Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis （Bory） is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplified fragment length polymorphism （AFLP） technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplification primers were used in this study. The primer combination E-TG/M-CCA detected a specific band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplified region （SCAR） primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identification of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.     

裙带菜配子体总RNA提取的比较研究

以裙带菜(Undaria pinnatifida)配子体为材料对比分析了4种方法,改进SDS法、CTAB法,Trizol试剂盒、RNAiso Reagent试剂盒法提取RNA的质量和纯度,并采用RT-PCR对mRNA反转录,对mRNA的可用性进行了检测.结果表明,两种试剂盒Trizol和RNAiso Reagent及其...     

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

Eighteen gametophytes includingL. japonica, L. ochotensis andL. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains ofLaminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species ofLaminaria. Contribution No. 4389 from the Institute of Oceanology, Chinese Academy of Sciences. This research was supported by the State's High-tech Project of China, Marine 863 project (No. 819-03-02) and (2001AA621090).     

三十烷醇对海带三个品系雌配子体克隆生长和发育的影响

以海带雌配子休克隆为实验材料,通过施用三十烷醇处理。结果表明,三个品系在供试条件下,LH36生长最好,LH30发育率最高。它们对TA的反应为浓度在0．5～1．0×10-5mg／L时,都有促生长作用,其中LH30反应最敏感,LH10次之。浓度在1．0×10-5mg／L时,发育率最高,其中LH30发育率为51．5％,LH10为18．0％。LH36对各浓度处理的发育反应,差异都不明显。     

Identification of a putative tetrasporophyte-specific gene in <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis> (Gracilariales,Rhodophyte)

A putative tetrasporophyte-specific gene, designated as SSH466 (GenBank accession No. DQ019223), was one of the genes identified in this work using suppression subtractive hybridization (SSH) method in Gracilaria lemaneiformis. The full length of the gene was obtained using SMART RACE strategy. Sequence analysis revealed that the gene had 1 019 nucleotides, including an open reading frame of 498 nucleotides encoding 166 amino acid residues, 158 nucleotides of 5′ untranslated region and 363 nucleotides of 3′ non-coding region. Protein motif and secondary structure prediction showed that there existed a transmembrane domain with a unique β-sheet. Thus, SSH466 protein might be a cross-membrane protein. Sequence homology search in the public GenBank databases did not reveal any significant match with SSH466. Virtual Northern blot analysis confirmed that it was a tetrasporophyte-specific gene.     

Cryopreservation of Gametophytes of Laminaria japonica （Phaeophyta） Using Encapsulation-Dehydration with Two-Step Cooling Method

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 molL-1 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‰ NaC1 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.     

龙须菜配子体发育和不同品系四分孢子体四分孢子形成过程显微结构观察

龙须菜配子体和四分孢子体在生殖结构的形成和发育的过程中,出现一系列的细胞形态和结构层面的变化。本实验以龙须菜雌雄配子体和不同品系龙须菜四分孢子体为实验材料,采用连续徒手切片的方法,观察龙须菜配子体发育和不同品系四分孢子体四分孢子形成过程。观察到,雌雄配子体发育经过幼苗期、毛细胞期、性成熟期,幼苗期和毛细胞期雌雄配子体间无明显差异,进入性成熟期后,产生生殖结构时才能将其区分开。四分孢子起源于四分孢子体表皮层细胞,初始为鲜红色直径约5~10 μm的细胞,逐渐生长形成成熟的直径约20~25 μm的四分孢子囊,并分裂为4个四分孢子聚集在四分孢子囊中,在藻体外,四分孢子释放变形成球形,龙须菜四分孢子的形成和发育过程伴随着髓部细胞中红藻淀粉颗粒的减少。其中"981"龙须菜形成较多的畸形四分孢子,可能是其表现低育的一个原因。龙须菜自基部到尖端的表皮层细胞层数、髓部细胞数目和体积均发生连续的变化。细胞连接广泛存在于龙须菜表皮层、皮层和髓部细胞之间,但各层细胞连接大小、数目、长度等都存在差异。释放的四分孢子可能会附着在四分孢子体上,固着器覆盖包绕四分孢子体甚至侵入到破损有伤口的四分孢子体内,形成四分孢子体和配子体在同一株上的世代混杂现象,这也可能是导致龙须菜表现遗传复杂性的原因。     

Study on the breeding technology of heterosis seedling of Laminaria and new combinations

After continuous breeding gametophyte of Laminaria, then breaking protonema, heterosis seedling is got by bilineal hybridization. The result shows that the weight-increasing rate of female and male gametophyte clone cells are 14.03% and 13.87%, after 16 d crossbreeding, the rate of ovulation is up to100%; the length of juvenile sporophyte after 40 d breeding is 2-3 cm. By fanning on the sea, a hybrid combination is primarily screened.     

海带雌性配子体克隆细胞的超低温保存实验

生物种质资源保存的超低温冷冻保存技术在国内外已进行了广泛的研究[1],在藻类种质保存工作方面,绿藻、硅藻、蓝藻[2~6]以及紫菜[7~9]的超低温保存工作也取得了良好的进展.海带配子体(克隆)是一种以有丝分裂方式进行细胞增殖的微小藻体,并可通过两性生殖或单性生殖发育成大型叶状体.