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Development of a female-specific RAPD marker for Sargassum thunbergii gender identification using bulked segregant analysis
Aquatic Botany 102 (2012) 79–81
Contents lists available at SciVerse ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Short communication
Development of a female-specific RAPD marker for Sargassum thunbergii gender identification using bulked segregant analysis Fuli Liu, Xiutao Sun, Wenjun Wang, Zhourui Liang, Feijiu Wang ∗ Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
a r t i c l e
i n f o
Article history: Received 15 December 2011 Received in revised form 11 May 2012 Accepted 12 May 2012 Available online 24 May 2012 Key words: Sargassum thunbergii Random amplified polymorphism DNA Gender identification Bulked segregant analysis
a b s t r a c t In the present paper, we report the development of a female-specific Random Amplified Polymorphism DNA (RAPD) marker for Sargassum thunbergii gender identification using bulked segregant analysis. Five male and five female mature individuals were selected as the samples, from which the genomic DNA were extracted and mixed to produce the male DNA pool and the female DNA pool respectively. The two DNA pools were subjected to RAPD analysis using a total of 100 decamer primers. Out of the 617 RAPD markers only one marker named F289 was polymorphic between the two DNA pools (only appeared in female DNA pool). Marker F289 was also only present in the five female individuals but absent in the five male individuals who made up the female and male DNA pool respectively. The female-specific character of marker F289 was further verified in the 12 male and 12 female individuals sampled randomly from the field. These results indicated that the marker F289 was a female-specific marker, which may link to the sex determination in S. thunbergii. Consequently, we concluded that F289 can be used as a reliable molecular marker for S. thunbergii gender identification. This marker will facilitate the artificial seedlings rearing based on sexual reproduction and also can be utilized in population genetics study as well as in sex-determining mechanism study for S. thunbergii. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Sargassum thunbergii is a common brown macroalgae in the intertidal and shallow sublittoral zone along the western North Pacific (Phillips, 1995). In China, it widely distributes in warm and temperate water from northern Liaodong Peninsula to southern Leizhou Peninsula (Tseng, 1983). Because of its large biomass and high productivity, S. thunbergii plays an important role in the coastal ecosystem (Tsukidate, 1984; Zhang et al., 2009). Sargassum thunbergii has no generation alternation in its life cycle, because it only has sporophyte generation, during which the sporophyte is dioecious. The male and female sporophytes are similar in morphology before sex mature, leading the difficulty to identify the gender according to their phenotypic characteristics. Generally, the solution is to wait until the sporophyte sex mature, then distinguish the male from the female according to the morphology differences between male and female receptacles (Wang et al., 2006). However, it is obvious that this method is time-consuming and easy influenced by the morphological plasticity. Gender identification of S. thunbergii is significant in its demographic study and sex determination study, especially in artificial seedlings rearing based on
∗ Corresponding author. Tel.: +86 0532 85838673; fax: +86 0532 85838673. E-mail address: [email protected] (F. Wang). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2012.05.001
sexual reproduction as well as in genetic breeding program (Wang et al., 2006; Li et al., 2009; Zhang et al., 2007; Wang and Liu, 2007). Therefore, it is necessary to develop an efficient method for S. thunbergii gender identification. Molecular markers have been proved to be a good tool for gender identifying in many animal, high plants as well as algae (Hormaza et al., 1994; Laporte et al., 1998; Isaacs et al., 2003; Sim et al., 2007; Guillenmin et al., 2012). In this paper, we report the development of a female-specific random amplified polymorphism DNA (RAPD) marker for Sargassum thunbergii gender identification using bulked segregant analysis (BSA).
2. Materials and methods 2.1. Sargassum thunbergii samples Sargassum thunbergii were sampled on 17 July 2011, in an intertidal zone of the Tai Ping Jiao Bay, Qingdao City, China. One lateral branch about 6–8 cm in length was torn off from the stem of one S. thunbergii individual. The branches were put into seawater and brought back to laboratory as quickly as possible. Then these branches with receptacles were brushed to clean away the surface epiphytes, rinsed thoroughly with sterile seawater and transferred to beakers containing 1000 mL sterile seawater. Then, these lateral branches were cultured under the conditions of 20 ◦ C, 40 mol photons m−2 s−1 and a photoperiod of 12:12 (light:dark)
80
F. Liu et al. / Aquatic Botany 102 (2012) 79–81
Fig. 2. RAPD banding patterns of FP and MP DNA pools as well as the females and the males making up the FP and MP DNA pools respectively amplified by primer S64. FP: female DNA pool; MP: male DNA pool; F1–F5: female individuals; M1–M5: male individuals; M: molecular marker; Arrow: the female-specific marker of 289 bp.
