Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m^-2 s^-...Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m^-2 s^-1 for 3 weeks were encapsulated in calcium alginate beads. The beads were dehydrated in 0.4 molLl sucrose prepared with seawater for 6 h, desiccated in an incubator set at 10℃ and 70% relative humidity for 4 h, pre-frozen at either -40℃ or -60℃ for 30 min, and stored in liquid nitrogen for 〉24 h. As high as 43% of survival rate was observed when gametophytes were thawed by placing the beads in 40℃ seawater and re-hydrated in 0.05 molL^-1 citrate sodium prepared using 30‰ NaCl 7 d later. More cells of male gametophytes survived the whole procedure in comparison with female gametophytes. The cells of gametophytes surviving the preservation were able to grow asexually and produce morphologically normal sporophytes.展开更多
Seagrass restoration as part of ocean ecosystem protection has been launched for many years all over the world, but intensive research on this subject in China has just begun in recent years. Seed broadcasting has bee...Seagrass restoration as part of ocean ecosystem protection has been launched for many years all over the world, but intensive research on this subject in China has just begun in recent years. Seed broadcasting has been widely accepted as the most potentially useful method for seagrass restoration over large areas. We examined the influence of key environmental factors on seed germination to help promote eelgrass bed restoration. Under anoxic conditions, the influence of temperature and salinity on the germination rate of eelgrass (Zostera marina L.) seeds was examined at different combinations of four temperatures (4, 9, 14, and 24℃) and nine salinities (5 to 45, increment of 5). The effect of significant interaction of temperature and salinity on germination rate was observed (ANOVA) (P<0.001). The highest germination rate (83.3 ± 3.5)% was reached in 8 weeks at 14℃ and salinity 5. Higher temperature significantly increased the germination rate at salinity 5 (P<0.001) during the whole observation period except for 24℃, while lower salinity significantly increased the germination rate at 14℃ (P<0.001). Although significant interaction was found between temperature and salinity (P<0.001), the influence of salinity was stronger than that of temperature for the germination of eelgrass seeds. These results provide useful information for the propagation of artificial seedlings for seagrass restoration in China.展开更多
Microsatellites or simple sequence repeats(SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp(Saccharina ...Microsatellites or simple sequence repeats(SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp(Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N_(50) of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.展开更多
Saccharina japonica is an economically important cold water brown alga extensively cultivated in China. It is cultivated upside down under a floating rope net with its holdfast and meristematic area facing sunlight an...Saccharina japonica is an economically important cold water brown alga extensively cultivated in China. It is cultivated upside down under a floating rope net with its holdfast and meristematic area facing sunlight and UV irradiation and its blade tip toward dark, and other worse cultivation environmental factors also make S . japonic a face more stresses. In this study, S . japonica transcriptomes corresponding to its four developmental stages were analyzed. In total, 7 800 genes predicted in the genome were transcribed. We found that 1 208 of the 7 800 expressed and 2 697 annotated were virus associating genes. Of 778 diff erentially expressed genes (DEGs), 372 were annotatable and 209 were virus associating. Such portion of virus associating genes indicated that the S . japonica genome contained a large portion of active virus originating genes. It was found that the transcripts abundance associated with sugar biosynthesis was about 2.13 folds of all the expressed, indicating that the biosyntheses of structural and storage sugars were very important cellular processes. The total abundance of genes involved in the biosynthesis of alginate and laminarin were similar among all developmental stages, however, that of genes involved in the biosynthesis of mannitol increased about 2-folds from mushroom and adult stages to mature and aging stages. This trend explained our observation that the content of alginate was almost constant at diff erent development stages, while that of mannitol increased sharply. In addition, we found that a set of defense and cell recurring genes highly expressed and many of them expressed diff erentially among stages. On average, the sum abundance of the transcripts of these genes at four stages were 3.40-and 4.96-folds of all the annotated and all the expressed, respectively. This indicated that S . japonica sporophytes persistently respond possible pathogen and environment stresses. The findings are important for timing S . japonica harvest and amending the current cultivation mode.展开更多
