# Transcriptomic monitoring of Douglas-fir heartwood formation

Didier Delourme, Laure Brémaud, Idelette Plazanet, Patrick Pélissier, Philippe Label, Nathalie Boizot, Christian Breton, Stéphanie Durand, Guy Costa

► To cite this version:

Didier Delourme, Laure Brémaud, Idelette Plazanet, Patrick Pélissier, Philippe Label, et al. Transcriptomic monitoring of Douglas-fir heartwood formation. BMC Genomic Data, 2023, 24 (1), pp.69. 10.1186/s12863-023-01172-z. http://hal-inrae.fr/hal-04534748

HAL Id: hal-04534748

https://hal.inrae.fr/hal-04534748

Submitted on 5 Apr 2024

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

Distributed under a Creative Commons Attribution 4.0 International License
# Transcriptomic monitoring of Douglas-fir heartwood formation

Didier Delourme, Laure Brémaud, Idelette Plazanet, Patrick Pélissier, Philippe Label, Nathalie Boizot, Christian Breton, Stéphanie Durand, Guy Costa

► To cite this version:

Didier Delourme, Laure Brémaud, Idelette Plazanet, Patrick Pélissier, Philippe Label, et al. Transcriptomic monitoring of Douglas-fir heartwood formation. BMC Genomic Data, 2023, 24 (1), pp.69. 10.1186/s12863-023-01172-z. http://hal-04534748

HAL Id: hal-04534748

https://hal.inrae.fr/hal-04534748v1

Submitted on 5 Apr 2024

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>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Transcriptomic monitoring of Douglas-fir heartwood formation

Didier Delourme¹, Laure Brémaud¹, Idelette Plazanet¹, Patrick Pélissier¹, Philippe Label², Nathalie Boizot³, Christian Breton³, Stéphanie Durand⁴ and Guy Costa¹*

**Abstract**

**Objectives** Molecular cues linked to heartwood formation open new (complementary) perspectives to genetic breeding programs of Douglas-fir, a tree species largely cultivated in Europe for the natural durability and civil engineering properties of its wood.

**Data description** RNAs from a single genotype of Douglas-fir, extracted from three distinct wood zones (outer sapwood, inner sapwood and transition zone) at four vegetative seasons to generate an extensive RNA-seq dataset used to apprehend the in-wood dynamic and seasonality of heartwood formation in this hardwood model species.

Previously published data collected on somatic embryos of the same genotype could be merged with the present dataset to upgrade grade the Douglas-fir reference transcriptome.

**Keywords** Douglas-fir transcriptome, Heartwood formation, Seasonality, Wood tissues, Outer sapwood, Inner sapwood, Transition zone

**Objective**

Douglas-fir (*Pseudotsuga menziesii*), a *Pinaceae* tree species originating from the North-Western parts of the United State and Canada, is currently considered in Europe as a major alternative to other endogenous conifers of economic importance [1] due to the natural durability and excellent building and civil engineering properties of its wood. Since its introduction in Europe, Douglas-firs have been selected for their growth qualities (trunk straightness, branching and rapid growth). To our knowledge, no comprehensive transcriptomic

study focussed on the heartwood formation of Douglas-fir has been published until now [2–6]. For that purpose, 24-year-old trees from a comparative plantation plot located in Gimel (CFBL, France), managed by FCBA and planted at 1111 stems per hectare, were harvested for RNA-Seq analysis. Four provenances (1039, 80,145, 80,189 and 80,315) were selected for our study and one tree per provenance was felled each season (spring, summer, fall and winter), except for provenance 80,189 for which 3 trees were felled in winter and in summer. Genotypes were confirmed though the use of 11 nuclear microsatellite sequences previously characterised in Douglas-fir [7]. Indeed, these microsatellite markers confirmed that each genotype shared the same genetic heritage (data not shown). In the end, the 80,189 genotype was further selected on the basis of wood quality related phenotypic traits such as the colour and the relative surface area of its heartwood. RNA extractions were carried out from three distinct zones (growth rings) corresponding to outer sapwood (OSW), inner sapwood (ISW) and the transition zone (TZ) [8], the link to the file that describes

<footnote>*Correspondence:
Guy Costa
guy.costa@unilim.fr
¹ Université de Limoges, LABCIS, UR 22722, 123 avenue Albert Thomas, Limoges F-87060, France
² UCA, INRAE, UMR PIAF, Clermont-Ferrand F-63000, France
³ UMR 0588 BioForA INRAE-ONF, Orléans F-45075, France
⁴ Université de Limoges, CAPTuR, UMR INSERM/CHU 1308, 2 Rue du Docteur Marcland, Limoges F-87025, France</footnote>
# Transcriptomic monitoring of Douglas-fir heartwood formation