The repeatable markers were further tested in the five male and five female individuals who make up the male and female DNA pools respectively as well as in 12 male and 12 female individuals sampled randomly. The tested markers that only appeared in male or female individuals were identified as the gender-specific markers. The polymorphic markers identified as gender-specific markers were purified from agarose gel, cloned into PMD-18T vector (TaKaRa, Tokyo, Japan) and sequenced (Sangon, Shanghai, China). The obtained sequences were deposited in NCBI database, and the homology analysis using BLAST program was carried out. 3. Results
Fig. 1. Morphology comparison between the mature male and female lateral branches. Scale bar: 10 mm.
until they sex mature. For the sex mature branches, while the males have the male receptacles which are relatively thinner and longer, the female have the female receptacles which are relatively thicker and shorter (Fig. 1); moreover, the male receptacles discharge sperms which immediately diffused in the seawater, whereas the female receptacles discharge eggs which attach to the female receptacles for a period of time until it was fertilized. According to the above characteristics, the male and female branches were separated from each other. Using the above method, five male and female branches were randomly screened out to produce the male and female DNA pool respectively; and another 24 (12 males and females respectively) branches were randomly sampled and used as the specimens to test the identified marker. 2.2. DNA extraction and bulked segregant analysis Genomic DNA was extracted from branches with a Plant Genomic DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China) according to manufacturer instructions. Genomic DNA from five male branches and five female branches were respectively mixed to generate the male DNA pool (MP) and the female DNA pool (FP). The two DNA pools were analyzed using RAPD marker system to screen polymorphic makers between MP and FP. A total of 100 random decamer primers (Sangon, Shanghai, China) were applied in RAPD analysis according to the method reported by Zhao et al. (2007). 2.3. Identification of the gender-specific marker The polymorphic makers between MP and FP were preliminarily supposed to be the gender-specific markers. Given the lower reproducibility of the RAPD maker system, the polymorphic markers were repeated twice at least in order to verify their reproducibility.
Among the total of 100 decamer primers used to amplify the two DNA pools, only five primers failed to amplify any products. The other 95 primers produced a total of 617 scored amplifying bands with an average of approximately 6 bands per primer. While all of other primers produced the same amplifying patterns between the male and female DNA pools, only one primer (S64, with the sequence 5 -CCTCCAGTGT-3 ) produced different amplifying pattern between the two DNA pools: a 289 bp DNA band was present in the female DNA pool and absent in the male DNA pool (Fig. 2). This polymorphic band was named F289 and preliminarily supposed to be the gender-specific marker, and its reproducibility was proved by three time repeats. When amplification reactions were carried out with DNA from the individuals making up the DNA pools, the amplification pattern of primer S64 kept consistent: the amplifying band F289 only appeared in female individuals and was absent in male individuals (Fig. 2). In addition, the marker F289 also only appeared in 12 female individuals and absented in the 12 male individuals (Fig. 3). These results indicated that the marker F289 was a female-specific marker, which may link to the sex determination in S. thunbergii. Consequently, we concluded that F289 can be used as a reliable molecular marker for S. thunbergii gender identification. The marker F289 was excised and purified from agarose gel, cloned into PMD-18T vector and sequenced. The obtained sequence has been submitted to NCBI database with the accession number of JQ269603. Homology analysis of the sequence using BLAST program did not reveal significant homology with the known sequences in the public database. 4. Discussion Molecular markers have been widely utilized in sex identification for many organisms such as animals and higher plants. However, there were only a few reports about sex identification with molecular marker in algae (Gracilaria gracilis, Martinez et al., 1999; Saccharina japonica, Liu et al., 2009; Undaria pinnatifida, Shan and Pang, 2010; G. chilensis, Guillenmin et al., 2012). All of these studies, including our study, show the potential application of molecular marker in algae gender identification. Since algae have complex life cycle styles and diversified reproductive strategies, it
F. Liu et al. / Aquatic Botany 102 (2012) 79–81
81
Fig. 3. RAPD banding patterns of 12 female and 12 male individuals amplified by primer S64. F1–F12: female individuals; M1–M12: male individuals; M: molecular marker; Arrow: the female-specific marker of 289 bp.
