IDENTIFICATION OF PORPHYRA HAITANENSIS (BANGIALES,RHODOPHYTA) MEIOSIS BY SIMPLE SEQUENCE REPEAT MARKERS1

The simple sequence repeat (SSR) marks were employed to identify the stage at which meiosis occurs in the life cycle of Porphyra haitanensis T. J. Chang et B. F. Zheng. More than 90% of F1 blades of heterozygous conchocelis produced by the cross between a red mutant (R, ♀) and the wildtype (W, ♂) were color sectored. Two parental colors (R and W) and two new colors (R′ and W′) appeared in linear sectors in the color‐sectored F1 blades. Two SSR primer pairs selected from a total of 52 primer pairs generated a specific paternal and maternal fragment, respectively. Co‐occurrence of these two bands was detected in heterozygous conchocelis and in the color‐sectored F1 blades with two to four sectors, such as R + W, R′ + W′, and R′ + R + W + W′. However, the single‐colored F1 blades exhibited only one band. In the sectors isolated from the color‐sectored F1 blades, R and R′ were the same, showing the maternal pattern, whereas W and W′ were the same, showing the paternal pattern. These data suggested that the two different bands from heterozygous conchocelis originated from the parents and segregated in the F1 blades, whereas the two new colors, R′ and W′, in the F1 blades were produced by the exchange and recombination of alleles of the parental colors during meiosis. These results indicated that meiosis of P. haitanensis occurs during the first two cell divisions of a germinating conchospore, and, therefore, the initial four cells constitute a linear genetic tetrad, leading to the formation of a color‐sectored blade.     

Glycerol‐3‐phosphate metabolism plays a role in stress response in the red alga Pyropia haitanensis

Glycerol‐3‐phosphate (G3P) has been suggested as a novel regulator of plant defense signaling, however, its role in algal resistance remains largely unknown. The glycerol kinase (also designated as NHO1) and NAD‐dependent G3P dehydrogenase (GPDH) are two key enzymes involved in the G3P biosynthesis. In our study, we cloned the full‐length cDNA of NHO1 (NHO1_Ph) and GPDH (GPDH_Ph) from the red alga Pyropia haitanensis (denoted as NHO1_Ph and GPDH_Ph) and examined their expression level under flagellin peptide 22 (flg22) stimulation or heat stress. We also measured the level of G3P and floridoside (a downstream product of G3P in P. haitanensis) under flg22 stimulation or heat stress. Both NHO1_Ph and GPDH_Ph shared high sequence identity and structural conservation with their orthologs from different species, especially from red algae. Phylogenetic analysis showed that NHO1s and GPDHs from red algae were closely related to those from animals. Under flg22 stimulation or heat stress, the expression levels of NHO1_Ph and GPDH_Ph were up‐regulated, G3P levels increased, and the contents of floridoside decreased. But the floridoside level increased in the recovery period after heat stress. Taken together, we found that G3P metabolism was associated with the flg22‐induced defense response and heat stress response in P. haitanensis, indicating the general conservation of defense response in angiosperms and algae. Furthermore, floridoside might also participate in the stress resistance of P. haitanensis.     

Metabolite changes during the life history of Porphyra haitanensis

Plant metabolomics is essentially the comprehensive analysis of complex metabolites of plant extracts. Metabolic fingerprinting is an important part of plant metabolomics research. In this study, metabolic fingerprinting of different stages of the life history of the red alga Porphyra haitanensis was performed. The stages included conchocelis filaments, sporangial branchlets, conchosporangia, discharged conchospores and conchosporangial branchlets after conchospore discharge. Metabolite extracts were analysed with ultra‐performance liquid chromatography coupled with electrospray ionisation quadrupole‐time of flight mass spectrometry. Analyses profiles were subjected to principal components analysis and orthogonal projection to latent structures discriminant analysis using the SIMCA‐P software for biomarker selection and identification. Based on the MS/MS spectra and data from the literature, potential biomarkers, mainly of phosphatidylcholine and lysophosphatidylcholine, were identified. Identification of these biomarkers suggested that plasma membrane phospholipids underwent major changes during the life history of P. haitanensis. The levels of phosphatidylcholine and lysophosphatidylcholine increased in sporangial branchlets and decreased in discharged conchospores. Moreover, levels of sphingaine (d18:0) decreased in sporangial branchlets and increased in discharged conchospores, which indicates that membrane lipids were increasingly synthesised as energy storage in sporangial branchlets, while energy was consumed in sporangial branchlets to discharged conchospores. A metabolomic study of different growth phases of P. haitanensis will enhance our understanding of its physiology and ecology.     