Didier Delourme¹, Laure Brémaud¹, Idelette Plazanet¹, Patrick Pélissier¹, Philippe Label², Nathalie Boizot³, Christian Breton³, Stéphanie Durand⁴ and Guy Costa¹*

## Abstract

**Objectives** Molecular cues linked to heartwood formation open new (complementary) perspectives to genetic breeding programs of Douglas-fir, a tree species largely cultivated in Europe for the natural durability and civil engineering properties of its wood.

**Data description** RNAs from a single genotype of Douglas-fir, extracted from three distinct wood zones (outer sap-wood, inner sapwood and transition zone) at four vegetative seasons to generate an extensive RNA-seq dataset used to apprehend the in-wood dynamic and seasonality of heartwood formation in this hardwood model species.

Previously published data collected on somatic embryos of the same genotype could be merged with the present dataset to upgrade grade the Douglas-fir reference transcriptome.

**Keywords** Douglas-fir transcriptome, Heartwood formation, Seasonality, Wood tissues, Outer sapwood, Inner sapwood, Transition zone

## Objective

Douglas-fir (*Pseudotsuga menziesii*), a *Pinaceae* tree species originating from the North-Western parts of the United State and Canada, is currently considered in Europe as a major alternative to other endogenous conifers of economic importance [1] due to the natural durability and excellent building and civil engineering properties of its wood. Since its introduction in Europe, Douglas-firs have been selected for their growth qualities (trunk straightness, branching and rapid growth). To our knowledge, no comprehensive transcriptomic

study focussed on the heartwood formation of Douglas-fir has been published until now [2–6]. For that purpose, 24-year-old trees from a comparative plantation plot located in Gimel (CFBL, France), managed by FCBA and planted at 1111 stems per hectare, were harvested for RNA-Seq analysis. Four provenances (1039, 80,145, 80,189 and 80,315) were selected for our study and one tree per provenance was felled each season (spring, summer, fall and winter), except for provenance 80,189 for which 3 trees were felled in winter and in summer. Genotypes were confirmed though the use of 11 nuclear microsatellite sequences previously characterised in Douglas-fir [7]. Indeed, these microsatellite markers confirmed that each genotype shared the same genetic heritage (data not shown). In the end, the 80,189 genotype was further selected on the basis of wood quality related phenotypic traits such as the colour and the relative surface area of its heartwood. RNA extractions were carried out from three distinct zones (growth rings) corresponding to outer sapwood (OSW), inner sapwood (ISW) and the transition zone (TZ) [8], the link to the file that describes

<footnote>*Correspondence:
Guy Costa
guy.costa@unilim.fr
¹ Université de Limoges, LABCIS, UR 22722, 123 avenue Albert Thomas, Limoges F-87060, France
² UCA, INRAE, UMR PIAF, Clermont-Ferrand F-63000, France
³ UMR 0588 BioForA INRAE-ONF, Orléans F-45075, France
⁴ Université de Limoges, CAPTuR, UMR INSERM/CHU 1308, 2 Rue du Docteur Marcland, Limoges F-87025, France</footnote>
the samples is given in Table 1 [9]. The major objective of our work is to characterise heartwood formation at the transcriptomic level.

Hence, we report here an update of the Douglas transcriptome project marked by the production of almost 400 million sequence reads focussed on wood and heartwood formation. This data complements the datasets describing the differentially expressed genes involved in somatic embryogenesis [10, 11]. Moreover, by combining both of these data (wood and somatic embryogenesis related cDNAs) with the data set already published on Douglas-fir seedlings [12–18], we propose to construct a new version of Douglas-fir reference transcriptome for the scientific community.

## Data description

A total of eight trees have been felled for this study, one in January and July, and three in April and October. In the field, wood slices were cut with a chainsaw and immediately frozen in liquid nitrogen. Total RNA was then extracted from freshly lyophilized disks. Three wood zones (growth rings) were harvested on each disk: the OSW that corresponds to the first ring under the cambial zone, the TZ, just located before heartwood, and the ISW representing the median ring between OSW and TZ. The rings of interest were isolated from the wood disc with a chisel, cut into fine bits and grinded with bench knife mill (4×5 s interspersed with a pause of 30 s) before being crushed into powder (granulometry of about 200 µm) by a ball milling machine (Labman Automation LTD, UK; Phenobois platform mill, INRAE Orléans) according to automated grinding sequences consisting of 6 grinding phases of 30 s interspersed with 50 s of pause.

