. RNA sequencing and gene expression analysis Leaf and root samples from 2x and 4x Citrandarin, in control and stressed conditions (90-mM stress modality), were ground to a fine powder in liquid nitrogen under ‘RNAse-free’ conditions. RNA extraction and quality control were performed as described in Bonnin et al., 2024 (Bonnin et al. 2024b). RNAseq libraries were prepared to obtain labelled and matched sequences using UDI indexes, following the protocol and recommendations from the Illumina Truseq kit. The libraries’ integrity and quality were checked using screentape D5000 TapeStation and qPCR. A first sequencing of the libraries was then performed to validate their quality as described in Bonnin et al., 2024 (Bonnin et al. 2024a). Library pooling was then built up using the TruSeq Stranded mRNA Sample Preparation kit from Illumina in accordance with Bonnin et al., 2024 (Bonnin et al. 2024a). Sequencing was performed on a 150-nt pair-end S4 flow cell lane at MGX in Montpellier. The clustering and sequencing steps were performed on an Illumina NovaSeq 6000 technique with the NovaSeq Reagent Kits using the Illumina TruSeq RNA protocol (Illumina Inc., San Diego, CA, USA).Our experimental design was described as follows for each tissue (roots or shoots): 2x and 4x Citrandarin hybrid / 2 modalities (control/stressed) / 3 biological replicates (Table1). Cutadapt (v4.1; STAR (2.7.10a) (Dobin et al., 2013) with the parameters ‘–outFilterMultimapScoreRange 0 –outFilterMultimapNmax 10 –outFilterMismatchNmax 10’ to align the reads on the reference genomes, made of the concatenation of the two CR parental genomes ( P. trifoliata and C. mandarin ). Finally, the featureCount function of the Subread tool (release 2.0.1), using the options -M and –fraction to take multimapping reads into account, was used to obtain count matrices as described in Bonnin et al., 2024 (Bonnin et al. 2024a). Genes with no conditions with a mean replicate count above 10 were considered little expressed and were removed from further analysis. A list of genes responding to ploidy and stress as well as their interactions was generated using the lrt test from the DESeq2 (Love, Huber & Anders 2014) package as described in Figure S7 . 2.5. Statistical analysis Variance was measured on the physiological and biochemical variables and the selected genes to determine whether the values varied differentially based on the ploidy level under stress compared to the control and/or differentially between the 2x and 4x under stress and control conditions. Given the small number of samples (<30), the non-parametric Kruskal–Wallis test was applied (p ≤ α, with α = 0.05) as well as the size effect Eta2, which defined the magnitude of the difference between groups (0.2 = small, 0.5 = medium, 0.8 = large) [23]. Pairwise comparison was then performed using Dunn’s test to measure the differences between groups, with statistical significance set at p < 0.05, and classify them.Multivariate analyses were conducted using the mixOmics R package (release 3.17) [58,59]. PLS-DA was conducted to evaluate the quality and the structure of the physiological/biochemical datasets and the selected genes. Statistical relationships between the genes (Y), selected according to the DESeq2 multifactorial model, and the biochemical/physiological variables (X) were measured using PLS analysis. Then variables were selected using sparse PLS following the tuning protocol of Mixomics (http://mixomics.org/case-studies/spls-liver-toxicity-case-study/). The SPLS tuning process was used to select 100 genes in leaves, 200 genes in roots, and 10 biochemical/physiological variables for both tissues. Finally, a relevance network was formed to improve the selection of the co-variating variables. A correlation cutoff of 0.9 was applied to select the highest positive and negative correlation between genes and biochemical/physiological variables. 3. RESULTS 3.1. Analysis of biochemical and physiological responses of 2x and 4x Citrandarins, and selection of discriminant variables At the whole plant level, both 2x and 4x leaves shared similar phenotypes (shape and size) and showed a complete lack of symptoms under salt stress (Figure S1) . In-depth analysis was performed at the physiological and biochemical levels. Photosynthetic performance, antioxidant metabolism, and mineral uptake were measured, in leaves and roots of 2x and 4x Citrandarins, under control (C) and salt stress (S) conditions. For each tissue, the influence of the ploidy, the stress, and the ploidy x stress, on the variations of all the parameters, were measured ( Tables 1 and 2) . Table 1: Statistical analysis of variation in biochemical and photosynthetic variables measured in leaves. Significance differences between groups were calculated ( p -value<0.05) using the Kruskal-Wallis test. Mean values and p- values measured the effect of ploidy (2x, 4x), treatment (control C, salt stress S), and interaction ploidy x stress. Significant p -values between means are in bold. Mean values and p- values “ploidy x stress” were highlighted in grey for variables selected by sPLS-DA. 2x 4x p -value C S p -value 2x-C 2x-S 4x-C 4x-S p -value F V F M 0.78 0.71 0.00 0.74 0.74 0.90 0.768ab 0.788b 0.719a 0.699a 0.0014 YII 0.62 0.60 0.66 0.61 0.62 0.45 0.59a 0.66a 0.63a 0.58a 0.1615 ETR 28.88 27.97 0.94 27.46 29.38 0.10 28.02a 29.73a 26.90a 29.03a 0.3720 SPAD 69.05 72.62 0.01 71.67 70.00 0.59 69.80ab 68.30a 73.53b 71.70b 0.0165 g s 0.04 0.04 0.61 0.06 0.02 0.00 0.05ab 0.02a 0.07b 0.02a 0.0005 P net 4.96 3.92 0.39 7.06 1.83 0.00 7.95b 1.98a 6.17ab 1.67a 0.0009 E 0.80 0.71 0.37 0.84 0.67 0.19 0.96a 0.65a 0.73a 0.70a 0.3742 P net /Ci 0.01 0.02 0.70 0.03 0.01 0.00 0.021a 0.006a 0.031b 0.006a 0.0008 P net /g s 80.19 121.63 0.18 115.55 86.28 0.49 71.50a 88.89ab 159.60b 83.67a 0.0451 ETR/ P net 10.27 11.67 0.81 4.43 17.51 0.00 5.37ab 15.17b 3.48a 19.85b 0.0005 P net /E 6.55 5.96 1.00 9.45 3.06 0.00 9.99b 3.11ab 8.92c 3.01a 0.0120 TPC 14.20 6.08 1.00 5.07 15.22 0.04 4.03a 24.38b 6.11ab 6.06ab 0.0026 tAsa 40.79 38.03 0.39 40.95 37.87 0.32 42.96a 38.61a 38.93a 37.13a 0.5186 Asa 7.40 7.62 1.00 5.01 10.01 0.01 2.94a 11.86b 7.08ab 8.16ab 0.0007 DHA 33.38 30.38 0.59 35.93 27.83 0.04 40.02a 26.75a 31.85a 28.90a 0.1279 Asa/DHA 0.26 0.26 1.00 0.15 0.37 0.01 0.075a 0.45b 0.28ab 0.29ab 0.0004 H 2 O 2 19.09 22.12 0.09 22.02 19.19 0.24 21.77a 16.41a 22.27a 21.98a 0.0818 MDA 4.71 4.94 1.00 2.52 7.14 0.00 2.84ab 6.59bc 2.19a 7.69c 0.0091 Proline 25.31 21.30 0.48 18.16 28.46 0.00 21.20ab 29.43b 15.11a 27.49b 0.0022 SOD 19.52 17.99 0.13 17.63 19.88 0.01 18.25ab 20.78b 17.01a 18.98ab 0.0021 CAT 1.92 2.49 1.00 1.36 3.05 0.00 1.70ab 2.14ab 1.03a 3.96b 0.0001 DHAR 0.14 0.36 0.01 0.19 0.31 0.24 0.06a 0.22ab 0.32b 0.39b 0.0027 APX 0.43 0.70 0.24 0.32 0.81 0.01 0.13a 0.73b 0.51ab 0.90b 0.0033 SFP 2.51 2.95 0.32 2.85 2.61 0.48 2.84a 2.18a 2.86a 3.03a 0.4126 Ca 2+ 36.28 32.21 1.00 28.25 40.24 0.00 34.10ab 38.45bc 22.40a 42.04c 0.0002 K + 20.56 24.26 0.56 27.03 17.80 0.00 23.49ab 17.63a 30.56b 18.00a 0.0016 Cl - 47.30 48.48 0.94 40.88 54.90 0.13 39.86a 54.74a 41.89a 55.07a 0.4829 Na + 5.99 6.37 0.90 6.27 6.09 0.90 5.25a 6.74a 7.29a 5.44a 0.1070 Chloride (Cl − ), sodium (Na + ), calcium (Ca 2+ ), and potassium (K + ) contents were expressed in mg·g−1. SOD, DHAR, APX and CAT activities were expressed in μmol·min−1·mg−1.protein. H 2 O 2 was expressed in nmol.g -1 FW. tAsa was expressed in μmol.g −1 . FW. Asa and Proline were expressed in μmol.g −1 . TPC was expressed in mg.g -1 FW. MDA was expressed in nmol.g −1 FW. g s was expressed in mol H 2 O. m −2 .s −1 . Chlorophyll content (SPAD) was expressed in SPAD units. P net was expressed in μmol.CO 2 .m -2 .s -1 . ETR was expressed in μmol.e-.m -2 .s -1 . E was expressed in H 2 O.m -2 .s -1 . Table 2: Statistical analysis of variation in biochemical and photosynthetic variables measured in roots. Significance differences between groups were calculated ( p -value<0.05) using the Kruskal-Wallis test. Mean values and p- values are given for ploidy (2x, 4x), treatment (control C, salt stress S), and interaction ploidy x stress. Significant p -values between means are in bold. Mean values and p- values “ploidy x stress” were highlighted in grey for variables selected by sPLS-DA. 2x 4x p -value C S p -value 2x-C 2x-S 4x-C 4x-S p -value TPC 4.57 4.88 0.8185 4.11 5.34 0.1783 3.85a 5.29a 4.37a 5.39a 0.5683 tAsa 37.51 43.14 0.4911 39.67 40.98 1.0000 37.51a 37.51a 41.83a 44.45a 0.9184 Asa 7.97 3.87 0.0020 4.18 7.65 0.1783 4.99bc 10.99c 3.38a 4.36ab 0.0001 DHA 29.34 40.04 0.1854 35.28 34.10 0.7068 32.11a 26.56a 38.45a 41.63a 0.5298 Asa/DHA 0.34 0.10 0.0014 0.14 0.30 0.4893 0.18ab 0.49b 0.094a 0.10a 0.0033 H 2 O 2 9.51 12.80 0.0945 9.29 13.03 0.1243 6.18a 12.84ab 12.39ab 13.21b 0.0677 MDA 7.13 5.65 0.8195 3.19 9.58 0.0020 2.84a 11.41b 3.54a 7.75ab 0.0007 Proline 29.77 29.18 0.9358 21.00 37.95 0.0024 22.27ab 37.28b 19.74a 38.63b 0.0058 SOD 19.17 18.78 0.6919 18.28 19.67 0.0237 18.31a 20.03a 18.25a 19.32a 0.1513 CAT 2.82 1.42 0.0658 1.34 2.89 0.0177 2.17ab 3.47b 0.52a 2.31ab 0.0015 DHAR 0.19 0.12 0.2368 0.11 0.21 0.0028 0.11a 0.28b 0.10a 0.14ab < 0.0001 APX 0.44 0.28 0.3075 0.24 0.48 0.0017 0.25a 0.63b 0.24a 0.32ab 0.0007 Ca 2+ 8.55 9.01 1.0000 8.78 8.78 0.3674 10.90a 6.20a 6.66a 11.36a 0.0615 K + 20.64 21.79 0.7819 24.00 18.43 0.1631 27.07b 14.20a 20.93ab 22.65b 0.0056 Mg 2+ 3.63 3.65 0.9027 4.41 2.87 0.0052 4.57b 2.70a 4.25b 3.04ab 0.0229 Na + 6.23 5.88 0.7796 3.11 9.01 0.0152 4.80ab 7.66ab 1.41a 10.36b 0.0121 P 2.43 2.39 0.7855 2.95 1.87 0.0023 3.06b 1.79a 2.84b 1.95ab 0.0097 K + /Na + 4.26 8.72 0.3739 10.83 2.15 0.0026 6.58ab 1.94a 15.08b 2.36a 0.0016 Cl - 55.13 59.26 0.4900 44.64 69.74 0.2437 30.22a 80.04a 59.07a 59.44a 0.1852 Chloride (Cl − ), phosphorus (P), sodium (Na + ), Magnesium (Mg 2+ ), calcium (Ca 2+ ), and potassium (K + ) contents were expressed in mg·g−1. DHAR, SOD, APX and CAT activities were expressed in μmol.min −1 .mg −1 .protein. TPC was expressed in mg.g -1 FW. H 2 O 2 was expressed in nmol.g -1 FW. tAsa was expressed in μmol.g −1 FW. AsA and DHA were expressed in mmol.g-1. Proline was expressed in μmol.g −1 . MDA was expressed in nmol.g −1 FW. Only a few of the physiological and biochemical parameters measured were differently regulated between the 2x and the 4x, regardless of the stress in leaf and root. Conversely, many of them were significantly affected by stress, regardless of the ploidy level. However, all of these variables were significant for “ploidy x stress”, except SOD in roots. This means that values were all different between 2x and 4x, as well as between the control or stress conditions. In leaves, 19 variables significant for “ploidy x stress”, out of a total of 28, were involved in photosynthetic activity ( F V F M , SPAD, g s , P net /Ci ), antioxidant metabolism (TPC/Asa, Asa/dehydroascorbate [DHA], deshydroascorbate reductase [DHAR], ascorbate peroxidase [APX]), and mineral uptake (Ca 2+ , K + ). In roots, 13 out of 19 variables significant for “ploidy x stress” were involved in antioxidant metabolism (Asa, Asa/DHA, H 2 O 2 , MDA, proline, CAT, DHAR, APX) and mineral uptake (K + , Na + , K + /Na + , Mg 2+ , P). Surprisingly, leaves of both 2x and 4x did not differ in their Na + and Cl - concentrations whatever the treatment (Figure S2) . However, Na + leaf/root ratio decreased significantly in 4x compared to 2x (ratio values of 5,24b for 4xC 0,61a for 4xS, respectively), and Cl - leaf/root ratio decreased significantly only in 2x (ratio values of 1,43b for 2xC and 0,72a for 2xS, respectively). Both ratios showed higher values in the control condition (Figure S4) .The next step was to identify the physiological and biochemical