# Whole genome expression in diploid and doubled diploid citrus seedlings in intra and interspecific contexts.

J. Dutra, Valérie Rodrigues, Tete Severien Barigah, P. Ollitrault, A. Gesteira, Raphaël Morillon

► To cite this version:

J. Dutra, Valérie Rodrigues, Tete Severien Barigah, P. Ollitrault, A. Gesteira, et al.. Whole genome expression in diploid and doubled diploid citrus seedlings in intra and interspecific contexts.. International Citrus Congress., Sep 2016, Foz Do Iguaçu, Brazil. SUSTAINABLE CITRICULTURE: The role of applied knowledge, 243 p., 2016. hal-01604107

HAL Id: hal-01604107

https://hal.science/hal-01604107

Submitted on 3 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /></figure>

Dutra J¹,⁴,⁵, Rodrigues V², Barigah T³, Ollitrault P⁴, Gesteira A⁵, Morillon R⁴

¹UESC, Campus Soane Nazaré, Km 16, Rodovia Ilhéus-Itabuna, 45662-900, Ilhéus- Bahia, Brasil ;

²UMR CMAEE Montpellier, France

³UMR PIAF INRA / UBP, 5 chemin de Beaulieu, 63 039 Clermont- Ferrand Cedex 02, France

⁴UMR Agap, Cirad, Station de Roujol, F-97170, Petit-Bourg, Guadeloupe;

⁵Embrapa Mandioca e Fruticultura, Rua Embrapa, s/n, Cruz das Almas, Bahia, Brasil, 44380-000

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

Introduction

Tetraploids may be allopolyploids or autotetraploid and may result either from sexual reproduction via 2n gametes or by duplication of the entire nucellar
chromosome set. Allopolyploids inherit different subgenomes after interspecific hybridization whereas subgenomes of autotetraploid arise from the same
species. Currently, genome expression changes in allotetraploids are considered to be more strongly affected by genome hybridization than by genome
ploidy changes. Thus, interactions among genomes are considered to play an important role in the development of new phenotypes in allopolyploids.
Studies aimed at identifying changes in genome expression patterns of autotetraploid are less numerous than for allotetraploids. Investigation in potato,
using microarrays showed that gene expression changes were very limited (Stupar et al., 2007). These results suggest that doubled diploids, that derive
all of their alleles from a single species, may undergo fewer alterations in their regulatory networks, resulting in very limited changes of gene expression
between diploid and doubled diploid genotypes (Stupar et al., 2007). Genome doubling can produce autoteraploid (with intraspecific diploid) but also
alloteraploid (from interspecific diploid). Gene expression changes observed in between diploid and doubled diploid genotypes could be attributed to
nuclear dosage and ploidy-driven cellular modifications leading to physiological changes such as cell size, division rate, or organellar composition but
also to modifications of instable regulation network or epigenetic marks in interspecific hybrids. In addition, the genome doubling confers better
adaptability to various environmental stresses included a higher tolerance to salinity (Saleh et al, 2008, Mouhaya, 2010, Ruiz et al, 2016) and water
deficit (Allario et al 2013; Oliveira et al 2016). In this work, using microarrays we investigated the genome expression in root of diploid of Trifoliate
Orange and Cleopatra mandarin, their respective doubled diploid, the diploid intergeneric hybrid Cleopatra mandarin × Trifoliate orange and its
respective doubled diploid. Also we investigated the hydraulic architecture of these plants. Our goal was to verify if the phenotypic differentiation due to
polyploidy led to changes regarding the plant hydraulic and if it could be related to specific genome expression.

Material & Methods: Genome expression and hydraulic architecture

Plant material: diploid (2x) Trifoliate orange (*Poncirus trifoliata*) and Cleopatra mandarin (*Citrus reshni*), their respective doubled diploid (4x), the 2x intergeneric hybrid Cleopatra mandarin × Trifoliate orange (*Citrus reshni* × *Poncirus trifoliata*) and its respective 4x were investigated in absence of stress. Plants were 18 months old.
Secondary root samples were harvested and RNA extracted. Gene expression in root of the different genotypes was investigated using 44K citrus Agilent microarrays.
Using a HPFM (High Pressure Flow Meter ) Carbon efficiency of root (CERt,) Specific conductances (Ktotal /leaf, Ktotal/cross sectional are, Kroot/leaf) were measured in the
different genotypes except 2x Cleopatra mandarin that was not included in the experimental design. Measurements were taken at ambient temperature according to Tyree et
al. (1995). The stem was cut at 3 cm above the soil surface, the stump was connected to the HPFM with a water-tight seal.