We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS(internal transcribed spacer) and 5S rDNA NTS(non-transcribed spacer) sequences.The ITS seq...We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS(internal transcribed spacer) and 5S rDNA NTS(non-transcribed spacer) sequences.The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera.In contrast,multiple highly variable regions were found in the ITS region of Enteromorpha flexuosa.In general,there were more variable sites in the NTS region than in the ITS region in the three species.The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high.However,a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.展开更多
Stocking density is widely recognized as a critical factor in aquaculture and a potential source of long-term stress.The influence of stocking density on growth and stress response of juvenile turbot（Scophthalmus max...Stocking density is widely recognized as a critical factor in aquaculture and a potential source of long-term stress.The influence of stocking density on growth and stress response of juvenile turbot（Scophthalmus maximus, ~3–75g, initial to final weight） was examined in fish held under low（LD, ~0.21–5.31 kg/m^2, initial to final density）,medium（MD, ~0.42–10.81 kg/m^2） and high stocking density（HD, ~0.63–14.27 kg/m^2） for 120 days in a recirculating aquaculture system（RAS）. In this trial, the growth curve for weight of juvenile turbot in RAS, all fitted by the Schnute model. No significant difference was found in growth performance among the three densities until at the final sampling（Day 120）. The final weight and body weight increase（BWI） in the HD group were significantly lower than in other groups（P〈0.05, weight：（75.83±2.49） g,（75.39±2.08） g,（65.72±2.86） g and BWI：（2 436.12±28.10）%,（2 421.29±4.64）%,（2 097.88±20.99）% in LD, MD and HD groups, respectively）. Similarly, the specific growth rate（SGR）, feed conversion ratio（FCR） and coefficient of variation for weight（CV_w） were adversely affected by high stocking density（P〈0.05）. However, there was no difference in survival and Fulton＇s condition factor（K） of turbot among the different groups. Physiological analyses demonstrated a clear increase in the plasma cortisol level and an obvious decrease in growth hormone（GH） concentration in the HD group on Day120（P〈0.05）. There was no significant effect of stocking density on plasma glucose, Cl– and protein levels. All these findings would provide a reference for selecting the optimal stocking density of juvenile turbot in RAS.展开更多
The effects of different feeds and feeding regimes on growth performance, flesh quality and fecal viscosity of Atlantic salmon （Salmo salar L.） in recirculating aquaculture systems （R.AS） were investigated. Fish ...The effects of different feeds and feeding regimes on growth performance, flesh quality and fecal viscosity of Atlantic salmon （Salmo salar L.） in recirculating aquaculture systems （R.AS） were investigated. Fish （initial body weight of 1677 g＋ 157 g） were fed with four commercial feeds （Nosan salmon-NS, Aller gold-AG, Skretting salmon-SS and Hart ye-HY） in two feeding regimes （80% and 100% satiation） for 78 d. The results showed that salmon specific growth ratio （SGR） and weight gain ratio （WGR） were significantly affected by feed type and feeding regime （P〈0.05）. Feed conversion ratio （FCR） varied between 0.93 and 3.40, which was significantly affected by feed type （P〈0.05）, and slightly improved with increased satiation degree. The activities of digestive enzymes including protease, lipase and amylase were also significantly affected by feed type and feeding regime （P〈0.05）, increasing with satiation degree. Flesh qualities for vitamin E, hydroxyproline （HYP）, liquid loss and muscle pH among all groups showed significant differences （P〈0.05）, ranging from 26.67 to 29.67, while no obvious difference was found in flesh color. Fecal viscosity for different treatments showed no significant difference, though improvement was found in 100% satiation group. From present experiment, it was concluded that both feed type and feeding regime can affect the important quality attributes of Atlantic salmon.展开更多
Long non-coding RNAs(lncRNAs)regulate a variety of biological processes,including sexual reproduction and differentiation.Saccharina japonica,a commercially important brown alga in China,shows remarkable sexual dimorp...Long non-coding RNAs(lncRNAs)regulate a variety of biological processes,including sexual reproduction and differentiation.Saccharina japonica,a commercially important brown alga in China,shows remarkable sexual dimorphism in haploid gametophytes.The sex of Saccharina japonica gametophytes is determined by UV sexual system.However,no results have been reported on the lncRNAs involved in the sex-related gene regulation of S.japonica.This study identified a number of lncRNAs and assessed their expression levels in male and female gametophytes.Among them,a total of 405 lncRNAs and 211 mRNAs showed differential expressions.Furthermore,the functions of target genes of differentially expressed lncRNAs(DELs)differed from those of differentially expressed genes(DEGs),suggesting that lncRNA may interact with other functional proteins,in addition to DEGs,to involve sex regulation in S.japonica.There were 32 and 90 potential cis-regulatory and trans-regulatory interactions between DELsDEGs,respectively.Five of these lncRNAs(LNC_002974,LNC_021059,LNC_038466,LNC_051584,and LNC_027400)interacted with putative male sex determination region(SDR)genes,suggesting that they act as regulators in gametophytes'sex regulation potentially.Findings from this study contribute to our understanding of the roles of lncRNAs in sex differentiation and lay the foundation for functional studies of candidate lncRNAs in the future.展开更多