variables that distinguished the specific responses of both ploidy levels under salt stress. First, partial least square discriminant analysis (PLS-DA) was conducted to highlight the overall structure of the samples according to all biochemical and physiological variables. Control and salt-treated 2x and 4x were discriminated on each side of the first component (PC1) in both leaf (Figure 1a) and root (Figure 1b) samples, representing 44% and 51% of the total variability, respectively. Figure 1. PLS-DA analysis of biochemical and physiological variables in 2x and 4x Citrandarin genotypes under control (C) and stress conditions (S), in leaves (a) and roots (b). Measurements were performed at W4 (90 mM NaCl) as described in Bonnin et al., 2024 (Bonnin et al. 2024a). Coloured dots represent biological replicates for each ploidy level and treatment. Clustered dots were defined using ellipses, such as at the 95% confidence level.The ploidy level was discriminated on the second component (PC2), representing 15% and 14% variability in leaves and roots, respectively. In the root samples, 2x and 4x controls were not differentiated from each other on the two first components. The discrimination of the two ploidy levels was possible by plotting the variance explained by components 2 and 3 (Data Sheet 1, Figure S4) . Thus, the PLS-DA demonstrated that, in each tissue, the physiological and biochemical responses of the Citrandarins were primarily affected by the applied treatments and that this response then varied according to the ploidy level.The most significant variables that explained the differences in the response between 2x and 4x were then identified using sparse PLS-DA. These variables are shown in bold characters in Figure 1 , and their means and p -values are highlighted in grey in Tables 1 and 2 . In leaves, 2x and 4x were differentiated according to their stomatal conductance ( g s ) (Figure 2a) and P net /Ci (Figure 2b) . Figure 2. Leaf photosynthetic performances, antioxidant system evaluation, and osmoprotection mechanism under control (C) and salt stress (S) conditions in leaves (L) and roots (R). Measurements of stomatal conductance ( g s ) (a), carboxylation efficiency ( P ne t/Ci ) (b), MDA content (c), DHAR (d), APX (e), and proline content (f) specific activity were performed, after four weeks of salt treatment (90 mM). Data represent three independent measurements (n=3). Significance of the values was calculated using the Kruskal–Wallis test ( p- values<0.05). Mean comparison and letter classification were calculated using Dunn test. Groups sharing the same letter are not significantly different.However, both g s and P net /Ci decreased in the 2x and 4x stressed plants compared to their control, but these variations were significant only in 4x. Moreover, both stressed 2x and 4x presented decreasing SPAD values compared to the control, but 4x had higher values than 2x, both in the control and stressed conditions (Table 1) . Variables related to antioxidant metabolism under stress were highly discriminant for 2x and 4x. Diploid and 4x stressed genotypes showed higher APX, TPC, Asa, and Asa/DHA values compared to the control, but the changes were significant only in 2x. DHAR activity increased in 2x under stress compared to the control, although it was not significant, whereas the DHAR activity was higher in the control condition in 4x compared to the 2x, and it remained stable under stress.In roots, 2x and 4x samples were differentiated mainly by 14 variables (Figure 1, Table 2) . Overall, the stressed 2x presented increased values for Asa, Asa/DHA, H 2 O 2 , MDA (Figure 2c) , DHAR (Figure 2d) , and APX (Figure 2e) compared to the control. On the contrary, the stressed 4x showed increased values for tAsa, DHA, CAT, and proline (Figure 2f) compared to the control. For three of them (tAsa, DHA, H 2 O 2 ), no significant change between groups was detected using the Kruskal–Wallis test (Table 2). However, a contrasted trend in the values of 2x and 4x was clearly observed. Taking a closer look at the data revealed that the non-significancy of this variation was due to a higher intra-variability of the stressed samples compared to the controls (Data Sheet 2) . Variation in parameters related to mineral uptake in roots was also discriminated in the 2x and 4x. Although roots of both 2x and 4x showed increased Na + contents under salt stress, compared to the control, it was significant only in 4x ( Figure 3a) . Stressed 2x had decreasing K + uptake, compared to the control, but not 4x (Figure 3b) . Consistent with this, 4x had a decreasing K + /Na + ratio (Figure 3c) . The change in Cl - content was not significant. However, it is worth noting that this ion increased significantly in 2x roots and remained stable in 4x roots (Figure 3d) . Figure 3. Mineral uptake in roots. Na + (a), K + (b), K + /Na + ratio (c) and (d) Cl - content, under control (C) and salt stress conditions (S). Measurements were performed after four weeks of salt treatment (90 mM). Data represent three independent measurements (n=3). Significance of the values was analysed using the Kruskal–Wallis test ( p- values<0.05). Mean comparison and letter classification were calculated using the Dunn test. Groups sharing the same letter are not significantly different. No letters were assigned when the p -value was not significant. 3.2. Analysis of the leaf and root transcriptomic datasets 3.2.1. Gene expression in leaves and roots of 2x and 4x Citrandarin under control and salt stress treatment A total of 1.037.569.398 pairs of paired end reads of 150 nucleotides were generated for the 24 samples, with a mean of 43.232.058 read pairs per sample. The rate of trimmed base was 8.3%, and after low-length reads removal, 1.026.827792 (99%) of the initial read pairs remained available for mapping. 96% of these reads were successfully mapped on the reference genomes using STAR, with a multimapping rate of 16.8%. A total of 88.6% of the mapped reads were assigned to known features and were used for counting. The transcripts were annotated on the two reference genomes, and the corresponding genes identified as Ptrif if present in the Poncirus genome, as well as CITRE if present in the C. reshni genome. Some genes were present in the two genomes and were thus identified with both Ptrif and CITRE identifier. For each gene, an Arabidopsis orthologue was identified using Best Blast Mutual Hits (BBMH). More details can be found in supplementary Data Sheet 2 and supplementary Figures S5 and S6 .From the total number of genes quantified by RNAseq (about 55.014 genes), 39.850 and 37.027 genes were identified as expressed in leaves and roots, respectively, after low count genes removal. From them, 9.930 and 6.243 genes were extracted, in leaf and root, respectively, using the multifactorial selection of DESeq2 (Figure S7) . They represent all the genes that were differentially regulated between the two ploidy levels, the stress versus the control treatment, and/or the interaction of both factors. Multifactorial PLS-DA analysis of these genes ( Figure 4 ) showed that the structures of leaf and root samples were highly similar to one other and to the structures observed using the physiological and biochemical variables ( Figure 1 ). For both tissues, the salt treatment was the first factor explaining the variability of the gene expression, regardless of the ploidy level. The first principal component (PC1) explained 72% and 50% of the variability in leaves (Figure 4a) and roots (Figure 4b), respectively. Diploid and 4x genotypes were discriminated on the second principal component (PC2), representing 7% and 21% of the variability in leaf (Figure 4a) and root tissues (Figure 4b), respectively. However, 2x and 4x samples were more differentiated in roots than in leaves, whatever the treatment. Taken together, our results show that in both leaf and root tissues, the difference in gene expression was first explained by the salt treatment and then by the ploidy level. Figure 4. PLS-DA analysis of transcriptomic data for 2x and 4x Citrandarins from leaves (a) and roots (b) under control (C) and salt stress (S) conditions. Samples were projected into the space spanned by the first component (PC1) and the second component (PC2). Samples are coloured according to the ploidy level and the treatment. Confidence ellipses were set to 95%.The genes regulated in leaves and roots were functionally investigated. First, three independent lists of genes were extracted from each tissue: “ploidy”-specific list of genes differentially regulated between the two ploidy levels, “stress”-specific list of genes differentially regulated under stress compared to the control treatment, and “ploidy x stress”-specific list of genes differentially regulated between the two ploidy levels and between the stress and control treatments. For each list, the number of genes up- and down-regulated in leaves and roots were significantly different ( Table 3; Data Sheet 2 ). Table 3: Number of up and down regulated genes in leaves and roots Total 9930 6243 “Ploidy “ genes 0 1363 “Stress “ genes 9886 4146 “Ploidy x stress” genes 44 701 In leaves, 99% of the genes were regulated in response to salt stress regardless of the ploidy level. No one gene was found to be differentially regulated only by ploidy, and only 44 genes were differentially regulated in 2x and 4x and in response to stress. In roots, 67% of the genes were regulated in response to the stress, 22% were differentially regulated in 2x and 4x, and 11% presented patterns of expression that changed between 2x and 4x under control or stress. Gene Ontology (GO) enrichment analysis of the genes from each list was then performed to identify which biological processes were significantly over-represented. Enrichment maps were then generated to visualize the top-level biological processes regulated by these genes. 3.2.2. Functional analysis of the genes regulated according to ploidy As described above, genes differentially regulated according to the ploidy level, regardless of the stress versus the control treatment, were identified only in roots (Table 3) . These genes were mainly involved in aromatic compound metabolism, including aldehyde, acetate, and nitrate metabolism, as well as pyridine nucleotide salvage processes (Figure 5; Data Sheet 2) . The aromatic compounds metabolism process included a high number of genes acting as transcription factors, such as AP2/ERF domain-containing proteins (13), NAC domain-containing proteins (16), and WRKY domain-containing proteins (11). Other transcription factors were identified such as MYBs, BHLHs, scarecrow-like proteins, MADS-box, and BZIP. Hormonal regulation was detected as well, which was modulated through the involvement of auxin responsive proteins, as well as ROS response with peroxidases and glutathione peroxidases. In comparison with the number of these regulatory genes, few of them participated in secondary metabolite biosynthesis such as the phenylalanine ammonia-lyase 2, anthranilate synthase, and caffeic acid 3-O-methyltransferase. Dirigent proteins, which play important roles in plant secondary biosynthesis, were also identified. Figure 5. Map enrichment of the “ploidy” genes list in roots. GO nodes are connected by edges if the genes belonging to these nodes shared GO similarity. Nodes belonging to very similar gene functions are clustered and labelled with a summarized name. Edges’ thickness between nodes is proportional to the degree of similarity between them. Node size is proportional to the number of expressed genes. Coloration increases from pink to red with the significancy ( p -value) of the GO enrichment. 