Quantification of heparin's antimetastatic effect by single‐cell force spectroscopy

In circulation, cancer cells induce platelet activation, leading to the formation of a cancer cell‐encircling platelet cloak which facilitates each step of the metastatic cascade. Since cancer patients treated with the anticoagulant heparin showed reduced metastasis rates and improved survival, it is supposed that heparin suppresses the cloak's formation by inhibiting the interaction between platelet's adhesion molecule P‐selectin with its ligands on cancer cells. To quantify this heparin effect, we developed a single‐cell force spectroscopy approach and quantified the adhesion (maximum adhesion force [F_A] and detachment work [W_D]) between platelets and human non‐small cell lung cancer cells (A549). A configuration was used in which A549 cells were glued to tipless cantilevers and force‐distance (F‐D) curves were recorded on a layer of activated platelets. The concentration‐response relationship was determined for heparin at concentrations between 1 and 100 U/mL. Sigmoid dose‐response fit revealed half‐maximal inhibitory concentration (IC₅₀) values of 8.01 U/mL (F_A) and 6.46 U/mL (W_D) and a maximum decrease of the adhesion by 37.5% (F_A) and 38.42% (W_D). The effect of heparin on P‐selectin was tested using anti‐P‐selectin antibodies alone and in combination with heparin. Adding heparin after antibody treatment resulted in an additional reduction of 9.52% (F_A) and 7.12% (W_D). Together, we quantified heparin's antimetastatic effect and proved that it predominantly is related to the blockage of P‐selectin. Our approach represents a valuable method to investigate the adhesion of platelets to cancer cells and the efficiency of substances to block this interaction.     

Inhibition effects of pesticides on glutathione‐S‐transferase enzyme activity of Van Lake fish liver

Glutathione‐S‐transferases (GSTs) have a function in xenobiotic metabolism. They are a significant multifunctional family with a wide variety of catalytic activities. In the current study, we determined in vitro inhibition effects of 2,4‐dichlorophenoxyacetic acid dimethylamine salt (2,4‐D DMA), haloxyfop‐P‐methyl, glyphosate isopropylamine, dichlorvos, and λ‐cyhalothrin on purified GST. For this purpose, GST were purified from Van Lake fish (Chalcalburnus tarichii Pallas) liver with 29.25 EU mg^?1 specific activity and 10.76% yield using GSH–agarose affinity chromatographic method. The pesticides were tested at various concentrations on in vitro GST activity. K_i constants were calculated as 0.17 ± 0.01, 0.25 ± 0.05, 3.72 ± 0.32, 0.42 ± 0.06, and 0.025 ± 0.004 mM, for 2,4‐D DMA, haloxyfop‐P‐methyl, glyphosate isopropylamine, dichlorvos, and λ‐cyhalothrin, respectively. λ‐Cyhalothrin showed a better inhibitory effect compared to the other pesticides. The inhibition mechanisms of λ‐cyhalothrin were competitive, while the other pesticides were noncompetitive.     