The variation of 90 Laminaria gametophyte clones representing the introduced Laminaria japonica （Group 1） and Laminaria Iongissima （Group 2）, the varieties of L. japonica （Group 3） and the varieties derived from...The variation of 90 Laminaria gametophyte clones representing the introduced Laminaria japonica （Group 1） and Laminaria Iongissima （Group 2）, the varieties of L. japonica （Group 3） and the varieties derived from interspecific hybrids （Group 4） was determined with 18 microsatellite markers. The allelic diversity and Nei＇s gene diversity of Group 1 were significantly higher than those of Group 2 （2.9 vs. 1.8 and 0.414 vs. 0.161, respectively）, demonstrating that the variation of the introduced L. japonica is richer than that of L. Iongissima. Both allelic diversity and Nei＇s gene diversity of Group 3 were lower than those of Group 1, indicating that only a portion of variation of L. japonica was incorporated into the varieties of L. japonica. Significant genetic differentiation was detected between four groups and between female （Population 1 ） and male （Population 2） gametophyte clones in each group. The variation among groups accounted for 39.95%, while that among populations accounted for 21.65% of the total. The genetic distance between Group 1 and Group 4 was obviously longer than that between Group 2 and Group 4 （0.686 vs. 0.291）, indicating that maternal gametophyte clone contributed more variation to the hybrids than the paternal gametophyte clone did.展开更多
文摘Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m^-2 s^-1 for 3 weeks were encapsulated in calcium alginate beads. The beads were dehydrated in 0.4 molLl sucrose prepared with seawater for 6 h, desiccated in an incubator set at 10℃ and 70% relative humidity for 4 h, pre-frozen at either -40℃ or -60℃ for 30 min, and stored in liquid nitrogen for 〉24 h. As high as 43% of survival rate was observed when gametophytes were thawed by placing the beads in 40℃ seawater and re-hydrated in 0.05 molL^-1 citrate sodium prepared using 30‰ NaCl 7 d later. More cells of male gametophytes survived the whole procedure in comparison with female gametophytes. The cells of gametophytes surviving the preservation were able to grow asexually and produce morphologically normal sporophytes.
基金supported by a Municipal Research and Developmental Program of Science and Technology of Yantai, Shandong Province, China (Grant No. 2009211)an Open Foundation of the State Oce-anic Administration of China (Grant No. 200905020-12)
文摘Seagrass restoration as part of ocean ecosystem protection has been launched for many years all over the world, but intensive research on this subject in China has just begun in recent years. Seed broadcasting has been widely accepted as the most potentially useful method for seagrass restoration over large areas. We examined the influence of key environmental factors on seed germination to help promote eelgrass bed restoration. Under anoxic conditions, the influence of temperature and salinity on the germination rate of eelgrass (Zostera marina L.) seeds was examined at different combinations of four temperatures (4, 9, 14, and 24℃) and nine salinities (5 to 45, increment of 5). The effect of significant interaction of temperature and salinity on germination rate was observed (ANOVA) (P<0.001). The highest germination rate (83.3 ± 3.5)% was reached in 8 weeks at 14℃ and salinity 5. Higher temperature significantly increased the germination rate at salinity 5 (P<0.001) during the whole observation period except for 24℃, while lower salinity significantly increased the germination rate at 14℃ (P<0.001). Although significant interaction was found between temperature and salinity (P<0.001), the influence of salinity was stronger than that of temperature for the germination of eelgrass seeds. These results provide useful information for the propagation of artificial seedlings for seagrass restoration in China.
基金financially supported by National High Technology Research and Development Program(863 Program)of China(2012AA10A406)National Science and Technology Supporting Program of China(2012 BAD55G01)+1 种基金Municipal Science and Technology Research and Development Project of Yantai(2013LGS002)Shandong Province Higher Educational Science and Technology Program(J15LE14)
文摘Microsatellites or simple sequence repeats(SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp(Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N_(50) of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.