could be supposed that the sex determination mechanism of algae is complex and diversified. So far, characterization of the sex determination process in algae is still in its infancy (Martinez et al., 1999; Guillenmin et al., 2012). These obtained molecular markers may benefit the further studies in algae sex determination mechanism study. In China, as it is the natural feed for holothurian and abalone, S. thunbergii wild resources were depleted with the fast development of holothurians and abalone aquaculture (Wang and Liu, 2007; Sun et al., 2010). In order to protect the wild resources and supply enough feeds for aquaculture, it is necessary to proliferate this alga by artificial cultivation. Nowadays, the artificial cultivation technology (float raft cultivation) and seedling rearing technology (based on sexual reproduction) has been established initially (Zou et al., 2005; Yuan et al., 2006; Wang et al., 2006; Li et al., 2009; Zhang et al., 2007; Wang and Liu, 2007; Sun et al., 2010). However, there is still no reliable method available to differentiate the dioecious sporophytes of S. thunbergii at an early developmental stage, leading the impossibility to address any sex related questions during artificial seedling rearing and genetic breeding. To our knowledge, marker F289 is the first molecular marker linked to the sex determination in S. thunbergii. It can be used as a reliable marker for S. thunbergii gender identification at any development stage, and then facilitate the studies of sex related questions in S. thunbergii. Acknowledgements This work was funded by the 863 Hi-Tech Research and Development Program of China (2012AA10A413) to Professor Feijiu Wang. The authors gratefully acknowledge the significant contributions of the anonymous reviewers. References Guillenmin, M.L., Huanel, O.R., Martinez, E.A., 2012. Characterization of genetic markers linked to sex determination in the haploid-diploid red alga Gracilaria chilensis. Journal of Phycology 48, 365–372. Hormaza, J.I., Dollo, L., Polito, V.S., 1994. Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis. Theoretical and Applied Genetics 89, 9–13.
Isaacs, S.M., Manivannan, N., Muralidharan, V., 2003. Identification of RAPD marker linked to a fertility restorer gene for pet-1 cytoplasm of sunflower (Helianthus annuus L.). Helia 26 (39), 67–74. Laporte, V., Merdinoglu, D., Saumitou-Laprade, P., Butterlin, G., Vernet, P., Cuguen, J., 1998. Identification and mapping of RAPD and RFLP markers linked to a fertility restorer gene for a new source of cytoplasmic male sterility in Beta vulgaris ssp. Maritima. Theoretical and Applied Genetics 96, 989–996. Li, M.Z., Ding, G., Zhan, D.M., Yu, B., Liu, W., Wu, H.Y., 2009. A method for early production of large-size Sargassum thunbergii seedling in north China. Progress in Fishery Sciences 30 (5), 75–82. Liu, Y.S., Li, L.H., Wu, W.K., Zhou, Z.G., 2009. A SCAR molecular marker specifically related to the female gametophytes of Saccharina (Laminaria) japonica (Phaeophyta). Journal of Phycology 45, 894–897. Martinez, E.A., Destombe, C., Quillet, M.C., Valero, M., 1999. Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis. Molecular Ecology 8, 1533–1538. Phillips, N., 1995. Biogeography of Sargassum (Phaeophyta) in the Pacific basin. In: Abbott, I.A. (Ed.), Taxonomy of Economic Seaweeds, vol. 5. California Sea Grant College System, pp. 107–145. Shan, T.F., Pang, S.J., 2010. Sex-linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta). Phycology Research 58, 171–176. Sim, M.C., Lim, P.E., Gan, S.Y., Phang, S.M., 2007. Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of Gracilaria changii (Rhodophyta). Journal of Applied Phycology 19, 763–769. Sun, X.T., Wang, F.J., Wang, W.J., Jiang, H.T., Wang, Y.M., Tang, T.Y., Li, S.Y., 2010. Large scale breeding of Sargassum thunbergii based on sexual reproduction. Marine Fisheries Research 31 (3), 84–91. Tseng, C.K., 1983. Common Seaweeds of China. Science Press, Beijing. Tsukidate, J., 1984. Studies on the regenerative ability of the brown algae, Sargassum muticum (Yendo) Fensholt and Sargassum tortile C. Agardh. Hydrobiologia 116/117, 393–397. Wang, F.J., Sun, X.T., Li, F., 2006. Studies on sexual reproduction and seedling-rearing of Sargassum thunbergii. Marine Fisheries Research 27 (5), 1–6. Wang, Z.F., Liu, J.G., 2007. The sexual