The extraction of RNAs was first performed according to the protocol described by [16] slightly modified by [17]. The RNAs are then precipitated with 10 M LiCl (1:4, v/v). After 2 days at 4 °C, the RNAs are sedimented by centrifugation at 10,000 g for 30 min. The supernatant is removed. The RNA pellet is then dissolved in SSTE buffer, washes 2 times with chloroform/Isoamyl alcohol.

After precipitation, RNA has been resuspended in 10 µl of DEPC-treated water.

The quantity and the purity of the total RNAs extracted were routinely determined with a micro-spectrophotometer (Nanodrop, ND-1000, ThermoScientific) and confirmed by electrophoresis using a Bioanalyzer 2100 (RNA 6000 Nano Kit, Agilent Technologies), a Bio-Rad Experion and a Fragment Analyzer (Advanced Analytical Technologies). Quality is given by the RQI (RNA Quality Index) for the Experion and by the RIN (RNA Integrity Number) for the other two.

The RNA-seq libraries were prepared using the Illumina strand-specific cDNA library kit according to the manufacturer's guidelines. A total of 47 wood libraries were obtained before being sequenced using Illumina HiSeq 2500 sequencing technology with a maximum read length of 2×125 bp, the link to access the sequences deposited at the NCIB is given in Table 1 [19].

Quality trimming RNA reads obtained from sequencing where quality assessed using FastQC V1.11.4. Illumina adapter sequences left in reads were removed using CutAdapt v1.2.1 (CutAdapt will search for a supplied list of adapters in all the reads, a minimum overlap of 15 bp between the adapter and the read is required). Low quality reads towards the 3' and 5' ends of the reads were trimmed with Trimmomatic v0.3; reads were scanned with a 4 base wide sliding window and leading or trailing bases with average phred quality score lower than 20 were dropped. Reads with a length lower than 50 bp were also discarded, the links of the FastQC files of the different tissues are given in Table 1 [20–22].

Transcriptome de novo assembly. Digital normalization and transcriptome de novo assembly was conducted using the Trinity 2.0.6 software with a default k-mer size of 25. Trinity is composed of three different modules: Inchworm, Chrysalis and Butterfly. Inchworm builds a k-mer dictionary from the reads, which will lead to the construction of contigs. Chrysalis connects all overlapped contigs into components using a de Bruijn graph approach. In a final step, Butterfly simplifies all the generated graphs to report full-length transcripts and their

<table><caption>**Table 1** Overview of data files/data sets</caption><thead><tr><td>Label</td><td>Name of data file/data set</td><td>File types (file extension)</td><td>Data repository and identifier (DOI or accession number)</td></tr></thead><tbody><tr><td>Data file 1</td><td>Experimental design</td><td>PDF file (.pdf)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/ELY4M6 [9]</td></tr><tr><td>Data set 1</td><td>Compressed RNA-seq files</td><td>Sequencing files (.fastq.bz2)</td><td>NCBI Sequence Read Archive: https://identifiers.org/ncbi/bioproject: PRJNA945886 [19]</td></tr><tr><td>Data file 2</td><td>MultiQC_trimmed_outer_sapwood</td><td>HTML file (.html)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/AGVBPI [20]</td></tr><tr><td>Data file 3</td><td>MultiQC_trimmed_inner_sapwood</td><td>HTML file (.html)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/DSMDCK [21]</td></tr><tr><td>Data file 4</td><td>MultiQC_trimmed_transition_zone</td><td>HTML file (.html)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/ERWUDJ [22]</td></tr></tbody></table>
the samples is given in Table 1 [9]. The major objective of our work is to characterise heartwood formation at the transcriptomic level.

Hence, we report here an update of the Douglas transcriptome project marked by the production of almost 400 million sequence reads focussed on wood and heartwood formation. This data complements the datasets describing the differentially expressed genes involved in somatic embryogenesis [10, 11]. Moreover, by combining both of these data (wood and somatic embryogenesis related cDNAs) with the data set already published on Douglas-fir seedlings [12–18], we propose to construct a new version of Douglas-fir reference transcriptome for the scientific community.