基金Supported by the Fundamental Research Funds for the Central Universities(No.201762017)
文摘Saccharina japonica is an economically important cold water brown alga extensively cultivated in China. It is cultivated upside down under a floating rope net with its holdfast and meristematic area facing sunlight and UV irradiation and its blade tip toward dark, and other worse cultivation environmental factors also make S . japonic a face more stresses. In this study, S . japonica transcriptomes corresponding to its four developmental stages were analyzed. In total, 7 800 genes predicted in the genome were transcribed. We found that 1 208 of the 7 800 expressed and 2 697 annotated were virus associating genes. Of 778 diff erentially expressed genes (DEGs), 372 were annotatable and 209 were virus associating. Such portion of virus associating genes indicated that the S . japonica genome contained a large portion of active virus originating genes. It was found that the transcripts abundance associated with sugar biosynthesis was about 2.13 folds of all the expressed, indicating that the biosyntheses of structural and storage sugars were very important cellular processes. The total abundance of genes involved in the biosynthesis of alginate and laminarin were similar among all developmental stages, however, that of genes involved in the biosynthesis of mannitol increased about 2-folds from mushroom and adult stages to mature and aging stages. This trend explained our observation that the content of alginate was almost constant at diff erent development stages, while that of mannitol increased sharply. In addition, we found that a set of defense and cell recurring genes highly expressed and many of them expressed diff erentially among stages. On average, the sum abundance of the transcripts of these genes at four stages were 3.40-and 4.96-folds of all the annotated and all the expressed, respectively. This indicated that S . japonica sporophytes persistently respond possible pathogen and environment stresses. The findings are important for timing S . japonica harvest and amending the current cultivation mode.
基金Supported by the National Key Technology Research and Development Program(No.2008BA49B01)the Key Innovation Program of Chinese Academy of Sciences(No.KZCX2-YW-209)
文摘We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS(internal transcribed spacer) and 5S rDNA NTS(non-transcribed spacer) sequences.The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera.In contrast,multiple highly variable regions were found in the ITS region of Enteromorpha flexuosa.In general,there were more variable sites in the NTS region than in the ITS region in the three species.The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high.However,a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.
基金The National Natural Science Foundation of China under contract Nos 31402315 and 31240012the Modern Agriculture Industry System Construction of Special Funds under contract No.CARS-50-G10+1 种基金the Key R&D Program of Jiangsu Province under contract No.BE2015328a foundation from the Key Laboratory of Mariculture&Stock Enhancement in North China’s Sea,Ministry of Agriculture,China
文摘Stocking density is widely recognized as a critical factor in aquaculture and a potential source of long-term stress.The influence of stocking density on growth and stress response of juvenile turbot（Scophthalmus maximus, ~3–75g, initial to final weight） was examined in fish held under low（LD, ~0.21–5.31 kg/m^2, initial to final density）,medium（MD, ~0.42–10.81 kg/m^2） and high stocking density（HD, ~0.63–14.27 kg/m^2） for 120 days in a recirculating aquaculture system（RAS）. In this trial, the growth curve for weight of juvenile turbot in RAS, all fitted by the Schnute model. No significant difference was found in growth performance among the three densities until at the final sampling（Day 120）. The final weight and body weight increase（BWI） in the HD group were significantly lower than in other groups（P〈0.05, weight：（75.83±2.49） g,（75.39±2.08） g,（65.72±2.86） g and BWI：（2 436.12±28.10）%,（2 421.29±4.64）%,（2 097.88±20.99）% in LD, MD and HD groups, respectively）. Similarly, the specific growth rate（SGR）, feed conversion ratio（FCR） and coefficient of variation for weight（CV_w） were adversely affected by high stocking density（P〈0.05）. However, there was no difference in survival and Fulton＇s condition factor（K） of turbot among the different groups. Physiological analyses demonstrated a clear increase in the plasma cortisol level and an obvious decrease in growth hormone（GH） concentration in the HD group on Day120（P〈0.05）. There was no significant effect of stocking density on plasma glucose, Cl– and protein levels. All these findings would provide a reference for selecting the optimal stocking density of juvenile turbot in RAS.