3.2.3. Functional analysis of the genes regulated under salt stress. As shown in Table 3, salt stress strongly affected the gene regulation in both tissues of Citrandarins. However, higher transcriptomic response was detected in leaves compared to roots (Table 3) . In leaves, genes regulated under salt stress were mainly enriched in processes related to primary metabolism (glycolysis), regulation of cell morphogenesis and cellular organization, ceramide catabolism, and photosynthesis (Figure 6.a; Data Sheet 2). More specifically, GO terms related to primary metabolism were linked to metabolism of amino acids (arginine, aspartate, glutamine, glycine, serine, methionine, histidine, lysine), carbohydrate and lipids (glycerolipids, fatty acids). Ceramide metabolism comprised sphingolipids regulation known to be involved in mineral ion homeostasis. Salt stress affected genes involved in cell organization in both 2x and 4x, including morphogenesis, circadian rhythm, cell morphogenesis, and cell shape. In these processes, various genes were found related to transcriptional, translational, and post-translational responses, as well as hormone regulation (ethylene and gibberellic acid mediated signalling pathway). Circadian rhythm involved several transcription factors such as LNK 1 and 3 (Ptrif.0008s1195, Ptrif.0008s2612 / CITRE_008G013120; Ptrif.0005S0739) and putative protein Reveille 1 and 7 (Ptrif.0006s1617, CITRE_003G020240). Cell organization involved chloride transport with regulation of several channel proteins. Five of them have Arabidopsis orthologues (Ptrif.0001s1927 / CITRE_001G021300 = AT1G55620; CITRE_002G011250 / Ptrif.0002s1085 = AT3G27170; CITRE_009G005200 / Ptrif.0009s0506 = AT5G26240; CITRE_005G001590 / Ptrif.0005s0200 = AT5G49890; CITRE_009G020650 = AT5G62290). Regulation of photosynthesis and chlorophyll biosynthesis were strongly affected by stress in Citrandarin. Among the genes involved in these processes, 25 chlorophyll a-b binding protein 2C, 19 genes related to photosystem I (PSI) units centre, and nine related to photosystem II (PSII) apparatus were identified.In Citrandarin roots, genes regulated under stress were mainly involved in the regulation of the circadian rhythm, as well as post-translational regulation, transmembrane oligo peptide transport, response to blue light, and phosphatidylinositol metabolism ( Figure 6b; Data Sheet 2) . Circadian rhythm involved some of the same genes identified in leaves, coding for transcription factors LNK 1 and 2, REVEILLE 7 and 8. It was highly regulated by transcription factors such as MYB42 (Ptrif.0003s0874 = AT4G12350), LATE ELONGATED HYPOCOTYL (LHY) (Ptrif.0003s3925/CITRE_003G043020 = AT1G01060), WD repeat-containing protein LWD1 (CITRE_002G009570 = AT1G12910), two-component response regulator-like APRR1 (CITRE_003G010580/Ptrif.0003s1054 = AT5G61380), PRR37 (Ptrif.0004s2702) and PRR95 (CITRE_003G043130), ELF4-LIKE 4 (CITRE_002G022970), and putative cold-regulated protein 27 (Ptrif.0008s0480 / CITRE_008G004840). Citrandarin’s roots under stress showed high post-translational regulation, involving many protein kinases and phosphates, as well as transmembrane oligopeptide transport, comprising Nitrate/Nitrite transporters (NRT/PTR Family). Response to blue light was regulated by some genes dependent of circadian rhythm such as Addagio (CITRE_002G020140 = AT1G68050), which is a E3 -ubiquitin ligase complex, and the putative transcription factor COR 27 (Ptrif.0008s0480 / CITRE_008G004840), involved in central circadian clock regulation. All the genes identified in the phosphatidylinositol metabolism were regulatory membrane lipids involved in plant development and cellular function. They belong to the phosphatidylinositol phosphate kinase family, including FAB1, FAB1C, FAB1D, PIP5K8, and PIP5K9.(Figure 6a)(Figure 6b) Figure 6. GO enrichment map of the “stress” genes list in leaves (a) and roots (b) of 2x and 4x Citrandarins. GO nodes are connected by edges if the genes belonging to these nodes shared GO similarity. Node size is proportional to the number of expressed genes. Coloration increases from pink to red with the significancy ( p -value) of the GO enrichment. Nodes belonging to very similar gene functions are clustered and labelled with a summarized name. Edges’ thickness between nodes is proportional to the degree of similarity between them. 3.2.4. Functional analysis of the genes differentially regulated between the 2x and 4x Citrandarins under stress More genes were differentially regulated between stressed 2x and 4x, in roots (702) than in leaves (44), when compared to their controls (Table 3) . In 2x and 4x roots, genes were significantly enriched in amino acid derivative biosynthesis and carbohydrate homeostasis, response to ROS, ion transport and defence response (Figure 7; Data Sheet 2) . The amino acid derivate comprises genes involved in secondary metabolism, with nicotianamine biosynthesis being the only enriched process. We also found genes involved in phenylpropanoid and lignan biosynthesis such as phenylalanine ammonia lyase 2, flavonol 3-O-glucosyltransferase, caffeic acid 3-O-methyltransferase, dirigent proteins, chitinases, and laccases. The amino acid derivative process was regulated by a high number of transcription factors belonging to various families: K-box, BHLH, WRKY, MADS MF2-like, WRKY, NAS, AP2/ERF, and so on. Response to ROS mainly involved genes coding for peroxidases and glutathione peroxidases. Ion transport was identified as well with genes coding for a vacuolar cation/proton exchanger (CITRE_001G021040 = AT1G55730), and K + and Mg 2+ transporter (Ptrif.0002s0617 = AT5G14880 and CITRE_008G002010 = AT2G04305, respectively). We also found two cation efflux proteins (Ptrif.0009s2337, Ptrif.0002s0137), a cation/H+ exchanger domain-containing protein (CITRE_006G006700), and two glutamate receptors (CITRE_001G000910 = AT2G29120, Ptrif.0001s0092). Finally, a chloride channel protein was identified (Ptrif.0001s2162 = AT5G33280).In leaves, no significant GO enrichment was detected, due to the low number of genes (Figure S8; Data Sheet 2) . Among them, some genes were related to photosynthetic processes such as the PSI reaction centre subunit (Ptrif.0007s2135), early light-induced proton (Ptrif.0009s0630 = AT3G22840), RuBisCO large subunit-binding protein subunit alpha (Ptrif.0008s1946 = AT2G28000), and protein PAM68 (CITRE_020g000090 = AT4G19100). Others participated in the carbohydrate metabolism process, including a bidirectional sugar transporter SWEET (Ptrif.0004s2461 = AT5G13170), pectinerase (Ptrif.0007s0712 = AT1G11580), and a phosphoglycerate kinase 2C chloroplastic (CITRE_009g024770 = AT1G56190). Figure 7. GO enrichment map for the “ploidy x stress” genes list in roots. GO nodes are connected by edges if the genes belonging to these nodes shared GO similarity. Node size is proportional to the number of expressed genes. Coloration increases from pink to red with the significancy ( p -value) of the GO enrichment. Nodes belonging to very similar gene functions are clustered and labelled with a summarized name. Edges’ thickness between nodes is proportional to the degree of similarity between them.To deepen the analysis of the “ploidy x stress” genes list, co-regulated genes that were up- or down-expressed in 2x and 4x Citrandarins were identified, as well as the biological processes in which they are involved. First, clustering analysis was performed to generate clusters containing the co-regulated genes in both leaf and root datasets. Second, groups of clusters having the same gene expression profile, up- or down-expression for 2x and 4x were formed, and GO enrichment analysis was performed on each of them. The enriched biological processes identified in each group are summarized in Table 4 .In leaves, five clusters of genes “ploidy x stress” were identified in total that were classified into four groups (Figure S9; Data Sheet 2) . Only genes from group 1 were up-regulated in 2x and down-regulated in 4x. In contrast to the low number of genes in this group (44), a significant enrichment in photosynthesis, as well as the biosynthesis of aromatic compounds with genes involved in methylation, was identified. In group 2, no GO enrichment could be detected because of the number of genes in this list: a trimethyltridecatetraene synthase involved in terpenoid synthesis and two stress-induced proteins (translocator protein, dehydrin). The three of them were significantly up-regulated in the stressed 4x. The 14 genes found in group 3 were not enriched in biological processes either. They were down-regulated in 2x and up-regulated in 4x. Among these genes, the following are worth mentioning: a stress-responsive gene AP2/ERF domain containing protein (Ptrif.0001s0412 = AT5G11590); and a sugar transporter SWEET15 (Ptrif.0004s2461 = AT5G13170) that regulates cell viability under high salinity. Group 4 was constituted of five genes, down-regulated in 2x, and up-regulated in 4x, and it was not GO enriched. Three of them participated in photosynthetic-related processes: a plastid redox-insensitive protein (CITRE_004g023680 = AT1G10522), a chaperonin protein binding to Rubisco (Ptrif.0008s1946 = AT2G28000), and a carboxyl-terminal-processing peptidase involved in PSII (Ptrif.0009s1689 = AT3G57680).In roots, 16 clusters ploidy x stress were identified (Figure S10; Data Sheet 2) and to classified in four groups according to their expression profile. Three of them had a significant GO enrichment (Table 4; Data Sheet 2). Genes from group 1 were enriched in processes related to the regulation of gene expression by RNA gene silencing, signalling, and zinc ion transport. Gene silencing comprised three genes participating in the production of small interfering RNAs: the putative nuclear RNA export factor SDE5 (Ptrif.0001s1299 = AT3G15390), the dicer-like protein 4 (Ptrif.0001s2576 = AT5G20320), and the probable RNA-dependent RNA polymerase 3 (Ptrif.0001s0822 = AT2G19910). They were over-expressed in the stressed 2x and down-expressed in the stressed 4x, as compared to their controls. Group 2 was enriched only in nicotianamine biosynthesis, comprising 74 genes that were down-expressed in 4x only. Group 3 was enriched in processes mainly related to cell wall metabolism, as well as biotic and abiotic stress response including ROS. It comprised 324 genes that were down-expressed in 2x and over-expressed in 4x. In group 4, 92 genes were over-expressed only in 4x and did not present any biological process enrichment. However, GO analysis of these genes showed that they regulated primary metabolism, amino acid, and carbohydrate, as well as the transport of ion and carbohydrate. 