*4.3. Quantitative Gene Expression*

Before assaying for gene expression, a temperature gradient protocol was implemented for each set of primers to establish the best experimental conditions. The e fficiency of the amplification and primer–dimer formation was assessed using a melting curve.

Real-time PCR was performed using SYBR Green master mix (Bio-Rad) and a forward and reverse primer pair (Table 1). Primers were designed from cDNA sequences of the *G. imbricata* transcriptome (BioProject record PRJNA309128). cDNA (2.5 μL) from apical parts of thalli in di fferent maturity stages were used as a template. Real time PCR reactions were carried out with four replicates of each sample in a real-time PCR MiniOpticon thermal cycler (Bio-Rad) using the following steps: initial denaturation at 98 ◦C for 1 min, amplification during 30 cycles at the pertinent temperature for 1 min (Table 1), 72 ◦C for 5 min, followed by a final extension at 72 ◦C for 10 min.

All gene expressions were normalised using methods to validate potential constitutive genes along with the GenNorm basic visual application following calculations described in [31]. Five housekeeping genes were selected and tested, as previously described [32]. Two of the five constituent genes were validated as housekeeping genes that encode a large subunit of ribosomal RNA and the elongation factor 1<sup>α</sup>. We used amplicons (~70 nt) selected from conserved regions of the large subunit of the ribosomal RNA of *Grateloupia turuturu* (DQ364073), *Halymenia schizymenioid* (DQ364067) and *Cryptonemia undulata* (AF419133) and from the elongation factor of *Chondrus crispus* (CO653259) and *Haematococcus pluvialis* (DV203478) to follow the expression of genes. Data were represented as relative to the expression in infertile thalli and were expressed as the mean ± SD from four separate experiments.


**Table 1.** Sequences of the forward (F) and reverse (R) primers, and temperature of annealing (Tm) for each gene: *Carbohydrate sulfotransferase* (*ST1* and *ST2*), *Galactose-6 sulfurylase* (*SY1* and *SY2*), *WD 40* and *Cytochrome P450.*

### *4.4. Gene Expressions for Development Stages of Thalli and Cystocarp Maturation*

To determine the transcript levels of two annotated *carbohydrate sulfotransferase* (henceforth *sulfotransferase, ST1* and *ST2*), and two *galactose-6 sulfurylase* (henceforth *sulfurylase, SY1* and *SY2*) genes, thalli of 100 mg each at di fferent development stages (fertilised, fertile and infertile thalli) were frozen in liquid nitrogen and stored at −80 ◦C until the RNA was isolated.

To test if gene expression was a ffected by cystocarp maturity, levels of transcript expression were measured in accordance with the di fferentiation and development of the cystocarps in the apex of the thalli. In other words, well-developed cystocarps, and mature cystocarps—henceforth, fully-developed cystocarps. The presence of cystocarps was always reported during the spring (April). Pooled thalli containing cystocarps at different stages of development were used as a control. Infertile thalli were also used as a control for total absence of cystocarps (winter period, December).

### *4.5. Gene Expression Encoding Stress Proteins for Cystocarp Maturity*

Levels of transcripts of two genes encoding stress proteins—i.e., *Cytochrome P450* (*Cyt P450*) and *WD40*—were determined by cystocarp maturity (well-developed and fully-developed cystocarps). The expressions of infertile thalli without cystocarps were used as controls.