## Data description

A total of eight trees have been felled for this study, one in January and July, and three in April and October. In the field, wood slices were cut with a chainsaw and immediately frozen in liquid nitrogen. Total RNA was then extracted from freshly lyophilized disks. Three wood zones (growth rings) were harvested on each disk: the OSW that corresponds to the first ring under the cambial zone, the TZ, just located before heartwood, and the ISW representing the median ring between OSW and TZ. The rings of interest were isolated from the wood disc with a chisel, cut into fine bits and grinded with bench knife mill (4×5 s interspersed with a pause of 30 s) before being crushed into powder (granulometry of about 200 µm) by a ball milling machine (Labman Automation LTD, UK; Phenobois platform mill, INRAE Orléans) according to automated grinding sequences consisting of 6 grinding phases of 30 s interspersed with 50 s of pause.

The extraction of RNAs was first performed according to the protocol described by [16] slightly modified by [17]. The RNAs are then precipitated with 10 M LiCl (1:4, v/v). After 2 days at 4 °C, the RNAs are sedimented by centrifugation at 10,000 g for 30 min. The supernatant is removed. The RNA pellet is then dissolved in SSTE buffer, washes 2 times with chloroform/Isoamyl alcohol.

After precipitation, RNA has been resuspended in 10 µl of DEPC-treated water.

The quantity and the purity of the total RNAs extracted were routinely determined with a micro-spectrophotometer (Nanodrop, ND-1000, ThermoScientific) and confirmed by electrophoresis using a Bioanalyzer 2100 (RNA 6000 Nano Kit, Agilent Technologies), a Bio-Rad Experion and a Fragment Analyzer (Advanced Analytical Technologies). Quality is given by the RQI (RNA Quality Index) for the Experion and by the RIN (RNA Integrity Number) for the other two.

The RNA-seq libraries were prepared using the Illumina strand-specific cDNA library kit according to the manufacturer's guidelines. A total of 47 wood libraries were obtained before being sequenced using Illumina HiSeq 2500 sequencing technology with a maximum read length of 2×125 bp, the link to access the sequences deposited at the NCIB is given in Table 1 [19].

Quality trimming RNA reads obtained from sequencing where quality assessed using FastQC V1.11.4. Illumina adapter sequences left in reads were removed using CutAdapt v1.2.1 (CutAdapt will search for a supplied list of adapters in all the reads, a minimum overlap of 15 bp between the adapter and the read is required). Low quality reads towards the 3' and 5' ends of the reads were trimmed with Trimmomatic v0.3; reads were scanned with a 4 base wide sliding window and leading or trailing bases with average phred quality score lower than 20 were dropped. Reads with a length lower than 50 bp were also discarded, the links of the FastQC files of the different tissues are given in Table 1 [20–22].

Transcriptome de novo assembly. Digital normalization and transcriptome de novo assembly was conducted using the Trinity 2.0.6 software with a default k-mer size of 25. Trinity is composed of three different modules: Inchworm, Chrysalis and Butterfly. Inchworm builds a k-mer dictionary from the reads, which will lead to the construction of contigs. Chrysalis connects all overlapped contigs into components using a de Bruijn graph approach. In a final step, Butterfly simplifies all the generated graphs to report full-length transcripts and their

<table><caption>**Table 1** Overview of data files/data sets</caption><thead><tr><td>Label</td><td>Name of data file/data set</td><td>File types (file extension)</td><td>Data repository and identifier (DOI or accession number)</td></tr></thead><tbody><tr><td>Data file 1</td><td>Experimental design</td><td>PDF file (.pdf)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/ELY4M6 [9]</td></tr><tr><td>Data set 1</td><td>Compressed RNA-seq files</td><td>Sequencing files (.fastq.bz2)</td><td>NCBI Sequence Read Archive: https://identifiers.org/ncbi/bioproject: PRJNA945886 [19]</td></tr><tr><td>Data file 2</td><td>MultiQC_trimmed_outer_sapwood</td><td>HTML file (.html)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/AGVBPI [20]</td></tr><tr><td>Data file 3</td><td>MultiQC_trimmed_inner_sapwood</td><td>HTML file (.html)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/DSMDCK [21]</td></tr><tr><td>Data file 4</td><td>MultiQC_trimmed_transition_zone</td><td>HTML file (.html)</td><td>Recherche Data Gouv repository: https://doi.org/10.57745/ERWUDJ [22]</td></tr></tbody></table>
alternatively spliced form. DeconSeq standalone version 0.4.3 was used to detect and remove sequence contaminations from the assembled transcriptome, using bacterial, fungal, plant, virus and other databases. DeconSeq was run with an alignment identity threshold of 95%.