Comparison of intestinal permeability and p‐glycoprotein effects on the intestinal absorption of enantiomers of 2‐(2‐hydroxypropanamido) benzoic acid in rats

The purpose of this study was to compare intestinal permeability between enantiomers of 2‐(2‐hydroxypropanamido) benzoic acid ((R )‐/ (S )‐ HPABA), a marine‐derived antiinflammatory drug, using an in situ single‐pass intestinal perfusion (SPIP) model in rats. Concentrations, isolated regions of small intestine, and p ‐glycoprotein (P‐gp) inhibitor were performed to investigate their influences on the intestinal absorption of (R )‐/ (S )‐ HPABA. In addition, a molecular docking method was performed to illustrate our prediction. The absorption rate coefficients (K _a ) and permeability values (P _eff ) of (R )‐/ (S )‐ HPABA were calculated. The permeability of (S )‐HPABA was significantly (P <  0.01) higher than that of (R )‐HPABA in jejunum, and ileum permeability of (R )‐/ (S )‐ HPABA appeared best in ileum; the investigated concentrations ranged from 20 to 80 μg/mL, K _a and P _eff values of (R )‐/ (S )‐ HPABA increased linearly; in the presence of P‐gp inhibitor (verapamil), P _eff values of two enantiomers were increased significantly; and the effect of P‐gp on absorption of (R )‐HPABA is stronger than that of (S )‐HPABA in ileum segment. Based on these results, carrier‐mediated transport or passive transport combined with carrier‐mediated transport seems to be the mechanism for intestinal absorption of (R )‐/ (S )‐ HPABA, and (R )‐/ (S )‐ HPABA may be recognized as the P‐gp substrate. In addition, the intestinal permeability of (S )‐HPABA is higher than that of (R )‐HPABA.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Optimization of conditions for cell cultivation of Porphyra haitanensis conchocelis in a bubble-column bioreactor

Summary Process conditions for cell cultures derived from conchocelis of female red macroalga Porphyra haitanensis were optimized in an illuminated 0.3-l bubble-column photobioreactor, using CO₂ in air as the sole carbon source during a 20-day cultivation period. It reached the highest growth rate when the initial cell density was 700 mg l⁻¹ (dry weight), the optional aeration rate was 1.2 v/v/min, inorganic nitrate concentration was 15 mM and inorganic phosphate concentration was 0.6 mM. This is the first reported bioreactor cultivation study of cell cultures derived from conchocelis of Porphyra haitanensis.     

Synthesis of 2‐amino‐3‐cyanopyridine derivatives and investigation of their carbonic anhydrase inhibition effects

The conversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻) to each other is very important for living metabolism. Carbonic anhydrase (CA, E.C.4.2.1.1), a metalloenzyme familly, catalyzes the interconversion of these ions (CO₂ and HCO₃⁻) and are very common in living organisms. In this study, a series of novel 2‐amino‐3‐cyanopyridines supported with some functional groups was synthesized and tested as potential inhibition effects against both cytosolic human CA I and II isoenzymes (hCA I and II) using by Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. The structural elucidations of novel 2‐amino‐3‐cyanopyridines were achieved by NMR, IR, and elemental analyses. K _i values of the novel synthesized compounds were found in range of 2.84–112.44 μM against hCA I and 2.56–31.17 μM against hCA II isoenzyme. While compound 7d showed the best inhibition activity against hCA I (K _i: 2.84 μM), the compound 7b demonstrated the best inhibition profile against hCA II isoenzyme (K _i: 2.56 μM).     

Observations on the division characterization of diploid nuclear in <Emphasis Type="Italic">Porphyra</Emphasis> (Bangiales,Rhodophyta)

Nuclear divisions of carpospores, conchocelis and conchospores of Porphyra yezoensis, P. haitanensis, P. katadai var. hemiphylla and P. oligospermatangia from China were investigated. The observations showed diploid chromosome numbers of 2n = 6 for P. yezoensis and P. oligospermatangia, and 2n = 10 for P. haitanensis and P. katadai var. hemiphylla. For all four species, somatic pairing of chromosome sets was observed in late prophase. Sister chromosomes separated at anaphase as mitosis took place in carpospores, conchocelis filamentous cells, conchosporangial branch cells and sporangial cells (conchospore formation). Chromosome configurations of tetrad and ring-shaped in conchospore germination were observed, demonstrating the occurrence of meiosis. The characteristics of diploid nuclear division in 2n = 6 species are the same as those of 2n = 10 species. The influence of somatic pairing on nuclear division of diploid cells in Porphyra was discussed.     