reproduction of Sargassum thunbergii and its application in seedling breeding. Oceanologia Et Limnologia Sini 38 (5), 453–457. Yuan, Y.D., Zhang, S.H., Sun, A.F., Liu, H.Y., 2006. Trial culture of Sargassum thunbergii with long line rafts. Transactions of Oceanology and Limnology 2, 125–128 (in Chinese with English abstract). Zhang, Q.S., Li, W., Liu, S., Pan, J.H., 2009. Size-dependence of reproductive allocation of Sargassum thunbergii (Sargassaceae, Phaeophyta) in Bohai Bay, China. Aquatic Botany 91, 194–198. Zhang, Z.Y., Li, X.L., Han, Y.X., Cao, S.Q., You, X.C., 2007. Indoor artificial seeding in seaweed Sargassum thunbergii. Journal of Dalian Fisheries University 22 (4), 255–259. Zhao, F.J., Wang, X.L., Liu, J.D., Duan, D.L., 2007. Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers. Journal of Applied Phycology 19, 409–416. Zou, J.X., Li, Y.Q., Liu, Y.X., Zhang, T.W., Wang, Y.M., 2005. Studies on biological features and raft culture of Sargassum thunbergii. Shangdong Fish 22, 25–28 (in Chinese).
Contents lists available at SciVerse ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Short communication
Development of a female-specific RAPD marker for Sargassum thunbergii gender identification using bulked segregant analysis Fuli Liu, Xiutao Sun, Wenjun Wang, Zhourui Liang, Feijiu Wang ∗ Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
a r t i c l e
i n f o
Article history: Received 15 December 2011 Received in revised form 11 May 2012 Accepted 12 May 2012 Available online 24 May 2012 Key words: Sargassum thunbergii Random amplified polymorphism DNA Gender identification Bulked segregant analysis
a b s t r a c t In the present paper, we report the development of a female-specific Random Amplified Polymorphism DNA (RAPD) marker for Sargassum thunbergii gender identification using bulked segregant analysis. Five male and five female mature individuals were selected as the samples, from which the genomic DNA were extracted and mixed to produce the male DNA pool and the female DNA pool respectively. The two DNA pools were subjected to RAPD analysis using a total of 100 decamer primers. Out of the 617 RAPD markers only one marker named F289 was polymorphic between the two DNA pools (only appeared in female DNA pool). Marker F289 was also only present in the five female individuals but absent in the five male individuals who made up the female and male DNA pool respectively. The female-specific character of marker F289 was further verified in the 12 male and 12 female individuals sampled randomly from the field. These results indicated that the marker F289 was a female-specific marker, which may link to the sex determination in S. thunbergii. Consequently, we concluded that F289 can be used as a reliable molecular marker for S. thunbergii gender identification. This marker will facilitate the artificial seedlings rearing based on sexual reproduction and also can be utilized in population genetics study as well as in sex-determining mechanism study for S. thunbergii. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Sargassum thunbergii is a common brown macroalgae in the intertidal and shallow sublittoral zone along the western North Pacific (Phillips, 1995). In China, it widely distributes in warm and temperate water from northern Liaodong Peninsula to southern Leizhou Peninsula (Tseng, 1983). Because of its large biomass and high productivity, S. thunbergii plays an important role in the coastal ecosystem (Tsukidate, 1984; Zhang et al., 2009). Sargassum thunbergii has no generation alternation in its life cycle, because it only has sporophyte generation, during which the sporophyte is dioecious. The male and female sporophytes are similar in morphology before sex mature, leading the difficulty to identify the gender according to their phenotypic characteristics. Generally, the solution is to wait until the sporophyte sex mature, then distinguish the male from the female according to the morphology differences between male and female receptacles (Wang et al., 2006). However, it is obvious that this method is time-consuming and easy influenced by the morphological plasticity. Gender identification of S. thunbergii is significant in its demographic study and sex determination study, especially in artificial seedlings rearing based on