## Limitations

* Due to the lethal sampling of Douglas-fir trees included in an on-going selection program, we only could have access to three biological repeats (trees) for two seasons (winter and summer) and one for the two other seasons.

* In some cases, especially for TZ, we have used two different rings of the same tree to have sufficient biomass for RNA extraction. In these cases, one ring was harvested at the base of the tree and the second at 1.30 m from the base of the trunk.

## Abbreviations

CFBL Coopérative Forestière Bourgogne Limousin

FCBA Forêt, Cellulose, Bois-construction et Ameublement

ISW Inner Sapwood

OSW Outer Sapwood

RNA-seq RiboNucleic Acid - sequencing

k-mer Substrings of length k

RQI RNA Quality Index

RIN RNA Integrity Number

TZ Transition Zone

## Acknowledgements

We acknowledge FCBA for allowing the collection of Douglas-fir samples. We thank the PHENOBOIS-INRAE platform for wood biomass disruption.

## Authors' contributions

G.C. designed the study; C.B., G.C., I.P., L.B. and P.P. carried out the experiments and the sequencing; S.D., D.D., and P.L. contributed to the data analysis; G.C., I.P., N.B. and C.B. contributed to tissue sampling. G.C., D.D. and L.B. wrote the paper with input from all co-authors. All authors have read and approved the manuscript.

## Funding

The project was supported by the foundation of the University of Limoges, the region Aquitaine-Limousin-Poitou-Charente (ALPC) and the Commission of European Communities through the Chair of Excellence Forest Resources and Wood Uses (http://www.fondation.unilim.fr/sylvalim) including a Ph.D. grant to Idelette Plazanet.

## Availability of data and materials

The RNA-seq sequences are available in the National Center for Biotechnology Information (NCBI) with the BioProject accession number https://identifiers.org/ncbi/bioproject:PRJNA945886. The other data accessed on the Recherche Data Gouv repository under doi:https://doi.org/10.57745/TZPTXE including [9, 20–22].

## Declarations

### Ethics approval and consent to participate

Douglas was discovered on Vancouver Island by Archibald Menzies in 1792. It is a coniferous belonging to the order Pinales, family Pinaceae, subfamily

Laricoideae, genus Pseudotsuga. Douglas, Pseudotsuga menziesii (Mirb.) Franco, is from the western part of North America, the subspecies menziesii (Douglas-fir) is present of the Pacific coast rainforests. Information on the voucher specimen is given at https://npgsweb.ars-grin.gov/gringlobal/taxon/taxonomydetail?id=310540. Douglas-fir is a widespread conifer in Europe, its wood is mainly used in exterior and interior construction, but also for decoration. The plant material used in this study comes from a cultivated plantation plot located in Gimel (CFBL, France), maintained by FCBA. FCBA has given permission to collect wood samples using local guidelines.

### Consent for publication

Not applicable.

### Competing interests

The authors declare no competing interests.

Received: 7 July 2023 Accepted: 10 November 2023

Published online: 20 November 2023

## References

1. Dyderski MK, Paz S, Frelich LE, Jagodzinski AM. How much does climate change threaten European forest tree species distributions? Glob Chang Biol. 2017;24(3):1150–63. https://doi.org/10.1111/gcb.13925.

2. Yang J, Kamdem DP, Keathley DE, Han K-H. Seasonal changes in gene expression at the sapwood-heartwood transition zone of black Locust (Robinia pseudoacacia) revealed by cDNA microarray analysis. Tree Physiol. 2004;24(4):461–74. https://doi.org/10.1093/treephys/24.4.461.

3. Yeh T-F, Chu J-H, Liu L-Y, Chen S-Y. Differential Gene profiling of the heartwood formation process in *Taiwania cryptomerioides* Hayata xylem tissues. Int J Mol Sci. 2020;21(3):960. https://doi.org/10.3390/ijms21030960.

4. Yoshida K, Futumara N, Nishiguchi M. Collection of expressed genes from the transition zone of Cryptomeria japonica in the dormant season. J Wood Sci. 2012;58:89–103. https://doi.org/10.1007/s10086-011-1234-6.

5. Chakraborty S, Britton M, Wegrzyn J, Butterfield T, Martinez-Garcia PJ, Reagan RL, Rao BJ, Leslie CA, Aradhaya M, Neale D, Woeste K, Dandekar AM. YeATS - a tool suite for analyzing RNA-seq derived transcriptome identifies a highly transcribed putative extensin in heartwood/sapwood transition zone in black walnut. F1000Res. 2015;17(4):155. https://doi.org/10.12688/f1000research.66172.