Long‐lasting enhancement of GABAA receptor expression in newborn dentate granule cells after early‐life febrile seizures

Febrile seizures (FS) are the most common type of seizures in childhood and are suggested to play a role in the development of temporal lobe epilepsy (TLE). Animal studies demonstrated that experimental FS induce a long‐lasting change in hippocampal excitability, resulting in enhanced seizure susceptibility. Hippocampal neurogenesis and altered ion channel expression have both been proposed as mechanisms underlying this decreased seizure threshold. The present study aimed to analyze whether dentate gyrus (DG) cells that were born after FS and matured for 8 weeks display an altered repertoire of ligand‐gated ion channels. To this end, we applied an established model, in which FS are elicited in 10‐day‐old rat pups by hyperthermia (HT). Normothermia littermates served as controls. From postnatal day 11 (P11) to P16, rats were injected with bromodeoxyuridine (BrdU) to label dividing cells immediately following FS. At P66, we evaluated BrdU‐labeled DG cells for coexpression with γ‐aminobutyric acid‐type A receptors (GABA_ARs) and N‐methyl‐D ‐aspartate receptors (NMDARs). In control animals, 40% of BrdU‐labeled cells coexpressed GABA_AR β2/3, whereas in rats that had experienced FS, 60% of BrdU‐labeled cells also expressed GABA_AR β2/3. The number of BrdU‐NMDAR NR2A/B coexpressing cells was in both groups about 80% of BrdU‐labeled cells. The results demonstrate that developmental seizures cause a long‐term increase in GABA_AR β2/3 expression in newborn DG cells. This may affect hippocampal physiology. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Purification of phycoerythrin from <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Ueda (Bangiales,Rhodophyta) using expanded bed absorption

R-phycoerythrin was purified by means of phenyl-sepharose expanded bed absorption and DEAE-sepharose ion-exchange chromatography from Porphyra yezoensis, one of the largest and important aquaculture species in China. Final R-phycoerythrin preparation was characterized by purity ratio above 4 and the homogeneity in native PAGE, respectively. The results of absorption spectrum, fluorescence spectrum and SDS-PAGE were in agreement with previous reports on R-PE. The yield of R-phycoerythrin was 0.82 mg g⁻¹ wet leafy gametophyte of P. yezoensis. This method is a high-protein recovery technology while reducing processing time, and is suitable for the large batch production of R-phycoerythrin, which will enhance the value of P. yezoensis in China, especially the inferior P. yezoensis which can not be used for flake processing.     

Isolation, characterization and expression of a cDNA encoding an elongation factor-1α from Porphyra yezoensis (Bangiales, Rhodophyta)

We report the isolation, characterization and expression of a cDNA encoding a polypeptide elongation factor‐1α (EF‐1α) from the marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and the sequence was analyzed. The clone contained an open reading frame for a protein of 449 amino acids which exhibits sequence similarity to the known EF‐1α. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra pur‐purea EF‐1αtef‐c (97%) than to the P. purpurea EF‐1αtef‐s (61%). The mRNA was detected both in the leafy gametophyte and the filamentous sporophyte.     

Pre‐dawn stomatal opening does not substantially enhance early‐morning photosynthesis in Helianthus annuus

Most C₃ plant species have partially open stomata during the night especially in the 3–5 h before dawn. This pre‐dawn stomatal opening has been hypothesized to enhance early‐morning photosynthesis (A) by reducing diffusion limitations to CO₂ at dawn. We tested this hypothesis in cultivated Helianthus annuus using whole‐shoot gas exchange, leaf level gas exchange and modelling approaches. One hour pre‐dawn low‐humidity treatments were used to reduce pre‐dawn stomatal conductance (g). At the whole‐shoot level, a difference of pre‐dawn g (0.40 versus 0.17 mol m^?2 s^?1) did not significantly affect A during the first hour after dawn. Shorter term effects were investigated with leaf level gas exchange measurements and a difference of pre‐dawn g (0.10 versus 0.04 mol m^?2 s^?1) affected g and A for only 5 min after dawn. The potential effects of a wider range of stomatal apertures were explored with an empirical model of the relationship between A and intercellular CO₂ concentration during the half‐hour after dawn. Modelling results demonstrated that even extremely low pre‐dawn stomatal conductance values have only a minimal effect on early‐morning A for a few minutes after dawn. Thus, we found no evidence that pre‐dawn stomatal opening enhances A.     