∗ Corresponding author. Tel.: +86 0532 85838673; fax: +86 0532 85838673. E-mail address: [email protected] (F. Wang). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2012.05.001
sexual reproduction as well as in genetic breeding program (Wang et al., 2006; Li et al., 2009; Zhang et al., 2007; Wang and Liu, 2007). Therefore, it is necessary to develop an efficient method for S. thunbergii gender identification. Molecular markers have been proved to be a good tool for gender identifying in many animal, high plants as well as algae (Hormaza et al., 1994; Laporte et al., 1998; Isaacs et al., 2003; Sim et al., 2007; Guillenmin et al., 2012). In this paper, we report the development of a female-specific random amplified polymorphism DNA (RAPD) marker for Sargassum thunbergii gender identification using bulked segregant analysis (BSA).
2. Materials and methods 2.1. Sargassum thunbergii samples Sargassum thunbergii were sampled on 17 July 2011, in an intertidal zone of the Tai Ping Jiao Bay, Qingdao City, China. One lateral branch about 6–8 cm in length was torn off from the stem of one S. thunbergii individual. The branches were put into seawater and brought back to laboratory as quickly as possible. Then these branches with receptacles were brushed to clean away the surface epiphytes, rinsed thoroughly with sterile seawater and transferred to beakers containing 1000 mL sterile seawater. Then, these lateral branches were cultured under the conditions of 20 ◦ C, 40 mol photons m−2 s−1 and a photoperiod of 12:12 (light:dark)
80
F. Liu et al. / Aquatic Botany 102 (2012) 79–81
Fig. 2. RAPD banding patterns of FP and MP DNA pools as well as the females and the males making up the FP and MP DNA pools respectively amplified by primer S64. FP: female DNA pool; MP: male DNA pool; F1–F5: female individuals; M1–M5: male individuals; M: molecular marker; Arrow: the female-specific marker of 289 bp.
The repeatable markers were further tested in the five male and five female individuals who make up the male and female DNA pools respectively as well as in 12 male and 12 female individuals sampled randomly. The tested markers that only appeared in male or female individuals were identified as the gender-specific markers. The polymorphic markers identified as gender-specific markers were purified from agarose gel, cloned into PMD-18T vector (TaKaRa, Tokyo, Japan) and sequenced (Sangon, Shanghai, China). The obtained sequences were deposited in NCBI database, and the homology analysis using BLAST program was carried out. 3. Results
Fig. 1. Morphology comparison between the mature male and female lateral branches. Scale bar: 10 mm.
until they sex mature. For the sex mature branches, while the males have the male receptacles which are relatively thinner and longer, the female have the female receptacles which are relatively thicker and shorter (Fig. 1); moreover, the male receptacles discharge sperms which immediately diffused in the seawater, whereas the female receptacles discharge eggs which attach to the female receptacles for a period of time until it was fertilized. According to the above characteristics, the male and female branches were separated from each other. Using the above method, five male and female branches were randomly screened out to produce the male and female DNA pool respectively; and another 24 (12 males and females respectively) branches were randomly sampled and used as the specimens to test the identified marker. 2.2. DNA extraction and bulked segregant analysis Genomic DNA was extracted from branches with a Plant Genomic DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China) according to manufacturer instructions. Genomic DNA from five male branches and five female branches were respectively mixed to generate the male DNA pool (MP) and the female DNA pool (FP). The two DNA pools were analyzed using RAPD marker system to screen polymorphic makers between MP and FP. A total of 100 random decamer primers (Sangon, Shanghai, China) were applied in RAPD analysis according to the method reported by Zhao et al. (2007). 2.3. Identification of the gender-specific marker The polymorphic makers between MP and FP were preliminarily supposed to be the gender-specific markers. Given the lower reproducibility of the RAPD maker system, the polymorphic markers were repeated twice at least in order to verify their reproducibility.