3.3. Identification of marker genes co-variating with biochemical/physiological variables In each leaf and root dataset, genes that could potentially contribute to the observed variation in biochemical and physiological responses in 2x and 4x were identified. Covariances between genes (X) and explanatory variables (Y)—that is to say, the biochemical and physiological variables—were measured using partial least square (PLS) analysis. Then, variables X and Y that were the most highly correlated (R>0.9) were selected by sparse PLS, and R values were used to generate an X-Y network.In leaves, 68 genes were found to be highly correlated to three variables related to photosynthesis ( P net , P net /Ci, g s ), two variables related to antioxidant metabolism (Asa, TPC), and two variables related to mineral uptake (K + , Ca 2+ ) (Figure 8; Data Sheet 2) . Interestingly, four of them ( P net /Ci , g s , Asa, TPC) were shown to significantly discriminate the 2x and 4x, control and stressed, by sPLS-DA. P net , P net /Ci, g s , and K + decreased in both the stressed 2x and 4x, compared to their respective controls. As shown above, they all decreased significantly in 4x under stress, compared to the control values, except for P net that decreased significantly in 2x. Conversely, Asa and TPC increased significantly only in the stressed 2x. Most of the genes were only regulated in 4x, comprising 54 genes ( Figure 8a ) when compared with the 16 genes in 2x ( Figure 8b ). All these genes were mostly down-regulated under stress, compared to the control, for both 2x and 4x. Among these genes, 20 have unidentified functions or are classified as hypothetical proteins. Genes of the whole network were significantly enriched in processes related to photosynthesis, response to light, and glycolysis ( Figure 8c ). Among these genes, significant protein–protein interactions were identified using their Arabidopsis orthologues (String database; ppi enrichment value = 3.4e -15 ). The corresponding network involved five genes belonging to the light-harvesting chlorophyll a/b binding (LHCB) gene family: LHCB2.4 (CITRE_002G016570 = AT3G27690), LHCB4.2 (Ptrif.0006s0854=AT3G08940), LHCB6 (CITRE_008G011420 = AT1G15820), LHCA1 (Ptrif.0004s0544 = AT3G54890), and LHCA4 (CITRE_003G010320 = AT3G47470). Two other genes were involved in glycolysis, a triose phosphate isomerase (Ptrif.0004s0004), and a glucose-6-phosphate isomerase (CITRE_005G020270 = AT4G24620). Among the other interesting genes identified in this list, a MYB4 transcription factor (CITRE_008g001720 = AT4G38620) was involved in the regulation of protection against UV, a protochlorophyllide reductase A (PORA) (Ptrif.0001s1312 = AT5G54190) acts as a photoprotectant during the transitory stage from dark to light, and a PSI subunit O (PSAO) (Ptrif.0008s1629 = AT1G08380) participates in the balancing of excitation energy between PSI and PSII ( Data Sheet 2 ).(Figure 8A_2x)(Figure 8A_4x)(Figure 8A_enrichment)(Figure 8B_2x)(Figure 8B_4x) (Figure 8B_enrichment) Figure 8. Networks of highly correlated biochemical/physiological variables and genes (R>0.9) in leaves (A) and roots (B). A unique network by tissue is presented here in two versions according to the respective gene regulation in 2x and 4x. Biochemical/physiological variables are represented in yellow. Genes differentially regulated under stress versus the control treatment, in 2x and/or 4x, are coloured in red (over-expressed) or green (down-expressed). Red and green edges represent positive and negative correlations, respectively, between each gene and physiological/biochemical variables. When genes were not differentially regulated in 2x or 4x, they are represented in a white circle with a grey edge.For each tissue, an enrichment map of all the genes of the 2x/4x network is presented. GO nodes are connected by edges if the genes belonging to these nodes shared GO similarity. Node size is proportional to the number of expressed genes. Coloration increases from pink to red with the significancy ( p -value) of the GO enrichment. Nodes belonging to very similar gene functions are clustered and labelled with a summarized name. Edges’ thickness between nodes is proportional to the degree of similarity between them.In roots, four biochemical/physiological variables related to antioxidant metabolism (APX, Asa, DHAR, MDA) were highly correlated to a total of 63 unique genes (Figure 8c). These four variables were identified as the most significant to differentiate 2x and 4x using sPLS-DA (Table 1). They increased in both stressed 2x and 4x, compared to the control, but were significantly changed only in 2x (Table 1) . These variables formed two networks with highly correlated genes—one related to MDA and the other related to ASA—APX and DHAR. On the 63 genes, 62 were regulated in 2x (Figure 8a) versus six in 4x ( Figure 9b ). Most of the genes were up-regulated (44 in 2x, 5 in 4x). Among all the co-expressed genes found in roots, 13 were unidentified proteins, against one in leaves. Glutamine transport was the unique GO process significantly enriched (Figure 8b), comprising only two hypothetical proteins (CITRE_001G000970, Ptrif.0001s0099). A more detailed examination of the genes within the entire network indicated they were associated with the regulation of the primary metabolism and transport (Data Sheet 1) . Some were specifically related to the phytohormone response pathway such as the NEDD8-activating enzyme E1 regulatory subunit (CITRE_002g015040 = AT1G05180), orthologue of Auxin Resistant 1 (AXR1), the WAT1 related-protein, and a vacuolar auxin transport (CITRE_004g023510/Ptrif004s2221 = AT1G70260). Two genes were involved in lignin biosynthesis, a serine/threonine-protein kinase PBL15 (Ptrif.0005s1066 = AT1G61590), and a cinnamyl alcohol dehydrogenase 6 (CAD6) (CITRE_007g006860 = AT4G37970). Interestingly, we evidenced a correlation between the MDA variable and a gene related to two histone H4 (CITRE_008g022040 = AT5G59970, Ptrif.0008s2155) (Data Sheet 2) . Moreover, several genes linked to MDA were associated with the ubiquitine pathway and proteasome degradation (CITRE_006g017110, Ptrif.005s2887, Ptrif.0003s0443). 4. DISCUSSION To evaluate the inheritance of salt tolerance in an interspecific context, the first investigation in 1941 was performed in a hybrid raised from the cross of Lycopersicon esculentum and Lycopersicon pimpinellifolium (Lyon 1941). In this hybrid, heterosis limited the decrease in stem elongation under saline conditions. It was later discovered that stem elongation was a dominant trait in hybrids with Solanum pennellii but not with Lycopercicon cheesmanii as the parent (Tal & Shannon 1983; Saranga, Zamir, Marani & Rudich 1991). This early study paved the way for the discovery of genetically complex traits (Shannon 1985). It revealed that heterosis dominance and additive effects were shared by other species, including citrus. In this later species, the evaluation of tolerance was challenging due to a large intergeneric and inter-individual variability (Shannon 1985).The present study attempts to address the complex challenge of elucidating the biological mechanisms regulated by a Citrandarin citrus hybrid in both diploid (2x) and tetraploid (4x) formats, with a particular focus on the distinct responses exhibited by each to salt stress. The 2x and 4x hybrid responses were investigated in leaf and root compartments by analysing physiological, biochemical, and transcriptomic results.To study the transcriptome, a specific strategy was applied for the annotation of the RNA-Seq data generated from each of the 2x and 4x hybrids. Then, various multivariate analyses were conducted to dissect the molecular mechanisms regulated by each of the 2x and 4x under stress. This approach enabled the identification of contrasting gene regulations between the two ploidy levels (“ploidy” genes), between the stress versus the control treatment (“stress” genes), and between 2x and 4x under stress compared to the control (“ploidy x stress” genes). The “ploidy x stress” genes were then further explored to highlight specific up- or down-regulated biological processes. Finally, marker genes were identified that were statistically associated with physiological and biochemical parameters that were investigated in the study. They all varied differentially between 2x and 4x under stress. An integrative analysis of these results is presented below. 4.1. Sequencing data treatment process for hybrids, constraints, and other possible strategies Processing RNAseq data from a hybrid genome having different ploidy levels is very challenging. There are several approaches to handle genome sequences from hybrids. A single reference genome coupled with diagnostic single nucleotide polymorphism (SNP) information or a pseudo genome could have been used (Huang, Holt, Kao, McMillan & Wang 2014). Despite the availability of information regarding these particular single-nucleotide polymorphisms (SNPs), this approach might not have been successful. Indeed, significant portions of the genome were not considered, including genes from the dispensable genome that may be of great importance.To address this issue, one solution would have been to create and use a pangenome graph using both reference sequences. This can be handled using tools such as Minigraph-Cactus (Hickey et al. 2024) for graph creation and vg mpmap (Garrison et al. 2018) for alignment. Recent studies in tomato (Y et al. 2022) and citrus (Huang et al. 2023; Droc et al. 2024) have shown that pangenomes containing many varieties can be generated. However, the construction of such a pangenome for the Cleopatra mandarin and the Trifoliate genome was beyond the scope of this study due to the complexity of the process. However, generating a de novo assembly would have had a negative impact on the generated GO enrichment analysis because any genes that do not appear in the assembled transcriptome (non-transcribed, low expression, assembly problems) would not have been included in the gene background when performing GO enrichment analysis. In the present study, we chose a third approach consisting of the concatenation of the two parental genomes to process the Citrandarins RNAseq data. This strategy was used with success on a V vinifera L . × V. rotundifolia hybrid (Shi et al. 2024). However, this approach may lead to some biases. In order to limit them, it is important to consider different parameters throughout the whole process: genes having close to identical sequences on both genomes may be impossible to distinguish, resulting in many multi-mapping reads. We handled this issue by accounting for multi-mapping reads in the parameters of both the mapping and counting tools. If we had removed multi-mapping reads, we may have biased the counts for some genes with close or identical sequences, leading to expressed genes appearing with low or null expression (false negatives). Also, the multi-mapping rate was not a major issue in our hands because it was less than 20%. Moreover, the tests that we performed were made on a per gene basis, and we assumed that if a count bias was introduced in one condition for a gene, the bias was similar in any other conditions. Overall, this very straightforward approach enabled us to identify highly significant and relevant biological processes and marker genes.The GO annotations were organized hierarchically and sometimes quite broadly. This means that terms relating to “sugar metabolism” often encompass several underlying processes, including the sucrose metabolism. So, even though sucrose is a major component in leaves, I may explain why genes involved in its metabolism may be annotated in global categories such as “carbohydrate metabolic process” or “primary metabolic process”, without a specific term “sucrose metabolic process” being used. Genes whose function related to sucrose are frequently automatically annotated in the context of carbohydrate metabolism, especially if specific sucrose information has not been integrated into the database. In summary, although sucrose is very important in citrus leaves, the genes regulating its metabolism can be annotated under general sugar metabolism terms. It is therefore possible to find “GO terms” relating to “sugar metabolism” without explicit mention of sucrose. 