Genetic analysis of the position of meiosis in <Emphasis Type="Italic">Porphyra haitanensis</Emphasis> Chang et Zheng (Bangiales,Rhodophyta)

Crossing experiments were carried out between artificial pigmentation mutants and the wild type in Porphyra haitanensis Chang et Zheng to ascertain where meiosis occurs in its life history by confirming whether the color segregation and the color-sectored blades appear in F₁ gametophytic blades developed from conchospores which are released from heterozygous conchocelis. Two red-type pigmentation mutants (R-10 and SPY-1) were used as the female parent. Their blades are red or red orange in color, thinner than the wild type and weak in elasticity, and have no denticles on their margins. The wild type (W) was used as the male parent; its blades are light brown in color, thick and good in elasticity, and have many marginal denticles. The F₁ gametophytic blades developed from conchospores which were released from heterozygous conchocelis produced in the crosses of R-10(♀)×W(♂) and SPY-1(♀)×W(♂) showed two parental colors (R and W) and two new colors (R', lighter in color than R; W', wild-type-like color and redder than W). Linear segregation of colors occurred in the F₁ blades, forming color-sectored blades with 2–4 sectors. In the color-sectored blades, R and R' sectors were thinner than W and W' sectors, and had weak elasticity and no denticles on their margins, whereas W and W' sectors were thick and had good elasticity and many marginal denticles. Of the F₁ gametophytic blades, 95.2–96.7% were color-sectored and only 3.3–4.8% were unsectored. These results indicate that meiosis of P. haitanensis occurs during the first two cell divisions of a germinating conchospore, and thus it is considered that the initial four cells of a developing conchosporeling constitute a linear genetic tetrad leading to the formation of a color-sectored blade. The new colors of R' and W' were recombinant colors due to the chromosome recombination during the first cell division in meiosis. It is considered that color phenotypes of the two mutants used in this paper were result of two (or more) recessive mutations in different genes, and that they also have mutations concerned with blade thickness and formation of marginal denticles, which are linked with the color mutations.     

Seasonal and within‐canopy variation in shoot‐scale resource‐use efficiency trade‐offs in a Norway spruce stand

Previous leaf‐scale studies of carbon assimilation describe short‐term resource‐use efficiency (RUE) trade‐offs where high use efficiency of one resource requires low RUE of another. However, varying resource availabilities may cause long‐term RUE trade‐offs to differ from the short‐term patterns. This may have important implications for understanding canopy‐scale resource use and allocation. We used continuous gas exchange measurements collected at five levels within a Norway spruce, Picea abies (L.) karst., canopy over 3 years to assess seasonal differences in the interactions between shoot‐scale resource availability (light, water and nitrogen), net photosynthesis (A_n) and the use efficiencies of light (LUE), water (WUE) and nitrogen (NUE) for carbon assimilation. The continuous data set was used to develop and evaluate multiple regression models for predicting monthly shoot‐scale A_n. These models showed that shoot‐scale A_n was strongly dependent on light availability and was generally well described with simple one‐ or two‐parameter models. WUE peaked in spring, NUE in summer and LUE in autumn. However, the relative importance of LUE for carbon assimilation increased with canopy depth at all times. Our results suggest that accounting for seasonal and within‐canopy trade‐offs may be important for RUE‐based modelling of canopy carbon uptake.     

