Assessment of Genetic Diversities of Selected Laminaria （Laminariales, Phaeophyta） Gametophytes by Inter-Simple Sequence Repeat Analysis

Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10 pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10 selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility.of applying ISSR markers to evaluate Laminaria germplasm diversities.     

白沙蒿不同地理种群遗传分化的ISSR分析

运用ISSR技术,对白沙蒿的5个种群进行遗传分化的分析。12个引物在108个个体中共扩增出222个位点,其中,多态位点为218个,多态位点百分率为98.20%,Shannon多样性指数为0.315 4,具有高的多态性。种群间的遗传分化系数（G_st）为0.076 7,与AMOVA分析的结果一致（Ф_st为7.96%）,表明绝大多数的遗传变异存在于种群内部。各种群间遗传一致度高达98%以上,遗传距离很小,基因流达3.008 2,均表现出白沙蒿各种群存在着广泛的基因交流,有着很小的遗传分化。     

ISSR Analysis of the Genetic Diversity of the Endangered Species Sinopodophyllum hexandrum （Royle） Ying from Western Sichuan Province, China

Slnopodophyllum hexandrum （Royle） Ying Is an Important medicinal and endangered species. Inter-simple sequence repeats （ISSR） analysis was conducted on seven natural populations from western Slchuan Province to Investigate the genetic diversity of S. hexandrum. Leaf samples of 140 Individuals were collected. Of the 139 discernible fragments generated by 12 selected primers （among 100 primers）, 54 appeared to be polymorphlc. The percentage of polymorphlc bands （PPB） was 38.85% at the species level, and PPB within a population ranged from 7.91% to 23.74%. Low levels of genetic variation （He = 0.092, Ho = 0.142） and high levels of genetic differentiation among the populations （Gst= 62.25%） was detected on the basis of results from POPGENE and analysis of molecular variance （AMOVA）, respectively. Furthermore, the limited gene flow （Nm = 0.361） may result from biological characteristics, such as self-pollination and short distance seed dispersal. Based on the genetic and ecological Information available for S. hexandrum, we propose some appropriate strategies for the conservation of the endangered medicinal species in this region, namely rescuing and conserving the core populations for in situ conservation and sampling and preserving more populations with fewer Individuals from each population for ex situ conservation.     

甘肃省冬虫夏草遗传多样性的ISSR分析

对采自甘肃省3个冬虫夏草主产区域6个具有代表性地方的18个样本进行ISSR分析。15条ISSR引物共扩增得到条带清晰并呈多态性的95条谱带,每一引物扩增获得的ISSR条带数在3－9条之间,扩增片段集中在300－3,000bp。基于遗传相似性系数（GS）、不加权成对群算术平均法（UPGMA）构建的系统树分析,可以看出：分离自同一株虫草上不同部位的样本无显著遗传差异;同一地点样本间的遗传分化较小;不同地域的样本间存在着较大的遗传分化,遗传多样性较丰富。同时6个地方的冬虫夏草明显地分为3个区域,而在同一区域不同     

黑龙江省主栽大豆品种遗传多样性的SSR分析

使用合丰25等来自13个育种单位的13份大豆（Glycinemax）品种对大豆20个连锁群上的100对SSR标记进行筛选,最终保留了扩增稳定且多态性较高的43对SSR标记,分析了黑龙江省83个主栽大豆品种的遗传多样性。结果表明,在所有供试材料中共鉴定出等位变异157个,每个位点2—7个,平均为3．65个。品种间遗传相似系数为0．216—0．937,平均为0．6384,表明黑龙江省大豆品种的遗传相似性较大,故拓宽黑龙江省大豆品种的遗传基础具有重要意义。     

Genetic Diversity of Kenaf (Hibiscus cannabinus) Evaluated by Inter-Simple Sequence Repeat (ISSR)

Understanding genetic diversity is very useful for scientific utilization for breeding. In this study, we estimated the genetic distances in a panel of 84 kenaf accessions collected from 26 countries and regions using ISSR markers. The results of UPGMA analysis showed that kenaf germplasm had abundant genetic variation, with genetic dissimilarity coefficients ranging from 0.01 to 0.62. The in-group dissimilarity coefficient (0.29) was observed in 84 kenaf accessions, and all the accessions could be divided into three groups: cultivars (L_1–1), relatively wild species (L_1–2 and L_1–3), and wild species (the others). Further in-group analysis in group L_1–1 (0.19) revealed that the kenaf cultivars could be divided into five subgroups with distinct regional characteristics. It is imperative that genes be exchanged among all kinds of tested varieties from different origins. The results provide a useful basis for kenaf germplasm research and breeding.     

The life cycle of Laminaria abyssalis (Laminariales,Phaeophyta) in culture

Laminaria abyssalis occurs in deep water in tropical latitudes of the Brazilian coast (19° 23 S, 38° 28 W to 22° 54 S, 42° 13 09 W). Its life cycle has been completed in the laboratory in seven months using different conditions of light and temperature. The gametophytic stage required for growth the low photon flux density of 1.2 ± 0.3 µmol m^–2 s^–1 and 18 °C, while the juvenile and adult sporophytes needed 15 µmol m^–2 s^–1 and 18 °C. The sporophytes became fertile at 23 °C. Our results showed that light and temperature are the main factors regulating the growth and life history of this species under the culture conditions tested.     