4.2. Regardless of the salt stress, the two ploidy levels differed by their photosynthetic performance and root antioxidant metabolism Several studies carried out on citrus have identified some morphological differences that could be related to the ploidy level. Among them, stunted growth is worth mentioning. Despite a growth delay, 4x plants are characterized by an increase in cell volume and more massive organs (Allario et al. 2011; Ruiz, Oustric, Santini & Morillon 2020). In our study, no growth difference could be observed between the 2x and 4x Citrandarins in one-year-old plants, at the end of the experiment. However, after one year, one remaining 4x replicate showed a lower growth than the 2x (unpublished results). Polyploid leaves are usually thicker and greener due to higher chlorophyll content [8]. In our experiment, the shape, width, and colour of the control leaves were not visually different between 2x and 4x Citrandarins. The shape of the leaves of both ploidy levels was very similar to the Trifoliate orange parent (Bonnin et al. 2023). Tetraploid citrus plants are known to have larger stomata, with lower density than their diploid counterparts (Ruiz et al. 2020). According to Allario (Allario et al. 2011), 4x citrus plants were characterized by a limited transpiration compared with 2x, which could lead to a more limited water consumption (Allario et al. 2011). Morillon and Chrispeels (Morillon & Chrispeels 2001) demonstrated that when stomata are closed—for instance, under salt stress conditions—the ratio of trans-cellular water flow/apoplastic flow is considered more preponderant than when stomata are open and the plant water flux is high (Morillon & Chrispeels 2001). Assuming that hydraulic conductivity of the plasma membrane is unchanged between 2x and 4x, then a greater cell size would favour cell-to-cell water exchange, as the main limitation would be the number of membranes to cross (Morillon, Liénard, Chrispeels & Lassalles 2001) . These differences probably increased the resistance to CO 2 diffusion to the site of carboxylation in the chloroplasts (Romero-aranda, Bondada, Syvertsen & Grosser 1997). This constitutive difference might give an advantage to 4x compared to 2x when both are subjected to salt stress. We will discuss this in more detail in 3.4 and 3.5.Although the relationship between phenotypic variation regarding the aerial part and stress tolerance in polyploid citrus has been widely investigated (Moya, Gómez-Cadenas, Primo-Millo & Talon 2003), the role of the root system is still poorly documented. In our study, we contrasted the root morphology between 2x and 4x Citrandarins. Tetraploid roots were shorter, thicker, and less branched; thus, they probably have a lower hydraulic conductance than their diploid counterpart (Allario et al. 2011). Similar results were observed in the parental genotypes (Bonnin et al. 2024b) as well as in other genotypes (Allario). These characteristics are known to improve salt stress tolerance in citrus {Citation}. These root specificities could be used further in a new rootstock selection programme.At the physiological and biochemical levels, few of the measured parameters varied between the two ploidy levels of the Citrandarin. The 2x leaves were characterized by a higher chlorophyll fluorescence parameter F v /F m , as well as a higher leaf greenness (measured by SPAD values) compared to 4x, unlike what was observed visually. Similar results have been obtained on 2x and 4x Rangpur lime ( Citrus limonia Osbeck ) seedlings (Allario et al. 2011). Interestingly, Rangpur lime is known to be one of the most tolerant citrus rootstocks, just after Cleopatra mandarin (Gómez-Cadenas et al. 2003; Al-Yassin 2004). In our experiment, roots of 4x Citrandarin had two times lower Asa and Asa/DHA when compared to 2x. The oxidation of glutathione and ascorbate leads to the elimination of H 2 O 2 . In the first reaction catalysed by APX, ascorbate acts as a reducing agent and oxidizes to monodehydroascorbate (MDHA). The DHA spontaneously produced by MDHA can be reduced to ascorbate by deshydroascorbate reductase (DHAR) using glutathione (GSH). During this process, glutathione is oxidized (GSSG). The cycle is completed when GR converts GSSG to GSH with the help of NAD(P)H (Caverzan et al. 2012). Higher values of Asa and Asa/DHA in 2x suggest that this genotype, contrary to 4x, has triggered detoxication strategy that could rely on Ascorbate scavenging activity (Pang & Wang 2010).At the gene expression gene level, no differential regulation was detected between the two ploidy levels in leaves. Similarly, only a few differentially expressed genes (0.8%) were identified in a transcriptomic study performed in 2x and 4x Ziyang xiangcheng ( Citrus junos Sieb. ex Tanaka ) leaves (Tan et al. 2015), and no variation was detected in Rangpur (Allario et al. 2011). Other types of regulation are likely to be involved such as post-transcriptional or epigenetic regulations. These regulatory processes have been observed in polyploid citrus species when compared to their diploid counterpart (Allario et al. 2011; Tan et al. 2015). In contrast to leaves, a significant number of genes (22%) were differentially regulated in roots between 2x and 4x Citrandarin. These genes participated in aromatic compound metabolism and comprised a high number of transcription factors. ROS response was also regulated involving peroxidase and glutathione peroxidases. In several studies, notably in citrus (Allario et al. 2011; Jude W. Grosser, Gary A. Barthe, Bill Castle, Frederick G. Jr. Gmitter, & Orie Lee 2015), it has been shown that polyploidy leads to better adaptation to various abiotic stresses, including salt stress (Oustric et al. 2019; Calvez et al. 2023). This adaptation relies on improved antioxidant capacities in 4x compared to its 2x counterpart, as it was already shown in the parents: Cleopatra mandarin and Trifoliate orange (Bonnin et al. 2023). In the Citrandarin hybrid, aromatic compound biosynthesis might be primed by signalling involving hormone and transcription factor, and this variation of this response could be related to the ploidy level. 4.3. Diploid and tetraploid Citrandarins shared strong and similar response to salt stress The consequences of salt stress are the result of three effects of excess salt: (1) osmotic stress (water deficit), (2) hyperosmotic stress (accumulation of ions to a toxic level in plant tissues), and (3) oxidative stress (production of ROS). Salinity negatively affects all aspects of plant development, including germination, vegetative growth, and reproductive development (Maas 1993; Maas & Grattan 1999; Munns & Tester 2008). The symptoms caused by excessive soil salinity are mainly characterized by a reduction in growth, fresh matter, dry matter, and other vegetative development parameters (EL Sabagh et al. 2021). In citrus, a reduction in plant growth has also been observed in plants subjected to salinity. In addition, studies show that salinity stress leads to a reduction in stem diameter, number of leaves per seedling, and root biomass (Singh, Saini & Behl 2004; Melgar, Syvertsen, Martínez & García-Sánchez 2008).In our study, the most striking result is undoubtedly the absence of visual stress symptoms in both the leaf and roots of the 2x and 4x Citrandarins. Even in the same stress conditions, the 2x Trifoliate orange parent was strongly affected (Bonnin et al. 2023). Symptoms of salt stress in leaves usually become visible at low salt concentrations in citrus (Maas 1993). The lower leaves withered and curled whereas the upper leaves showed a certain degree of leaf margin withering. As salt stress increased, the withering and curling of the leaves of the whole increased, especially in the lower leaves, where the petiole showed drying and abscission. The leaf damage culminates in leaf abscission when many of the whole plant’s leaves and total petioles dried up and fell off. Although both ploidy levels had leaves similar to those of their Trifoliate orange parents, this absence of visual stress symptoms was quite similar to that of their Cleopatra mandarin parents (Bonnin et al. 2023).Under salt stress, plants can reduce their exposure to salinity by changing their roots’ growth direction to avoid a saline environment (halotropism). Primary and lateral root length and the number of lateral roots were consistently reduced due to high salt concentration in soil media (Shelden & Munns 2023). A few studies demonstrated the influence of root morphology in citrus subjected to salt stress (Allario et al. 2011). The greater seedling height, stem diameter, and root constituents (length, fresh and dry weight) in Volkamer lemon than in sour orange explained, at least in part, its better tolerance (Othman, Hani, Ayad & St Hilaire 2023). In our study, we found no evidence of any phenotypic changes in the root architecture of the plants tested, probably because of the short duration of the stress applied, the plants’ age, and the size of the pots.Unlike the absence of visual stress symptoms, 2x and 4x Citrandarins showed a strong and similar response to stress at the physiological, biochemical, and gene levels. In leaves, variables related to photosynthesis were similarly affected in the stressed 2x and 4x, as shown by the significant decrease in photosynthetic efficiency ( P net ), transpiration ( P net /E), and stomatal conductance ( g s ), as compared to the control. The regulation of transpiration and its associated metabolic adjustments have been shown to be an adaptive response to salinity, leading to reduction in Cl − accumulation in tolerant genotypes (Brumós et al. 2009a; Brumós, Talón, Bouhlal & Colmenero-Flores 2010). Regulation of the photosynthetic process was detected at the gene level, too, as well as chlorophyll biosynthesis, primary metabolism, and cell morphogenesis. This result suggests a change in cell metabolism in leaves under salt stress. Indeed, many studies reported a decrease in starch, sugar, and amino acid production under salt stress (Arbona et al. 2005; Acosta-Motos et al. 2017). In roots, the gene regulation was about two times higher under salt stress than in leaves, compared to the control, involving circadian rhythm regulation. This process could have an indirect effect on cell size and root structure modification. These results are in agreement with a study on Carrizo citrange ( C. sinensis (L.) Osb . × P. trifoliata (L.) Raf.) and Cleopatra mandarin ( C. reshni Hort. ex Tan .) (Brumós et al. 2009a). The authors showed that the tolerant rootstock induced wider stress responses related to gene expression while repressing central metabolic processes, such as photosynthesis and carbon utilization.Salt stress is known to trigger generation of ROS (Gueta-Dahan, Yaniv, Zilinskas & Ben-Hayyim 1997). Overproduction of ROS triggers proteins, lipids, carbohydrates, and deoxyribonucleic acid damage. To prevent the oxidative damage induced by salt stress, the activation of enzymatic and non-enzymatic antioxidant compounds leads to the detoxification of ROS and thus helps plants maintain growth under stress conditions (Acosta-Motos et al. 2017). Oxidative stress in plants leads to the induction of several enzymes, such as superoxide dismutase (SOD) (EC 1.15.1.1), catalase (CAT) (EC 1.11.1.6), and APX (EC 1.11.1.11) [35]. These enzymes are responsible for the conversion of superoxide ion to hydrogen peroxide (H 2 O 2 ) and its subsequent reduction to H 2 O. In our study, SOD, CAT, and APX activity increased significantly under salt stress in both leaves and roots, as well as MDA and proline contents. Several studies reported an induction in antioxidant enzyme activity under water deficit and salt stress conditions (Piqueras, Hernández, Olmos, Hellín & Sevilla 1996; Gueta-Dahan et al. 1997; Lourkisti, Froelicher, Morillon, Berti & Santini 2022). Whereas DHAR activity increased significantly in Citrandarin root only, 4x had a constitutively higher DHAR in its leaves than 2x. The