High homocysteine levels prevent via H2S the CoCl2‐induced alteration of lymphocyte viability

High homocysteine (HCy) levels are associated with lymphocyte‐mediated inflammatory responses that are sometimes in turn related to hypoxia. Because adenosine is a potent lymphocyte suppressor produced in hypoxic conditions and shares metabolic pathways with HCy, we addressed the influence of high HCy levels on the hypoxia‐induced, adenosine‐mediated, alteration of lymphocyte viability. We treated mitogen‐stimulated human lymphocytes isolated from healthy individuals and the human lymphoma T‐cell line CEM with cobalt chloride (CoCl₂)to reproduce hypoxia. We found that CoCl₂‐altered cell viability was dose‐dependently reversed using HCy. In turn, the HCy effect was inhibited using DL‐propargylglycine, a specific inhibitor of the hydrogen sulphide (H₂S)‐synthesizing enzyme cystathionine‐γ‐lyase involved in HCy catabolism. We then addressed the intracellular metabolic pathway of adenosine and HCy, and the role of the adenosine A_2A receptor (A_2AR). We observed that: (i) hypoxic conditions lowered the intracellular concentration of HCy by increasing adenosine production, which resulted in high A_2AR expression and 3′, 5′‐cyclic adenosine monophosphate production; (ii) increasing intracellular HCy concentration reversed the hypoxia‐induced adenosinergic signalling despite high adenosine concentration by promoting both S‐adenosylhomocysteine and H₂S production; (iii) DL‐propargylglycine that inhibits H₂S production abolished the HCy effect. Together, these data suggest that high HCy levels prevent, via H₂S production and the resulting down‐regulation of A_2AR expression, the hypoxia‐induced adenosinergic alteration of lymphocyte viability. We point out the relevance of these mechanisms in the pathophysiology of cardiovascular diseases.     

Crystallographic studies for the chiral recognition of the novel chiral stationary phase derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid

Chiral discrimination observed in high‐performance liquid chromatography (HPLC) with the novel chiral stationary phase (CSP‐18C6I) derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid [(+)‐18C6H₄] was investigated by X‐ray crystallographic analysis of the complex composed of the R‐enantiomer of 1‐(1‐naphthyl)ethylamine (1‐NEA) and (+)‐18C6H₄. Mixtures of 1‐NEA (the R‐ or S‐enantiomer) and (+)‐18C6H₄ were dissolved in methanol‐water (1:1) solution and allowed to stand for crystallization. The R‐enantiomer crystallized with (+)‐18C6H₄ as a co‐crystal, although the S‐enantiomer did not. This result was in good agreement with the enantiomer elution order of 1‐NEA in CSP‐18C6I. The apparent binding constants (K_a) of the enantiomers to the (+)‐18C6H₄ obtained from ¹H‐NMR experiments also supported the above‐mentioned result. The X‐ray crystal structure of the 1:1 complex of the R‐enantiomer and (+)‐18C6H₄ indicated the four sets of hydrogen bond association between the naphthylethylammonium cation and oxygen of polyether ring or carbonyl group of (+)‐18C6H₄. Chirality 11:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

IDENTIFICATION OF CROSS‐FERTILIZED CONCHOCELIS USING CLEAVED AMPLIFIED POLYMORPHIC SEQUENCE MARKERS IN CROSS‐EXPERIMENTS OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

As a part of the construction of a Porphyra yezoensis Ueda genetic linkage map, we conducted intraspecific cross‐experiments and subsequent screening of cross‐fertilized conchocelis by cleaved amplified polymorphic sequence (CAPS) analysis. The cross‐experiments were carried out between males of the wildtype (KGJ) and females of the recessive green mutant (TU‐2) using two methods, controlled and random crosses. A total of 42 and 186 wildtype‐colored conchocelis colonies were obtained from the former and latter experiments, respectively. Among those, 49 DNA samples (14% and 23% obtained from the former and latter crosses, respectively) showed biparental CAPS patterns in the two gene regions (EF‐1α open reading frame [ORF] region and V‐ATPase). This study represents the first report in which the cross‐fertilized conchocelis of P. yezoensis has been directly confirmed by molecular marker. The combination of the simple DNA extraction and CAPS analysis may be applicable in genetic studies of other macroalgae that are monoecious and/or grow slowly in laboratory culture.