6. Yang H, An W, Gu Y, Peng J, Jiang Y, Li J, Chen L, Zhu P, He F, Zhang F, Xiao J, Liu M, Wan X. Integrative metabolomic and transcriptomic analysis reveals the mechanism of specific color formation in *Phoebe Zhennan* heartwood. Int J Mol Sci. 2022;23(21):13569. https://doi.org/10.3390/ijms232113569.

7. Slavov GT, Howe GT, Yakovlev I, Edwards KJ, Krutovskii KV, Tuskan GA, Carlson JE, Strauss SH, Adams WT. Highly variable SSR markers in Douglas-fir: mendelian inheritance and map locations. Theor Appl Genet. 2004;108(5):873–80. https://doi.org/10.1007/s00122-003-1490-y.

8. Beritognolo I, Magel E, Abdel-Latif A, Charpentier JP, Jay-Allemand C, Breton C. Expression of genes encoding chalcone synthase, flavanone 3-hydroxylase and dihydroflavonol 4-reductase correlates with flavanol accumulation during heartwood formation in Juglans nigra. Tree Physiol. 2002;22(5):291–300. https://doi.org/10.1093/treephys/22.5.291.

9. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. 2023. "Experimental design.pdf", Transcriptomic monitoring of Douglas-fir heartwood formation, https://doi.org/10.57745/ELY4M6, Recherche Data Gouv, V2.

10. Gautier F, Eliášová K, Leplé JC, Vondráková Z, Lomenech AM, Le Metté C, Label P, Costa G, Trontin JF, Teyssier C, Lelu-Walter MA. Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of *Pseudotsuga menziesii* [Mirb]. BMC Plant Biol. 2018;18(1):164. https://doi.org/10.1186/s12870-018-1337-y.

11. Gautier F, Label P, Eliášová K, Leplé JC, Motyka V, Boizot N, Vondráková Z, Malbeck J, Trávníčková A, Le Metté C, Lesage-Descauses MC, Lomenech AM, Trontin JF, Costa G, Lelu-Walter MA, Teyssier C. Cytological, biochemical and molecular events of the Embryogenic State in Douglas-fir
alternatively spliced form. DeconSeq standalone version 0.4.3 was used to detect and remove sequence contaminations from the assembled transcriptome, using bacterial, fungal, plant, virus and other databases. DeconSeq was run with an alignment identity threshold of 95%.

## Limitations

* Due to the lethal sampling of Douglas-fir trees included in an on-going selection program, we only could have access to three biological repeats (trees) for two seasons (winter and summer) and one for the two other seasons.

* In some cases, especially for TZ, we have used two different rings of the same tree to have sufficient biomass for RNA extraction. In these cases, one ring was harvested at the base of the tree and the second at 1.30 m from the base of the trunk.

## Abbreviations

CFBL Coopérative Forestière Bourgogne Limousin

FCBA Forêt, Cellulose, Bois-construction et Ameublement

ISW Inner Sapwood

OSW Outer Sapwood

RNA-seq RiboNucleic Acid - sequencing

k-mer Substrings of length k

RQI RNA Quality Index

RIN RNA Integrity Number

TZ Transition Zone

## Acknowledgements

We acknowledge FCBA for allowing the collection of Douglas-fir samples. We thank the PHENOBOIS-INRAE platform for wood biomass disruption.

## Authors' contributions

G.C. designed the study; C.B., G.C., I.P., L.B. and P.P. carried out the experiments and the sequencing; S.D., D.D., and P.L. contributed to the data analysis; G.C., I.P., N.B. and C.B. contributed to tissue sampling. G.C., D.D. and L.B. wrote the paper with input from all co-authors. All authors have read and approved the manuscript.

## Funding

The project was supported by the foundation of the University of Limoges, the region Aquitaine-Limousin-Poitou-Charente (ALPC) and the Commission of European Communities through the Chair of Excellence Forest Resources and Wood Uses (http://www.fondation.unilim.fr/sylvalim) including a Ph.D. grant to Idelette Plazanet.

## Availability of data and materials

The RNA-seq sequences are available in the National Center for Biotechnology Information (NCBI) with the BioProject accession number https://identifiers.org/ncbi/bioproject:PRJNA945886. The other data accessed on the Recherche Data Gouv repository under doi:https://doi.org/10.57745/TZPTXE including [9, 20–22].