Cleopatra mandarin parent’s better adaptation to salt stress, compared to Trifoliate orange, was related to a higher activity in Asa, CAT, APX, and DHAR, in addition to an improved proline synthesis and ion homeostasis [17]. Many publications demonstrated the role of proline in osmoprotection, ROS scavenging, and 1 O 2 quencher to protect the photosynthetic apparatus under salt stress (Hoque et al. 2007; Anjum et al. 2014; El Moukhtari, Cabassa-Hourton, Farissi & Savouré 2020). Proline is known as the central component of plant adaptation against salt stress. Therefore, proline biosynthesis or accumulation could mean a better ability to fight against osmotic pressure induced by salt stress.Salt stress induces excessive accumulation of Na + and Cl - and consequent deficiency of other ions, such as Ca 2+ and K + (Parida & Das 2005; Ottow et al. 2005; Hao et al. 2021). Increasing Na + content induces competitive inhibition of K + . To ensure their physiological activity, plant cells generally maintain a relatively high concentration of K + (80-100 mM) and a relatively low concentration of Na + (25-50 mmol. L -1 ) in the cytoplasm (Zhu 2003; Zhang et al. 2017; Almeida, Oliveira & Saibo 2017). In plant cells, Ca 2+ levels are also reduced through competitive inhibition of Na + . The regulation of osmotic pressure in plants subjected to high Na + concentrations is partly ensured by the compensatory decrease in Ca 2+ (Hao et al. 2021). In addition, an increase in Na + concentration in plant cells beyond the threshold value of 25-50 mmol.L -1 can reduce membrane potential and promote Cl - uptake under the effect of a chemical gradient. Excessive Cl - concentration leads to a reduction in chlorophyll content and destruction of the cell membrane and organelle structure, which also hampers plant growth (Rodríguez’, Roberts, Jordan & Drew 1997). The rapid uptake of Cl - by plants at the onset of salt stress may promote osmotic regulation of the root system in glycophytes. Due to the interaction between Na + and NH 4 + and/or between Cl - and NO 3 - , salinity leads to nitrogen (N) and phosphorus (P) deficiency in plants. This ultimately reduces growth and yield in salt-stressed plants (Patade, Suprasanna & Bapat 2008). Tissues’ ability to compartmentalize excess Na + and Cl - to avoid toxic accumulation of these ions within the cytoplasm is crucial to their tolerance. In Citrandarin, the decrease in Mg 2+ and P content and the K/Na + ratio in roots are associated with an increase in Ca 2+ and a decrease in K + in leaves under salt stress, suggesting that salt stress induced hyperosmotic stress (ion imbalance). The symptoms of citrus’ sensitivity to salinity are strongly linked to chloride accumulation in leaves (Brumós et al. 2009a; Hussain, Luro, Costantino, Ollitrault & Morillon 2012). Indeed, in 2x and 4x Citrandrins, salt stress led to a significant accumulation of Na + in roots, unlike in the leaves. Under salt stress conditions (50 mM NaCl), Cleopatra mandarin ( Citrus reshni Hort. Ex Tan. ) shows the lowest reduction in growth induced by the effect of NaCl (Zekri & Parsons 1992; Cámara-Zapata, García-Sánchez, Martinez, Nieves & Cerdá 2004). On the other hand, rough lemon ( Citrus jambhiri Lush ), Trifoliate orange, and their hybrid, citrange Carrizo ( Citrus sinensis (L.) Osb. X Poncirus trifoliata (L.) Raf. ) are defined as “Cl - includers” and are more sensitive to the effects of salt stress (Gómez-Cadenas et al. 2003; Al-Yassin 2004). Based on this criterion, the genotypes were classified based on tolerance. Among the ten genotypes tested, Cleopatra mandarin was identified as the most tolerant and Trifoliate orange was the most sensitive. Our results showed that subjected to 90 mM NaCl, Citrandarins showed no significant accumulation of Cl - ion in the leaves or roots, possibly due to a greater capacity to exclude chloride inherited from its mandarin parents, regardless of the ploidy level.Polyols (sugar alcohols, such as mannitol and sorbitol) often act as osmoprotectants in plants. When a plant is exposed to salt stress, the increased concentration of salts in the soil induces osmotic pressure, which can lead to cell dehydration. By synthesizing polyols, the plant can adjust its internal osmolarity to preserve water balance, protect cell membranes, and mitigate the effects of oxidative stress. Therefore, the activation of genes linked to polyol biosynthesis is an adaptive response to improve tolerance to salt stress. In a study on boron deficiency in citrus (Liu et al. 2022), activation of polyol biosynthesis was also likely observed in response to boron-deficiency-induced stress. Although the initial signals differ (salt stress versus boron deficiency), plants often mobilize similar defence mechanisms to cope with various abiotic stresses. In both cases, polyol synthesis can serve to (1) regulate cell osmolarity and thus preserve cell hydration and structure and (2) protect against oxidative stress by acting as stabilizing agents and free radical scavengers. If our GO analyses show an over-representation of genes involved in polyol biosynthesis in citrus plants subjected to salt stress (90 mM NaCl), this suggests that these plants mobilize this defence mechanism to mitigate the effects of stress. Although sucrose is an important leaf constituent, polyols complement sucrose’s role in osmotic adjustment and protection against salt-induced damage. It is quite conceivable that, as in the boron deficiency study, polyol accumulation is a strategy shared by plants to respond to various types of stress. In both situations, although the nature of the stress is different, polyol production could represent a convergent response to protect cells and stabilize metabolism. The enrichment of polyol biosynthesis-related genes in our GO analyses under salt stress probably indicates that citrus plants activate a defence mechanism aimed at improving their tolerance by adjusting osmolarity and protecting their cells. This mechanism, which is also found under boron-deficient conditions, illustrates plants’ ability to mobilize similar responses in the face of various abiotic stresses. 4.4. Stressed 2x and 4x Citrandarins had contrasting photosynthetic activity, antioxidant metabolism, and ion homeostasis In this section, we addressed the questions “ What are the specific variables that differentiate the stress response of the 2x and 4x Citrandarins? ” and “ What can we infer from them about their respective adaptability to salt stress? ” Numerous studies have been conducted to understand the mechanisms by which certain plants thrive in saline conditions (Flowers & Colmer 2008; Acosta-Motos et al. 2017). Under salt stress conditions, the PSII maximum quantum yield (YII; F v /F m ), photochemical extinction coefficient (qP), and electron transport rate (ETR) decrease whereas non-photochemical extinction (qN) increases (Netondo, Onyango & Beck 2004). Salt stress also significantly decreases the net photosynthetic rate ( P net ), PSII photochemical efficiency ( F v /F m ), stomatal conductance ( g s ), and intercellular CO 2 concentration ( C i ) (Martin & Ruiz-Torres 1992). The F v /F m ratio decreases under salt stress conditions as minimum fluorescence ( F o ) increases, and maximum fluorescence ( F m ) decreases (Wang, Li & Yuan 2007). In our study, photosynthesis ( P net ) decreased significantly in the stressed 2x only, as compared to its control. On the other hand, 4x showed a significant decrease in PSII photochemical efficiency ( F v /F m ratio) and in stomatal conductance ( g s ) under salt stress, compared to the control, as well as an increase in P net /Ci and ETR/P net . Photosynthetic activity was down-regulated at the gene level in 4x only. An increase in ETR/ P net ratio indicates an imbalance between the electron flow and the CO 2 assimilation during photosynthesis (López-Climent, Arbona, Pérez-Clemente & Gómez-Cadenas 2008). When coupled with the elevated oxygenase activity of Rubisco, this disturbance might contribute to other physiological processes rather than to CO 2 assimilation reactions. Therefore, the concomitant increase in ETR/ P net and decrease in P net /Ci , significant in 4x only, might indicate a loss of photosynthetic efficiency under salinity. This occurrence has been largely observed in wheat (Tabatabaei & Ehsanzadeh 2016) and citrus (Lourkisti et al. 2022). However, it has been shown that an increase in ETR/ Pnet is not necessarily an indicator of sensitivity because alternative electron sinks could be used in citrus plants (Ribeiro, Machado, Santos & Oliveira 2009). Among these mechanisms, non-photochemical chlorophyll fluorescence quenching dissipates excess energy as heat. All the 2x and 4x Citrandarins seem to have set up specific photoprotective mechanisms to limit the photo-inhibition induced by the salt stress as it had been observed for their parents’ CL (Bonnin et al. 2023). These results suggest that 4x responded to salt stress earlier than 2x and triggered earlier stress response mechanisms. This could be explained by different water use efficiency (WUE) in 4x and 2x.Consistent with this, water regulation under salt stress was more efficient in 4x than in 2x, as the significant decrease in P net /g s (intrinsic WUE) and P net /E (instantaneous WUE) showed, as compared to the control. These two ratios are mainly influenced by stomatal behaviour. They indicate a relationship between carbon gain and water loss called WUE (Martin & Ruiz-Torres 1992; Zhang et al. 2019). This result could be related to the specific morphological characteristics in 4x (section 3.2). Larger stomata size and lower stomatal density are associated with thicker leaves and lower roots. Hydraulic conductivity tends to limit transpiration and water consumption (Allario et al. 2011; Lourkisti et al. 2021). This also probably increased the resistance to CO 2 diffusion to the site of carboxylation in the chloroplasts (Morillon & Chrispeels 2001; Morillon et al. 2001). A progressive decrease in osmotic potential (Ψπ) in 4x followed by a progressive and synchronous decrease in g s without a comparable decrease in photosynthesis suggest a better tolerance to salt stress. Therefore, the improved WUE in the stressed 4x Citrandarin could be of great interest if selected in rootstocks.The importance of the differential regulation of photosynthetic activity between 2x and 4x under stress was corroborated by the correlation analysis between genes, including physiological and biochemical data. In the leaf network, most of the 68 genes identified were found to be correlated with variables related to photosynthesis (P net , P net /Ci, g s ) and mineral uptake (K + , Ca 2+ ). As described above, all these variables decreased significantly under salt stress in 4x only. Most of the co-variating genes were down-regulated in the stressed 4x compared to the control. With a few exceptions, these genes are unique to one of the Trifoliate orange or the Cleopatra mandarin genomes ( Data Sheet 2 ). Potassium (K + ) has been found to mitigate the impact of salt stress on photosynthesis by alleviating the proton diffusion potential in thylakoids (Che et al. 2022). Che et al. (2022) demonstrated that K + could reverse the decrease in plasma membrane ATPase activity induced by salt stress. Moreover, it could improve PSII activity and PSI cyclic electron flux. In line with this recent finding, our result suggests that K + homeostasis plays a significant role in the 4x Citrandarin’s stress tolerance, especially in the alleviation of photosynthetic damages. These results were supported by the identification of five genes belonging to the LHCB gene family (LHCB2.4, LHCB4.2, LHCB6, LHCA1, LHCA4). In green plants, these genes encode for light-harvesting Chl a/b-binding (LHC) proteins that collect and transfer light energy to the reaction centres of PSII (Xue et al. 2024). In this network, we also found transcription factors involved in photosynthesis regulation, such as MYB4, ABF3 (ABSCISIC AC-ID-INSENSITIVE 5-like protein 6), and HSFA3 (heat stress transcription factor A-3). Giving the well-known importance of Ca 2+ signalling in salt stress response, its presence in the correlation network tends to validate our hypothesis. Indeed, salt stress, loss of cell turgor, and cell volume change are always associated with activation of Ca 2+ channels. Altered levels of intracellular Ca 2+ induced by salt stress enable Ca 2+ to play the role of secondary messenger in signalling. By binding to and activating Ca 2+ sensors, it triggers a cascade of signals.The antioxidant system was differentially regulated between 2x and 4x Citrandarins under salt stress. In leaves of the 4x plants, a significant increase in MDA and proline content as well as CAT-specific activity was measured. Stomatal closure induced by salt stress can lead to a decrease in intracellular CO 2 concentration ( Ci ) and the formation of singlet oxygen ( 1 O 2 ) (Apel & Hirt 2004). Indeed, the excitation of chlorophyll at PSII leads to a triplet state of chlorophyll, which reacts with oxygen to produce 1 O 2 . The production of 1 O 2 can lead to inhibition of PSII (Zahra et al. 2022). Lipid peroxidation (reflected by the increase of MDA) (Shalata & Neumann 2001; Gaweł, Wardas, Niedworok & Wardas 2004; Del Rio, Stewart & Pellegrini 2005), inevitable under salt stress, can be counteracted in 4x by increasing proline content, which acts as an ROS scavenger (Campos et al. 2011; Rehman et al. 2021) and an osmoprotectant (Ghoulam, Foursy & Fares 2002; Dutta, Neelapu, Wani & Challa 2018; El Moukhtari et al. 2020)), and CAT activity (Khelifa, M’hamdi, Rejeb, Belbahri & Souayeh 2011). This might explain the absence of leaf symptoms in 4x (no burned, curly, yellowish leaves). Because 2x and 4x Citrandarin shared the symptom of lack of leaves, they must both trigger antioxidant defence mechanisms. In contrast to 4x, in Asa content, Asa/DHA ratio, APX specific activity, and TPC content increased significantly in 2x leaves under salt stress. All these variables are linked to antioxidant metabolism. The first three variables are linked to the ascorbate-glutathione cycle (Pang & Wang 2010; Caverzan et al. 2012). It is well known that phenolic compounds contribute to ROS mitigation (Parvin et al. 2022). Our study showed that the Asa content was correlated with the TPC content, and both were highly correlated with two genes, one amine oxidase and one glycosyltransferase 2-like. These results suggest that 2x, unlike 4x, might preferentially trigger the ascorbate glutathione cycle and aromatic compound metabolism to fight oxidative stress in leaves under salt stress. Our study showed that they both have contrasting but efficient antioxidant mechanisms to counteract salt stress symptoms in leaves. As in leaves, the antioxidant metabolism was differentially regulated in roots of both 2x and 4x Citrandarins under salt stress. MDA content and CAT and DHAR activity increased in 2x whereas proline content increased significantly only in 4x under salt stress. In 4x, the induction of early osmoprotection (proline biosynthesis) could alleviate oxidative stress (MDA increase). This has already been demonstrated in the parental genotype (Bonnin et al. 2023) and other citrus species (Campos et al. 2011), including polyploids (Fasih Khalid et al. 2020). At the gene level, roots regulated processes related to ROS response, ion transport, and secondary metabolism, such as nicotianamine biosynthesis. Most enriched clusters belong to the same gene expression pattern group (Group 3), up-regulated in 4x, unlike in 2x. They were mainly enriched in genes related to cell wall metabolism and cellular response to ROS. Eight genes coding for peroxidases and a gene coding for CAT (Ptrif.0005s2888) were identified. Taken together, these results tend to suggest that in 4x, stress pressure is efficiently mitigated by early osmoprotection associated with antioxidant mechanisms. As they act in concert, these mechanisms are activated to a lesser extent in 4x than in 2x. In other words, both mechanisms could help prevent oxidative stress induced by salt exposure in 4x whereas a stronger antioxidant response seems to be required to achieve the same result in 2x (i.e. the absence of stress symptoms). It has been shown that these stress response strategies could be related to cell wall maintenance (Le Gall et al. 2015) and rely on the management of ion transport management (Foyer & Noctor 2005; Brumós et al. 2009b; Almeida et al. 2017).The correlation network analysis confirmed antioxidant metabolism’s role in the 2x and 4x Citrandarins’ stress response. It showed that it is was regulated, at least in part, at the transcriptional level. Four variables involved in the antioxidant metabolism (MDA, Asa, APX, DHAR) were found to be highly correlated to 63 genes. Although all these genes were regulated in 2x, mostly up-regulated when compared to the control, few of them were regulated in 4x. Many of these genes are still uncharacterized, explaining the unique detected enrichment in glutamine transport, with few genes involved, but we still identified some interesting genes related to phytohormone response pathway, especially ABA and Auxin. An important study reported phytohormones’ crucial role in salt stress alleviation in plants (Achard et al. 2006; Golldack, Li, Mohan & Probst 2014; Fahad et al. 2015; Yang & Guo 2018). Germination studies have shown that treatment with indole acetic acid (IAA) recovers the germination capacity of the plants tested (Gulnaz, Iqbal & Azam 1999). Foliar application of IAA can also mitigate salt-induced negative effects by (i) increasing the accumulation of essential inorganic nutrients, (ii) maintaining membrane permeability, (iii) increasing chlorophyll content, and (iv) allowing Na + concentration and electrolyte leakage to decrease (Kaya, Tuna & Yokaş 2009). It has been demonstrated that ABA biosynthesis and/or accumulation could limit the negative effect of salinity on photosynthesis and assimilate translocation and hence growth (Popova, Stoinova & Maslenkova 1995; Jaschke, Peuke, Pate & Hartung 1997). ABA production in roots could help decrease Na + and Cl - concentrations and increase K + and Ca 2+ concentrations (Gurmani, Bano, Khan, Din & Zhang 2011). It would improve xylem water potential and plant water uptake (Fricke, Akhiyarova, Veselov & Kudoyarova 2004). It may also enable proline accumulation and soluble sugar content, which are crucial for adaptation in a saline environment (Chen et al. 2002). In salt-stressed plants, it is the osmotic component of salt stress rather than ionic toxicity that induces an increase in ABA concentration (Zhang, Jia, Yang & Ismail 2006). The increase in ABA concentration in the root xylem enables the plant to close these stomata and thus limit water loss through the leaves (Babu, Singh & Gothandam 2012; Atkinson & Urwin 2012). ABA also regulates the expression of genes involved in the response to salt stress and water deficit (Soma, Takahashi, Yamaguchi-Shinozaki & Shinozaki 2021; Hussain 2022). Our results show clear links among the MDA, Asa, DHAR, and APX variables and genes related to phytohormones. Hormonal signalling activated by osmotic stress and MDA production might trigger an antioxidant response in 2x Citrandarin. The increase in MDA, an oxidative stress marker, in 2x roots as well as the increase in DHAR and APX activity showed that it was more affected than the 4x. This hypothesis is sustained by the presence of Asa in the network, as this molecule is essential to APX catalytic activity. Therefore, these results highlighted the absence of oxidative stress in 4x. We demonstrated in a previous article on the Citrandarin’s two parents that the Cleopatra mandarin genotype has a better salt stress tolerance than the Trifoliate orange (Bonnin et al. 2023). This better tolerance was the result of combined ion storage capacity at the root zone, efficient osmoprotection mechanisms, and better antioxidant system activity (Bonnin et al. 2023, 2024a). Citrandarin had inherited its Cleopatra mandarin parent’s salt stress tolerance capacity, and its 4x genotype had even better potential. In the future, it could be interesting to investigate this hybrid’s response to salt stress under a higher salt concentration.Ben Yahmed et al., (2015) (Ben Yahmed et al. 2015) showed Cl - translocation from roots to leaves was limited in mandarin accessions having few leaf symptoms, such as Cleopatra and ‘Shekwasha’. Accessions with the most severe foliar symptoms, such as ‘Fuzhu’, ‘Willowleaf’, ‘Beauty’, ‘King of Siam’, and ‘Nasnaran’, have greater Cl - translocation from roots to leaves (Cl - leaf/root ratio). Other studies (Brumós et al. 2009a) have shown that in Cleopatra mandarins, salt stress did not lead to the accumulation of toxic levels of chlorides in shoots. Finally, a study on the high-salinity-tolerant rootstock (Foral-5) (López-Climent et al. 2008) showed that the combination of an efficient Cl - exclusion mechanism and an active photosynthetic system can improve salt stress tolerance in this genotype. Here, a significant decrease in the Na + leaf/root ratio was observed in 4x Citrandarin and a significant decrease in the Cl - leaf/root ratio was observed in 2x, but no significant differences in leaf Na + and Cl - concentrations between the two genotypes were observed. In roots, Na + content and K + /Na + ratio increased significantly in 4x under salt stress.Gene analysis highlighted a significant enrichment in ion transport. In previous work (Bonnin et al. 2023), we demonstrated that Cleopatra mandarin and Trifoliate orange 4x genotypes tend to accumulate more Na + and Cl - ions in their roots than their 2x relatives (Bonnin et al. 2023). In citrus, salinity-sensitive genotypes accumulate more Cl - and Na + in the leaves, whereas salinity-tolerant genotypes restrict Cl - and Na + in the roots. This limits Cl - transport from the roots to the aerial parts and allows the plants to better adapt to salinity (Moya et al. 2003; Brumós et al. 2009a; Sykes 2011; Hussain et al. 2012; Cirillo et al. 2019). The 4x Cleopatra mandarin’s adaptive strategy also relied on the regulation of gene expression in relation to ion homeostasis (Bonnin et al. 2024a). As expected, Trifoliate orange and Cleopatra mandarin genotypes shared many genes that varied in ploidy level and the stress treatment to ion transport. The highest number of clusters containing GO terms related to ion transport was found in Cleopatra mandarin roots (Bonnin et al. 2024a). Thanks to the clustering analysis, relevant genes were identified related to the ion transport in Citrandarin. More specifically, we found a chloride channel protein (Ptrif.0001s2162 = AT5G33280) and a cation efflux protein that were down-regulated in 4x and 2x under salt stress. However, this down-regulation is significant in 4x only. We also found three genes coding for a vacuolar cation/proton exchanger (CITRE_001G021040 = AT1G55730), a magnesium transporter (CITRE_008G002010), and an ADP/ATP translocase (Ptrif.0004s2576), which were significantly up-regulated in 4x, unlike in 2x, under salt stress. Finally, our results highlighted a cation efflux protein cytoplasmic domain-containing protein (Ptrif.0009s2337) and a potassium transporter (Ptrif.0002sS0617 = AT5G14880), which were significantly down-regulated in the stressed 4x, unlike in 2x, compared to the control. A cation/H + exchanger domain-containing protein (CITRE_006G006700) was significantly up-regulated in the stressed 2x only compared to the control. Our results suggest that both ploidy levels of the Citrandarin genotypes may have inherited their Cleopatra mandarin parent’s adaptation strategy. This strategy could be based on improved ion restriction at the root level, preventing the accumulation of toxic ions in leaves, where they can impair photosynthesis. A better ion storage capacity and hence limitation of toxic ion transport in the root could explain the Citrandarin’s salt stress tolerance. 