Results Root genome expression in 2x and 4x genotypes of Trifoliate Orange, Cleopatra mandarin, and in the intergeneric hybrid Cleopatra mandarin Trifoliate orange – Hydraulic architecture

Root genome expression

* Root gene expression was investigated by comparing 2x and 4x couples. Just a few genes presented a significant differential expression between 2x and 4x Trifoliate Orange (data not showed). In Cleopatra, mandarin very limited number of genes (< 0.1%) presented a gene expression change > 2 fold (Fig.1).

* Large changes of expression were observed when comparing the 2x intergeneric hybrid Cleopatra mandarin × Trifoliate orange and its respective 4x, 2826 being over expressed and 2378 genes being down-regulated (expression change > 2 fold).

<figure><img src="image_7.png" /><figcaption>**Figure 1:** Comparison of gene expression in 2x versus 4x genotypes.
Analysis was performed using Genespring software.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 2:** Specific conductances (Ktotal/leaf, Ktotal/cross sectional are, Kshoot/leaf, Kroot/leaf) and CERt were measured in the different genotypes. Different letters indicate a statistical difference between 2x and 4x belonging to the same genotype (t-test, P ≤ 0.05).</figcaption></figure>

Hydraulic architecture

*Hydraulic architecture was investigated (CERt, Specific conductances: Ktotal/leaf, Ktotal/cross sectional are, Kroot/leaf) and did not reveal large changes between 2x and 4x couples, except CERt between 2x and 4x Trifoliate orange and Kroot/leaf between 2x and 4x hybrid Cleopatra mandarin × Trifoliate orange (Fig. 2)

Conclusions

* Diploid and 4x Trifoliate orange and Cleopatra mandarin did not present changes of gene expression.

* Large changes of gene expression were observed when comparing the 2x intergeneric hybrid Cleopatra mandarin × Trifoliate orange and its respective 4x suggesting that this doubled diploid of an intergeneric cross behaves as an allotetraploid regarding it genome expression.

* When comparing gene expression in 2x or in 4x, large changes of expression were also noted.

* Investigations of CERt and specific conductances (Ktotal/leaf, Ktotal/cross sectional are, Kroot/leaf) did not reveal much difference between 2x and 4x of Trifoliate orange except for CERt. Unfortunately we did not have 2x Cleopatra mandarin in experimental design, and comparisons between 2x and 4x was thus not possible. Regarding the comparison between the 2x intergeneric hybrid Cleopatra mandarin × Trifoliate orange and its respective 4x, only Kroot/leaf was significantly different. This suggest that tetraploidy do not induces large changes in hydraulic architecture in absence of stress. Since doubled diploid were showed to present greater traits of adaption to stress, it would be necessary to do such physiological measurement under stress conditions to decipher the impact of polyploidy.

References:

- Allario T, Brumos J, Colmenero-Flores JM, Iglesias DJ, Pina JA, Navarro L, Talon M, Ollitrault P, Morillon R. (2013) Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production. Plant Cell Environ. 36: 856-68.

- Mouhaya W, Allario T, Brumos J, Andrés F, Froelicher Y, Luro F, Talon M, Ollitrault P, Morillon R (2010) Sensitivity to high salinity in tetraploid citrus seedlings increases with water availability and correlates with expression of candidate genes. Functional Plant Biology, 37: 674-685

- Oliveira TM, Ben Yahmed J, Dutra J, Maserti BE, Talon M, Navarro L, Ollitrault P, Gesteira A, Morillon R. (2016) Better tolerance to water deficit in doubled diploid 'Carrizo citrange' compared to diploid seedlings is associated with more limited water consumption and better H₂O₂ scavenging. Acta Physiologia Plantarum. In press.

- Ruiz M, Quiñones A, Martínez- Cuenca, M-R; Aleza P, Morillon R, Navarro L, Primo-Millo E, Martinez Alcantara B. (2016). Tetraploidy improves salinity tolerance in Carrizo citrange seedlings (Citrus sinensis L. Osb. x Poncirus trifoliata L. Raf.). Journal of Plant Physiology. In press.

- Saleh B., Allario T., Dambier D., Ollitrault P. Morillon R. (2008). Tetraploid citrus rootstocks are more tolerant to salt stress than diploid. Comptes Rendus de Biologie de l'Académie des sciences 331: 703-710.

- Stupar RM, Bhaskar PB, Yandell BS, et al. 2007. Phenotypic and transcriptomic changes associated with potato autopolyploidization Genetics 176, 2055-2067

- Tyree, M.T., Patiño, S., Bennink, J., Alexander, J., 1995. Dynamic measurements of root hydraulic conductance using a high-pressure flowmeter in the laboratory and field. J Exp Bot. 46, 83-94

Fundings: J. Dutra was granted by CAPES, Brazil. This work was funded by a French CTPS project AAP12n°3/C2012-01" and by Feder Guadeloupe "Cavalbio" project.