Mariculture of Laminaria japonica (Laminariales, Phaeophyceae) using protoplast regeneration

The conditions for culture of viable protoplasts from Laminaria japonica were investigated and the regenerative processes were observed in detail. As a result of culturing at four water temperatures (5, 10, 15, and 18°C), we found that low water temperature was better for survival, division and rhizoidal formation of protoplast‐derived cells. Only epidermis‐derived protoplasts developed into normal sporophytes through a direct developmental process. Protoplast‐derived cells divided after 5 days and 2–10 celled germlings formed the first rhizoids after 15 days. Only initial sporophytes with the first rhizoids grew to normal sporophytes with multilayered blades, stipes and holdfasts. When these young sporophytes were transplanted into the sea, they grew to normal fertile sporophytes.     

Development of 18 polymorphic microsatellite DNA markers of Laminaria japonica (Phaeophyceae)

Eighteen polymorphic microsatellite DNA markers were developed for Laminaria japonica, a brown alga cultured intensively in China. These markers are independent from each other and are moderately variable in L. japonica. The number of alleles and gene diversity detected in 53 gametophyte clones representing the varieties of L. japonica cultured once or currently in China range from two to nine and from 0.355 to 0.768, respectively. These markers will certainly facilitate the management and exploitation of the germplasm resource of L. japonica conserved indoor as gametophyte clones and the determination of the genetic diversity of L. japonica naturally distributed.     

基于ISSR分子标记的枇杷材料亲缘关系分析

为了判断川早枇杷与其他枇杷材料间的亲缘关系,探讨ISSR分析鉴定枇杷变异的有效性。以川早枇杷等13份枇杷品种(品系)为材料,通过DNA提取、ISSR-PCR反应体系建立、引物筛选及PCR扩增,使用Quantity One软件对扩增条带进行统计分析,计算遗传相似系数,并进行UPGMA聚类分析。结果表明:用14条ISSR引物进行扩增,共获得165条带,条带长度为300～1 500 bp; 日本茂木与川早枇杷的遗传相似系数最高,为0.7920,早钟六号和晚钟与川早枇杷的遗传相似系数分别为0.7717和0.7444,当遗传相似系数为0.7160时,13份枇杷材料可以被分为4类。因此,相对于早钟六号,川早枇杷材料在遗传物质上发生了变异,具备成为枇杷新品种的条件。     

Growth and reproductive responses of Laminaria longicruris (Laminariales,Phaeophyta) to nutrient enrichment

A series of comparative culture experiments were conducted in order to determine responses of Laminaria longicruris male and female gametophytes and juvenile sporophytes to several temperatures (5, 10, 15, 20 °C), light levels (10, 35, 75 µmol m^–2 s^–1) and media nitrogen concentrations (0, 20, 100 µM ammonium-nitrogen). Responses were measured as numbers of male and female gametophytes producing gametangia and number of sporophytes produced following fertilization. Both male and female gametogenesis was reduced at 5 and 20 °C versus 10 and 15 °C. At 20 °C gametogenesis inhibition was greater with higher levels of ammonium-nitrogen concentration (100 µM). Sporophyte production was more sensitive to light, temperature and nitrogen concentration than gametogenesis. Production of sporophytes was inhibited completely at 20 °C. At lower temperatures, increasingly higher nutrient concentrations produced greater inhibition of production of sporophytes.     

A SCAR MOLECULAR MARKER SPECIFICALLY RELATED TO THE FEMALE GAMETOPHYTES OF SACCHARINA (LAMINARIA) JAPONICA (PHAEOPHYTA)1

PCR amplification was employed to identify female or male gametophyte associated markers in Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (=Laminaria japonica Aresch.). One pair of the primers, P5, was screened from five pairs designed based on a specific sequence (GenBank accession no. AB069714 ) of Marchantia polymorpha Y chromosome, resulting in a differential band ～500 bp in size between female and male gametophytes of Rongfu strain of S. japonica. According to the SCAR (sequence‐characterized amplified regions) strategies, one pair of primers, P51, was designed on the basis of the sequence of this band that was only present in female gametophytes. A SCAR marker, designated FRML‐494 (494‐bp Female‐Related Marker of S. japonica, GenBank accession no. EU931619 ), was developed successfully by PCR amplification using the designed P51 primer pair. The SCAR marker was verified to be present only in female gametophytes of another variety 901 of this kelp that was a hybrid between S. japonica as paternal and S. longissima (Miyabe) C. E. Lane, C. Mayes, Druehl et G. W. Saunders (=Laminaria longissima Miyabe) as maternal, suggesting that the FRML‐494 marker was specifically related to female gametophytes of the genus. This marker is the first molecular tool reported for sex identification in kelps. This study was beneficial for identifying gametophyte gender during vegetative growth and for judging whether the monogenetic sporophytes came from exclusive male or female gametophytes, as well as for further research on sex determination at the molecular level in kelps.     