4.5. Genetic complexity resulting from both hybridization and ploidy The somatic hybridization of citrus via protoplast fusion has become an integral component of a variety of improvement programmes all over the world (Dambier et al. 2022). Most of the citrus germplasms are diploid (Gmitter, Wu, Rokhsar & Talon 2020). Nevertheless, these breeding strategies allow for ploidy manipulation, enabling the generation of several hundred interspecific and intergeneric allotetraploid hybrids. New phenotypes are often produced, exceeding the range of variability existing in the diploid gene pool (Gancel et al. 2005; Gancel, Grimplet, Sauvage, Ollitrault & Brillouet 2006; Jacquemond 2005). The use of somatic hybridization for the improvement of citrus rootstock theoretically allows for the combination of all dominant traits from complementary heterozygous parents and the production of citrus with various traits of interest (Dambier et al. 2011). Moreover, the formation of polyploids contributes to the improvement of citrus adaptation to environmental stresses. Causes of novel variations in polyploids are not yet well understood. However, it is believed that these variations could contribute to increased changes in dosage-regulated gene expression, altered regulatory interactions, and rapid genetic and epigenetic changes (Xu et al. 2014).Studies on citrus hybrids have shown dominance of mandarin traits, but those studies were focused on the very specific biosynthesis of volatile compounds in leaves (Gancel et al. 2005, 2006; Gancel, Alter, Dhuique-Mayer, Ruales & Vaillant 2008). In a previous study, we showed that Cleopatra mandarin and Trifoliate orange parents had very contrasting behaviour under salt stress conditions (Bonnin et al. 2024b). In that study, we showed that 2x Trifoliate orange had developed stress symptoms. Conversely, Citrandarin did not show any leaves symptoms induced by salt stress, similar to Cleopatra mandarin genotypes and to 4x Trifoliate orange (Bonnin et al. 2023). These results suggest that Citrandarin inherited its greater tolerance of salt stress from its Cleopatra mandarin parents. We demonstrated that Cleopatra mandarin’s better adaptation resulted from combined efficient antioxidant metabolism and osmoprotection. Our results also highlighted the improved capacity of the 2x and 4x Cleopatra mandarin genotype to restrict ion transport to the roots, unlike the 2x Trifoliate orange genotype (Bonnin et al. 2023, 2024a).Like its Cleopatra mandarin parent, each 2x and 4x Citrandarin had developed a distinct, complex strategy to cope with salt stress. Both strategies relied on efficient antioxidant and osmoprotection mechanisms in the leaves and roots. Stronger root response in 4x contributed to minimized leaf exposure to the salt stress effect. In other words, 2x demonstrated a stronger ROS response in leaves whereas 4x relied on an adaptative strategy focused on roots, associating antioxidant defence with ion transport management. This supports the idea of a probable dominance effect of Cleopatra mandarin traits in the stressed Citrandrin.At the gene level, in the 2x and 4x Citrandarin gene networks, we noticed that the Cleopatra mandarin and Trifoliate orange parents had approximately the same number of ortholog genes. However, the regulation of these genes was different between 2x and 4x. Moreover, the total number of orthologous genes in both genotypes was roughly equivalent in all conditions tested (ploidy, stress, ploidy x stress). Ploidy effect had a different influence on the expression of Cleopatra mandarin vs Trifoliate orange ortholog genes in roots. A higher number of Cleopatra mandarin orthologs genes were down-regulated under the ploidy effect. In Trifoliate orange, the ploidy effect was translated to an equivalent number of up- and down-regulated ortholog genes. The stress effect also had different contrasted gene expression profiles in Trifoliate orange and Cleopatra mandarin orthologs. In the roots, a higher number of Cleopatra mandarin orthologs genes were up-regulated whereas Trifoliate orange had higher number of down-regulated orthologs genes. Interestingly, in Trifoliate orange leaves, the stress tended to affect the same number of up- and down-regulated orthologs genes. In Cleopatra mandarin leaves, we found more down-regulated orthologs than up-regulated ones. The effect of the interaction between stress and ploidy level, like the stress effect, differentially affected expression level of Trifoliate orange and Cleopatra mandarin root orthologs. To sum up, Citrandarin expressed a higher number of Cleopatra mandarin ortholog genes, in both leaves and roots, that were up-regulated under the effect of the interaction between stress and ploidy.Overall, our results show potential dominance of mandarin genes, leading to a better salt stress tolerance in Citrandarin hybrids. Tetraploidy might enhance Citrandarin’s ability to cope with salt stress due to a better antioxidant response associated with better ion homeostasis. 5. CONCLUSIONS Tetraploid citrus intergeneric hybrids may hold great potential for rootstock breeding programmes and germplasm enhancement. Both 2x and 4x Citrandarins seems to have inherited better salt stress adaptation capacity from their Cleopatra mandarin parent. This complex adaptation response was close to the Cleopatra mandarin parent response. The present work presents a new perspective on the 4x Citrandarin hybrid’s salt stress tolerance capacity. Future extensive work is necessary to evaluate this hybrid’s potential for use as rootstocks. Moreover, if Trifoliate orange is sensitive to salt stress, it is tolerant to cold and Tristeza virus. It will be interesting to see whether we found combined parent tolerance capacity in the Citrandarin hybrids and whether the 4x overcomes its counterpart’s tolerance.Up to now, the identification of genetic determinants of salt stress response originating from a large diversity of perennial crops including citrus has remained limited. Only a few studies have been conducted to investigate alleles whose effects are modulated by salt stress in this species. Most of the existing studies focus on model plant Arabidopsis. Genome wide association studies (GWAS) are increasingly used to explore genetic diversity and the numerous recombination events present in the germplasm collections. Mass analysis techniques, such as those used in GWAS, allow for a simultaneous test of thousands of genetic variants across several genomes, to highlight the ones that are associated with the studied trait. Looking ahead, the use of this technique might help decipher key regulatory nodes that can be targeted to improve breeding programmes. Considering the number of molecular factors regulated in response to the salt stress signal, valuable tools could be used to deepen our understanding of salt stress tolerance. Our team is currently investigating DNA methylation triggered by salt stress in 2x and 4x Citrandarins. Preliminary results suggest a possible implication of some epigenetic regulations in Citrandarin hybrids under salt stress. Further, we aim to develop a model of multiomics association, which could be considered a powerful tool for investigating multiple or complex traits related to salt stress tolerance.As a perennial crop that has been largely studied in terms of agronomy, physiology, biochemistry, and genetics, citrus could be considered a model. 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All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the “Collectivité de Corse” and CIRAD Montpellier UMR AGAP Team Seapag. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The original contributions presented in this paper are included in the article and in the supplemental data. Further inquiries can be directed to the corresponding author/s. The raw sequencing data have been deposited at NCBI under the accession bioproject ”PRJNA1156330”. Parent PO reference genome (V1.3) and annotation (V1.3.1) were downloaded from phytozome (Peng et al., 2020). Information can also be found here https://phytozome-next.jgi.doe.gov/info/Ptrifoliata_v1_3_1. CL Genome was assembled as part of the pre-HLB and a primary version of his upcoming annotation was used. The CL genome sequencing is part of a more global project. Information can be found in Droc et al. 2024 [25]. Acknowledgments: We would like to express our sincere gratitude to Pierre Mournet and Hélène Vigne for sharing their knowledge in sequencing techniques. We also would like to thank Pascal Barantin for helping with lab procedures and plant watering, and Marie Denis for her advices regarding the tuning of the parameters for the PLS and the sPLS. Finally we would like to thanks the “Collectivité territoriale de Corse” for having founding this work. Conflicts of Interest: The authors declare no conflicts of interest. The research presented in this article was conducted in the absence of any commercial or financial relationships that could be considered as conflict of interest. Information & Authors Information Version history V1 Version 1 09 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords citrus genetic variation oxidative stress salt stress tetraploidy water relations Authors Affiliations Marie Bonnin Universite de Corse Pasquale Paoli View all articles by this author Alexandre Soriano Institut National de Recherche pour l'Agriculture l'Alimentation et l'Environnement Centre Occitanie-Montpellier View all articles by this author Radia Lourkisti Universite de Corse Pasquale Paoli View all articles by this author Julie Oustric Universite de Corse Pasquale Paoli View all articles by this author Lenny Calvez Institut National de Recherche pour l'Agriculture l'Alimentation et l'Environnement Centre Occitanie-Montpellier View all articles by this author Maëva Miranda Institut National de Recherche pour l'Agriculture l'Alimentation et l'Environnement Centre Occitanie-Montpellier View all articles by this author Nathalie Leonhardt CEA CNRS BIAM UMR7265 Aix Marseille Université View all articles by this author Patrick Ollitrault Institut National de Recherche pour l'Agriculture l'Alimentation et l'Environnement Centre Occitanie-Montpellier View all articles by this author Liliane Berti Universite de Corse Pasquale Paoli View all articles by this author Jeremie Santini Universite de Corse Pasquale Paoli View all articles by this author Raphael Morillon Institut National de Recherche pour l'Agriculture l'Alimentation et l'Environnement Centre Occitanie-Montpellier View all articles by this author Bénédicte Favreau 0000-0003-2786-9374 [email protected] Institut National de Recherche pour l'Agriculture l'Alimentation et l'Environnement Centre Occitanie-Montpellier View all articles by this author Metrics & Citations Metrics Article Usage 280 views 165 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Marie Bonnin, Alexandre Soriano, Radia Lourkisti, et al. 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