Among the total of 100 decamer primers used to amplify the two DNA pools, only five primers failed to amplify any products. The other 95 primers produced a total of 617 scored amplifying bands with an average of approximately 6 bands per primer. While all of other primers produced the same amplifying patterns between the male and female DNA pools, only one primer (S64, with the sequence 5 -CCTCCAGTGT-3 ) produced different amplifying pattern between the two DNA pools: a 289 bp DNA band was present in the female DNA pool and absent in the male DNA pool (Fig. 2). This polymorphic band was named F289 and preliminarily supposed to be the gender-specific marker, and its reproducibility was proved by three time repeats. When amplification reactions were carried out with DNA from the individuals making up the DNA pools, the amplification pattern of primer S64 kept consistent: the amplifying band F289 only appeared in female individuals and was absent in male individuals (Fig. 2). In addition, the marker F289 also only appeared in 12 female individuals and absented in the 12 male individuals (Fig. 3). These results indicated that the marker F289 was a female-specific marker, which may link to the sex determination in S. thunbergii. Consequently, we concluded that F289 can be used as a reliable molecular marker for S. thunbergii gender identification. The marker F289 was excised and purified from agarose gel, cloned into PMD-18T vector and sequenced. The obtained sequence has been submitted to NCBI database with the accession number of JQ269603. Homology analysis of the sequence using BLAST program did not reveal significant homology with the known sequences in the public database. 4. Discussion Molecular markers have been widely utilized in sex identification for many organisms such as animals and higher plants. However, there were only a few reports about sex identification with molecular marker in algae (Gracilaria gracilis, Martinez et al., 1999; Saccharina japonica, Liu et al., 2009; Undaria pinnatifida, Shan and Pang, 2010; G. chilensis, Guillenmin et al., 2012). All of these studies, including our study, show the potential application of molecular marker in algae gender identification. Since algae have complex life cycle styles and diversified reproductive strategies, it
F. Liu et al. / Aquatic Botany 102 (2012) 79–81
81
Fig. 3. RAPD banding patterns of 12 female and 12 male individuals amplified by primer S64. F1–F12: female individuals; M1–M12: male individuals; M: molecular marker; Arrow: the female-specific marker of 289 bp.
could be supposed that the sex determination mechanism of algae is complex and diversified. So far, characterization of the sex determination process in algae is still in its infancy (Martinez et al., 1999; Guillenmin et al., 2012). These obtained molecular markers may benefit the further studies in algae sex determination mechanism study. In China, as it is the natural feed for holothurian and abalone, S. thunbergii wild resources were depleted with the fast development of holothurians and abalone aquaculture (Wang and Liu, 2007; Sun et al., 2010). In order to protect the wild resources and supply enough feeds for aquaculture, it is necessary to proliferate this alga by artificial cultivation. Nowadays, the artificial cultivation technology (float raft cultivation) and seedling rearing technology (based on sexual reproduction) has been established initially (Zou et al., 2005; Yuan et al., 2006; Wang et al., 2006; Li et al., 2009; Zhang et al., 2007; Wang and Liu, 2007; Sun et al., 2010). However, there is still no reliable method available to differentiate the dioecious sporophytes of S. thunbergii at an early developmental stage, leading the impossibility to address any sex related questions during artificial seedling rearing and genetic breeding. To our knowledge, marker F289 is the first molecular marker linked to the sex determination in S. thunbergii. It can be used as a reliable marker for S. thunbergii gender identification at any development stage, and then facilitate the studies of sex related questions in S. thunbergii. Acknowledgements This work was funded by the 863 Hi-Tech Research and Development Program of China (2012AA10A413) to Professor Feijiu Wang. The authors gratefully acknowledge the significant contributions of the anonymous reviewers. References Guillenmin, M.L., Huanel, O.R., Martinez, E.A., 2012. Characterization of genetic markers linked to sex determination in the haploid-diploid red alga Gracilaria chilensis. Journal of Phycology 48, 365–372. Hormaza, J.I., Dollo, L., Polito, V.S., 1994. Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis. Theoretical and Applied Genetics 89, 9–13.