Microsatellite DNA Variation of the Gametophyte Clones Isolated from Introduced Laminaria japonica （Phaeophyta） and L. Iongissima of China and Varieties Derived from them

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.     

Genetic variation among wild and cultivated populations of the Chinese medicinal plant Coptis chinensis (Ranunculaceae)

To examine if the cultivation process has reduced the genetic variation of modern cultivars of the traditional Chinese medicinal plant, Coptis chinensis, the levels and distribution of genetic variation was investigated using ISSR markers. A total of 214 C. chinensis individuals from seven wild and three cultivated populations were included in the study. Seven ISSR primers were used and a total of 91 DNA fragments were scored. The levels of genetic diversity in cultivated populations were similar as those in wild populations (mean PPL = 65.2% versus PPL = 52.4%, mean H = 0.159 versus H = 0.153 and mean I = 0.255 versus I = 0.237), suggesting that cultivation did not seriously influence genetic variation of present-day cultivated populations. Neighbour-joining cluster analysis showed that wild populations and cultivated populations were not separated into two groups. The coefficient of genetic differentiation between a cultivar and its wild progenitor was 0.066 (G(st)), which was in good accordance with the result by amova analysis (10.9% of total genetic variation resided on the two groups), indicating that cultivated populations were not genetically differentiated from wild progenitors. For the seven wild populations, a significant genetic differentiation among populations was found using amova analysis (45.9% of total genetic variation resided among populations). A number of causes, including genetic drift and inbreeding in the small and isolated wild populations, the relative limited gene flow between wild populations (N(m) = 0.590), and high gene flow between cultivars and their wild progenitors (N(m) = 7.116), might have led to the observed genetic profiles of C. chinensis.     

Temperature requirements for growth and maturation of the warm temperate kelp Eckloniopsis radicosa (Laminariales,Phaeophyta)

The annual kelp Eckloniopsis radicosa is distributed along Japanese coasts and occurs within the area with a February isotherm ranging 15–18°C and August isotherm ranging 25–28°C. In this study, the effects of temperature on the gametophyte growth and maturation, and the young sporophyte growth of E. radicosa were examined and the results are discussed in relation to the distribution of other warm‐adapted kelp species and the potential effects of climate change. The optimal temperature ranges for growth of male and female gametophytes were 23–27°C and 20–26°C, respectively. The upper survival temperature for gametophyte growth was 31°C for males and 30°C for females, respectively. The optimal temperature range for maturation of female gametophytes was ≤23°C. The optimal temperature range for growth of young sporophytes was 14–22°C. It was clarified that E. radicosa has the most warm‐adapted characteristics for growth and maturation of gametophytes among members of the Laminariales studied so far. The natural seawater temperature ranges during the growth and maturation seasons for gametophytes of E. radicosa, as well as the growth season for young sporophytes near to the northern and southern distribution limits (Izu‐Oshima: 14.9–24.5°C, Ichiki‐kushikino: 17.1–29.6°C), agreed with the experimentally determined temperature requirements. The warm‐adapted gametophyte stage and annual lifecycle are major factors enabling survival of E. radicosa in warm waters near tropical regions along the Japanese coast.     

Association of Genetic Structure and Diversity in Iranian Wild Germplasms of Mentha longifolia L. Based on Phenotypical,Biochemical, and Molecular Markers

Mentha longifolia L. is well-known to be one of the most pervasive wild-growing species of the Lamiaceae family, which has extensive beneficial properties in the fields of pharmacology and biological products. In the present study, the correlation between Inter-simple sequence repeat (ISSR) markers and morpho-chemical parameters of twenty different M. longifolia accessions (MLACs) were assessed. The geographic information system (GIS) has been employed to interpret the original habitat of the accessions in Iran. ISSR analysis indicated a remarkable difference in the studied accessions, segregated them into three main groups, constructed by an unweighted pair-group method with arithmetic (UPGMA) and principal coordinate analysis (PCoA). A total of 89 bands were generated by 12 ISSR primers, among which 82 (91.97 %) of them were polymorphic. The cluster analysis based on agro-morphological data scattered MLACs into two main groups. The essential oils (EOs) were analyzed through GC/FID/MS, and four chemotypes were characterized according to the major constituents. Pulegone ranged from 0.17 to 69.50 % was the main oil constituent with the highest content. Also, HPLC-PDA was employed to identify and to quantify the phenolic compounds in the MeOH extracts of MLACs. Heatmap cluster based on phenolic compounds produced three main categories of accessions. The components identified in the extracts were rosmarinic acid, rutin, vanillic acid, ferulic acid, chlorogenic acid, caffeic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and p-coumaric acid, which among them rosmarinic acid (RA) varied from 39.16 to 261.55 mg/100 g (DW) as a predominant constituent. Subsequently, multiple regression analyses between ISSR fragments and morpho-chemical data illustrated considerable relationships in the plant materials. The high variation and correlation observed in metabolic and phenotypic traits of MLACs establish an adequate source to conduct reserves conservation programs.