基金supported by the National Key Technologies R&D Program(2011BAD13B04)the earmarked fund for Modern Agro-industry Technology Research System(CARS-48)the National Natural Science Foundation of China(No.31240012)
文摘The effects of different feeds and feeding regimes on growth performance, flesh quality and fecal viscosity of Atlantic salmon （Salmo salar L.） in recirculating aquaculture systems （R.AS） were investigated. Fish （initial body weight of 1677 g＋ 157 g） were fed with four commercial feeds （Nosan salmon-NS, Aller gold-AG, Skretting salmon-SS and Hart ye-HY） in two feeding regimes （80% and 100% satiation） for 78 d. The results showed that salmon specific growth ratio （SGR） and weight gain ratio （WGR） were significantly affected by feed type and feeding regime （P〈0.05）. Feed conversion ratio （FCR） varied between 0.93 and 3.40, which was significantly affected by feed type （P〈0.05）, and slightly improved with increased satiation degree. The activities of digestive enzymes including protease, lipase and amylase were also significantly affected by feed type and feeding regime （P〈0.05）, increasing with satiation degree. Flesh qualities for vitamin E, hydroxyproline （HYP）, liquid loss and muscle pH among all groups showed significant differences （P〈0.05）, ranging from 26.67 to 29.67, while no obvious difference was found in flesh color. Fecal viscosity for different treatments showed no significant difference, though improvement was found in 100% satiation group. From present experiment, it was concluded that both feed type and feeding regime can affect the important quality attributes of Atlantic salmon.
基金supported by the National Natural Science Foundation of China(No.132072962)the Agricultural Industrial Technology System in Shandong Province(No.SDAIT-26)+3 种基金the‘First Class Fishery Discipline’Programme[(2020)3]in Shandong Provincethe Special Talent Programme‘Yishi Yiyi’in Shandong Province,Chinathe National Key R&D Program of China(No.2018YFD0901500)the Shandong Province Agriculture Seed Project(No.2021LZ GC004)。
文摘Long non-coding RNAs(lncRNAs)regulate a variety of biological processes,including sexual reproduction and differentiation.Saccharina japonica,a commercially important brown alga in China,shows remarkable sexual dimorphism in haploid gametophytes.The sex of Saccharina japonica gametophytes is determined by UV sexual system.However,no results have been reported on the lncRNAs involved in the sex-related gene regulation of S.japonica.This study identified a number of lncRNAs and assessed their expression levels in male and female gametophytes.Among them,a total of 405 lncRNAs and 211 mRNAs showed differential expressions.Furthermore,the functions of target genes of differentially expressed lncRNAs(DELs)differed from those of differentially expressed genes(DEGs),suggesting that lncRNA may interact with other functional proteins,in addition to DEGs,to involve sex regulation in S.japonica.There were 32 and 90 potential cis-regulatory and trans-regulatory interactions between DELsDEGs,respectively.Five of these lncRNAs(LNC_002974,LNC_021059,LNC_038466,LNC_051584,and LNC_027400)interacted with putative male sex determination region(SDR)genes,suggesting that they act as regulators in gametophytes'sex regulation potentially.Findings from this study contribute to our understanding of the roles of lncRNAs in sex differentiation and lay the foundation for functional studies of candidate lncRNAs in the future.
基金The Key Laboratory of Mariculture of Chinese Ministry ofEducation, Ocean University of China (200405)Provincial Departmentof Science and Technology of Shandong (2005GG3205190).
文摘The variation of 90 Laminaria gametophyte clones representing the introduced Laminaria japonica （Group 1） and Laminaria Iongissima （Group 2）, the varieties of L. japonica （Group 3） and the varieties derived from interspecific hybrids （Group 4） was determined with 18 microsatellite markers. The allelic diversity and Nei＇s gene diversity of Group 1 were significantly higher than those of Group 2 （2.9 vs. 1.8 and 0.414 vs. 0.161, respectively）, demonstrating that the variation of the introduced L. japonica is richer than that of L. Iongissima. Both allelic diversity and Nei＇s gene diversity of Group 3 were lower than those of Group 1, indicating that only a portion of variation of L. japonica was incorporated into the varieties of L. japonica. Significant genetic differentiation was detected between four groups and between female （Population 1 ） and male （Population 2） gametophyte clones in each group. The variation among groups accounted for 39.95%, while that among populations accounted for 21.65% of the total. The genetic distance between Group 1 and Group 4 was obviously longer than that between Group 2 and Group 4 （0.686 vs. 0.291）, indicating that maternal gametophyte clone contributed more variation to the hybrids than the paternal gametophyte clone did.