## Declarations

### Ethics approval and consent to participate

Douglas was discovered on Vancouver Island by Archibald Menzies in 1792. It is a coniferous belonging to the order Pinales, family Pinaceae, subfamily

Laricoideae, genus Pseudotsuga. Douglas, Pseudotsuga menziesii (Mirb.) Franco, is from the western part of North America, the subspecies menziesii (Douglas-fir) is present of the Pacific coast rainforests. Information on the voucher specimen is given at https://npgsweb.ars-grin.gov/gringlobal/taxon/taxonomydetail?id=310540. Douglas-fir is a widespread conifer in Europe, its wood is mainly used in exterior and interior construction, but also for decoration. The plant material used in this study comes from a cultivated plantation plot located in Gimel (CFBL, France), maintained by FCBA. FCBA has given permission to collect wood samples using local guidelines.

### Consent for publication

Not applicable.

### Competing interests

The authors declare no competing interests.

Received: 7 July 2023 Accepted: 10 November 2023

Published online: 20 November 2023

## References

1. Dyderski MK, Paz S, Frelich LE, Jagodzinski AM. How much does climate change threaten European forest tree species distributions? Glob Chang Biol. 2017;24(3):1150–63. https://doi.org/10.1111/gcb.13925.

2. Yang J, Kamdem DP, Keathley DE, Han K-H. Seasonal changes in gene expression at the sapwood-heartwood transition zone of black Locust (Robinia pseudoacacia) revealed by cDNA microarray analysis. Tree Physiol. 2004;24(4):461–74. https://doi.org/10.1093/treephys/24.4.461.

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8. Beritognolo I, Magel E, Abdel-Latif A, Charpentier JP, Jay-Allemand C, Breton C. Expression of genes encoding chalcone synthase, flavanone 3-hydroxylase and dihydroflavonol 4-reductase correlates with flavanol accumulation during heartwood formation in Juglans nigra. Tree Physiol. 2002;22(5):291–300. https://doi.org/10.1093/treephys/22.5.291.

9. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. 2023. "Experimental design.pdf", Transcriptomic monitoring of Douglas-fir heartwood formation, https://doi.org/10.57745/ELY4M6, Recherche Data Gouv, V2.

10. Gautier F, Eliášová K, Leplé JC, Vondráková Z, Lomenech AM, Le Metté C, Label P, Costa G, Trontin JF, Teyssier C, Lelu-Walter MA. Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of *Pseudotsuga menziesii* [Mirb]. BMC Plant Biol. 2018;18(1):164. https://doi.org/10.1186/s12870-018-1337-y.

11. Gautier F, Label P, Eliášová K, Leplé JC, Motyka V, Boizot N, Vondráková Z, Malbeck J, Trávníčková A, Le Metté C, Lesage-Descauses MC, Lomenech AM, Trontin JF, Costa G, Lelu-Walter MA, Teyssier C. Cytological, biochemical and molecular events of the Embryogenic State in Douglas-fir
(Pseudotsuga menziesii [Mirb]). Front Plant Sci. 2019;10:118. https://doi.org/10.3389/fpls.2019.00118.

12. Wegrzyn JL, Lee JM, Tearse BR, Neale DB, TreeGenes. A forest tree genome database. Int J Plant Genomics. 2008;2008:412875. https://doi.org/10.1155/2008/412875.

13. Lorenz WW, Ayyampalayam S, Bordeaux JM, Howe GT, Jermstad KD, Neale DB, Rogers DL, Dean JFD. Conifer DBMagic: a database housing multiple de novo transcriptome assemblies for 12 diverse conifer species. Tree Genet Genomes. 2012;8(6):1477–85. https://doi.org/10.1007/S11295-012-0547-Y.

14. Müller T, Ensminger I, Schmid KJ. A catalogue of putative unique transcripts from Douglas-fir (Pseudotsuga menziesii) based on 454 transcriptome sequencing of genetically diverse, drought stressed seedlings. BMC Genomics. 2012;13:673. https://doi.org/10.1186/1471-2164-13-673.

15. Howe GT, Yu J, Knaus B, Cronn R, Kolpak S, Dolan P, Lorenz WW, Dean JF. A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation. BMC Genomics. 2013;28(14):137. https://doi.org/10.1186/1471-2164-14-137.

16. Little SA, Boyes IG, Donaleshen K, von Aderkas P, Ehlting J. A transcriptomic resource for Douglas-fir seed development and analysis of transcription during late megagametophyte development. Plant Reprod. 2016;29:273–86. https://doi.org/10.1007/s00497-016-0291-9.