Isaacs, S.M., Manivannan, N., Muralidharan, V., 2003. Identification of RAPD marker linked to a fertility restorer gene for pet-1 cytoplasm of sunflower (Helianthus annuus L.). Helia 26 (39), 67–74. Laporte, V., Merdinoglu, D., Saumitou-Laprade, P., Butterlin, G., Vernet, P., Cuguen, J., 1998. Identification and mapping of RAPD and RFLP markers linked to a fertility restorer gene for a new source of cytoplasmic male sterility in Beta vulgaris ssp. Maritima. Theoretical and Applied Genetics 96, 989–996. Li, M.Z., Ding, G., Zhan, D.M., Yu, B., Liu, W., Wu, H.Y., 2009. A method for early production of large-size Sargassum thunbergii seedling in north China. Progress in Fishery Sciences 30 (5), 75–82. Liu, Y.S., Li, L.H., Wu, W.K., Zhou, Z.G., 2009. A SCAR molecular marker specifically related to the female gametophytes of Saccharina (Laminaria) japonica (Phaeophyta). Journal of Phycology 45, 894–897. Martinez, E.A., Destombe, C., Quillet, M.C., Valero, M., 1999. Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis. Molecular Ecology 8, 1533–1538. Phillips, N., 1995. Biogeography of Sargassum (Phaeophyta) in the Pacific basin. In: Abbott, I.A. (Ed.), Taxonomy of Economic Seaweeds, vol. 5. California Sea Grant College System, pp. 107–145. Shan, T.F., Pang, S.J., 2010. Sex-linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta). Phycology Research 58, 171–176. Sim, M.C., Lim, P.E., Gan, S.Y., Phang, S.M., 2007. Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of Gracilaria changii (Rhodophyta). Journal of Applied Phycology 19, 763–769. Sun, X.T., Wang, F.J., Wang, W.J., Jiang, H.T., Wang, Y.M., Tang, T.Y., Li, S.Y., 2010. Large scale breeding of Sargassum thunbergii based on sexual reproduction. Marine Fisheries Research 31 (3), 84–91. Tseng, C.K., 1983. Common Seaweeds of China. Science Press, Beijing. Tsukidate, J., 1984. Studies on the regenerative ability of the brown algae, Sargassum muticum (Yendo) Fensholt and Sargassum tortile C. Agardh. Hydrobiologia 116/117, 393–397. Wang, F.J., Sun, X.T., Li, F., 2006. Studies on sexual reproduction and seedling-rearing of Sargassum thunbergii. Marine Fisheries Research 27 (5), 1–6. Wang, Z.F., Liu, J.G., 2007. The sexual reproduction of Sargassum thunbergii and its application in seedling breeding. Oceanologia Et Limnologia Sini 38 (5), 453–457. Yuan, Y.D., Zhang, S.H., Sun, A.F., Liu, H.Y., 2006. Trial culture of Sargassum thunbergii with long line rafts. Transactions of Oceanology and Limnology 2, 125–128 (in Chinese with English abstract). Zhang, Q.S., Li, W., Liu, S., Pan, J.H., 2009. Size-dependence of reproductive allocation of Sargassum thunbergii (Sargassaceae, Phaeophyta) in Bohai Bay, China. Aquatic Botany 91, 194–198. Zhang, Z.Y., Li, X.L., Han, Y.X., Cao, S.Q., You, X.C., 2007. Indoor artificial seeding in seaweed Sargassum thunbergii. Journal of Dalian Fisheries University 22 (4), 255–259. Zhao, F.J., Wang, X.L., Liu, J.D., Duan, D.L., 2007. Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers. Journal of Applied Phycology 19, 409–416. Zou, J.X., Li, Y.Q., Liu, Y.X., Zhang, T.W., Wang, Y.M., 2005. Studies on biological features and raft culture of Sargassum thunbergii. Shangdong Fish 22, 25–28 (in Chinese).