17. Hess M, Wildhagen H, Junker LV, Ensminger I. Transcriptome responses to temperature, water availability and photoperiod are conserved among mature trees of two divergent Douglas-fir provenances from a coastal and an interior habitat. BMC Genomics. 2016;17(1):682. https://doi.org/10.1186/s12864-016-3022-6.

18. Chang S, Puryear J, Cairney J. A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep. 1993;11(2):113–6. https://doi.org/10.1007/BF02670468.

19. NCBI Sequence Read Archive. 2023. https://identifiers.org/ncbi/biopobject:PRJNA945886.

20. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. 2023. "MultiQC_trimmed_outer_sapwood.html", Transcriptomic monitoring of Douglas-fir heartwood formation, https://doi.org/10.57745/AGVBPI, Recherche Data Gouv, V2.

21. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. MultiQC_trimmed_inner_sapwood.html. Transcriptomic Monit Douglas-fir Heartwood Formation. 2023. https://doi.org/10.57745/DSMDCK. Recherche Data Gouv, V2.

22. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. MultiQC_trimmed_transition_zone.html. Transcriptomic Monit Douglas-fir Heartwood Formation. 2023. https://doi.org/10.57745/ERWUDJ. Recherche Data Gouv, V2.

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<figure><img src="image_3.png" /></figure>
(Pseudotsuga menziesii [Mirb]). Front Plant Sci. 2019;10:118. https://doi.org/10.3389/fpls.2019.00118.

12. Wegrzyn JL, Lee JM, Tearse BR, Neale DB, TreeGenes. A forest tree genome database. Int J Plant Genomics. 2008;2008:412875. https://doi.org/10.1155/2008/412875.

13. Lorenz WW, Ayyampalayam S, Bordeaux JM, Howe GT, Jermstad KD, Neale DB, Rogers DL, Dean JFD. Conifer DBMagic: a database housing multiple de novo transcriptome assemblies for 12 diverse conifer species. Tree Genet Genomes. 2012;8(6):1477–85. https://doi.org/10.1007/S11295-012-0547-Y.

14. Müller T, Ensminger I, Schmid KJ. A catalogue of putative unique transcripts from Douglas-fir (Pseudotsuga menziesii) based on 454 transcriptome sequencing of genetically diverse, drought stressed seedlings. BMC Genomics. 2012;13:673. https://doi.org/10.1186/1471-2164-13-673.

15. Howe GT, Yu J, Knaus B, Cronn R, Kolpak S, Dolan P, Lorenz WW, Dean JF. A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation. BMC Genomics. 2013;28(14):137. https://doi.org/10.1186/1471-2164-14-137.

16. Little SA, Boyes IG, Donaleshen K, von Aderkas P, Ehlting J. A transcriptomic resource for Douglas-fir seed development and analysis of transcription during late megagametophyte development. Plant Reprod. 2016;29:273–86. https://doi.org/10.1007/s00497-016-0291-9.

17. Hess M, Wildhagen H, Junker LV, Ensminger I. Transcriptome responses to temperature, water availability and photoperiod are conserved among mature trees of two divergent Douglas-fir provenances from a coastal and an interior habitat. BMC Genomics. 2016;17(1):682. https://doi.org/10.1186/s12864-016-3022-6.

18. Chang S, Puryear J, Cairney J. A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep. 1993;11(2):113–6. https://doi.org/10.1007/BF02670468.

19. NCBI Sequence Read Archive. 2023. https://identifiers.org/ncbi/biopobject:PRJNA945886.

20. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. 2023. "MultiQC_trimmed_outer_sapwood.html", Transcriptomic monitoring of Douglas-fir heartwood formation, https://doi.org/10.57745/AGVBPI, Recherche Data Gouv, V2.

21. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. MultiQC_trimmed_inner_sapwood.html. Transcriptomic Monit Douglas-fir Heartwood Formation. 2023. https://doi.org/10.57745/DSMDCK. Recherche Data Gouv, V2.

22. Delourme D, Brémaud L, Plazanet I, Pélissier P, Label P, Boizot N, Breton C, Durand S, Costa G. MultiQC_trimmed_transition_zone.html. Transcriptomic Monit Douglas-fir Heartwood Formation. 2023. https://doi.org/10.57745/ERWUDJ. Recherche Data Gouv, V2.

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ready to submit your research? Choose BMC and benefit from:

* fast, convenient online submission

* thorough peer review by experienced researchers in your field

* rapid publication on acceptance

* support for research data, including large and complex data types

* gold Open Access which fosters wider collaboration and increased citations

* maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

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