海带配子体克隆的扩增培养及多样性分析
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摘要
海带是我国重要的大型经济藻类,随着海带配子体无性繁殖系的建立,目前,海带配子体已经在种质保存,遗传育种和育苗方面发挥着重大作用,并且也是对海带进行遗传变异分析的重要材料。
本实验在对海带配子体进行扩增培养的过程中,研究了在静置培养条件下,营养盐添加量,光照强度,光照时间以及温度参数对配子体克隆扩增培养的影响,并且以长海带和海带的配子体克隆为材料,对两个群体进行了RAPD分析。主要结论如下:
静置培养的瞬时生长率明显低于充气悬浮培养,不能满足生产性大规模培养的需要,但可满足实验室工作的需要,在起始培养密度较低的条件下,N6.0ppm,P1.0ppm,每周更换培养液一次,可满足其生长需要。
静置培养条件下,温度对海带配子体克隆的生长有明显影响,10-15℃为最适生长温度,25℃条件下出现致死。
4500lx光强条件下海带配子体生长最快,但是出现细胞色素淡化现象,说明配子体细胞在此光强下受到损伤,因此在实际工作中不宜采用此光强,还应以2500lx光强进行培养,培养光时以24h/d为宜。
通过筛选,共获得19条随机引物,扩增得到252个位点,其中多态性位点228个,多态百分率为90.5%,其中有的位点在两个群体中的出现频率差异极大。根据遗传距离构建系统树发现,不但不同种的海带在系统树上能够得到很好的区分,而且也能够很好的区分同种海带的雌、雄配子体,表明RAPD标记应用于海带配子体细胞系的鉴定是有效的。
两群体的基因多样性水平较高。两群体间存在较为明显的遗传分化。不同海带的雌、雄配子体间均存在明显的遗传分化,长海带群体不同性别的配子体间的分化程度高于海带群体不同性别配子体间的分化程度,对于同种海带而言,雌配子体的基因多样性水平明显比雄配子体的基因多样性水平高。
Laminaria is a group of macroalgae, which are very important. As the development of the cloning technique of Laminaria gametophytes, gametophyte clones can be widely used in germplasm preservation, genetic breeding and seedling raising and in genetic variation analysis.
In this study, the effects of the amount of nutrient salts, illumination intensity and time and temperature on the vegetative amplification of Laminaria gametophytes were determined. In addition, the genetic diversity of the gametophyte clones isolated from L. japonica and L. lonngissima was analyzed with RAPD markers and compared each other.
The major findings were listed as the following:
Instantaneous growth rate of Laminaria gametophyte clones at static cultivation was obviously lower than that at suspension cultivation. Therefore, static cultivation was not appropriate for the large scale vegetative amplification of Laminaria gametophyte clones. However, at a low initial density, the medium containing 6.0ppm N and 1.0ppm P and the replacing frequency of one time each week, static cultivation met the need of laboratory use.
At static cultivation, temperature affected the vegetative amplification of Laminaria gametophyte clones obviously. The optimum temperature for the amplification of Laminaria gametophyte clones ranged from 10 to 15℃. It was found that 25℃was lethal to Laminaria gametophyte clones.
Laminaria gametophyte clones showed the highest growth rate under an illumination intensity of 4500lx. However, the decrease of cytochrome was observed at such intensity, indicating that Laminaria gametophyte clones were not tolerant to high illumination intensity. It was suggested that the illumination intensity of 2500lx should be used in large scale amplification of Laminaria gametophyte clones, the optimal illumination time is 24h/d.
Nineteen random primers were obtained after primary selection. Among 252 loci, 228 were polymorphic. The ratio of polymorphic loci was 90.5%. Some loci showed obvious differentiation between clones with different species origins and genders. The phylogenetic tree constructed based on the genetic distances depicted that Laminaria gametophyte clones isolated from different species and those with different genders can be easily discriminated. RAPD marker was proved to be effective for determining the genetic diversity of Laminaria gametophyte clones.
Nei’s gene diversity of L .longissima gametophyte clones and Laminaria japonica gametophyte clones were relatively high. There was obvious genetic differentiation between the clones isolated from two species. The variation levels between male and female gametophyte clones within a species and among the whole collection of clones were different obviously. The differentiation between different genders of the gametophyte clones from L. longissima was obviously higher than that of L. japonica. For Laminaria of the same species, female gametophytes have significantly higher genetic diversity than their male analogs.
本实验在对海带配子体进行扩增培养的过程中,研究了在静置培养条件下,营养盐添加量,光照强度,光照时间以及温度参数对配子体克隆扩增培养的影响,并且以长海带和海带的配子体克隆为材料,对两个群体进行了RAPD分析。主要结论如下:
静置培养的瞬时生长率明显低于充气悬浮培养,不能满足生产性大规模培养的需要,但可满足实验室工作的需要,在起始培养密度较低的条件下,N6.0ppm,P1.0ppm,每周更换培养液一次,可满足其生长需要。
静置培养条件下,温度对海带配子体克隆的生长有明显影响,10-15℃为最适生长温度,25℃条件下出现致死。
4500lx光强条件下海带配子体生长最快,但是出现细胞色素淡化现象,说明配子体细胞在此光强下受到损伤,因此在实际工作中不宜采用此光强,还应以2500lx光强进行培养,培养光时以24h/d为宜。
通过筛选,共获得19条随机引物,扩增得到252个位点,其中多态性位点228个,多态百分率为90.5%,其中有的位点在两个群体中的出现频率差异极大。根据遗传距离构建系统树发现,不但不同种的海带在系统树上能够得到很好的区分,而且也能够很好的区分同种海带的雌、雄配子体,表明RAPD标记应用于海带配子体细胞系的鉴定是有效的。
两群体的基因多样性水平较高。两群体间存在较为明显的遗传分化。不同海带的雌、雄配子体间均存在明显的遗传分化,长海带群体不同性别的配子体间的分化程度高于海带群体不同性别配子体间的分化程度,对于同种海带而言,雌配子体的基因多样性水平明显比雄配子体的基因多样性水平高。
Laminaria is a group of macroalgae, which are very important. As the development of the cloning technique of Laminaria gametophytes, gametophyte clones can be widely used in germplasm preservation, genetic breeding and seedling raising and in genetic variation analysis.
In this study, the effects of the amount of nutrient salts, illumination intensity and time and temperature on the vegetative amplification of Laminaria gametophytes were determined. In addition, the genetic diversity of the gametophyte clones isolated from L. japonica and L. lonngissima was analyzed with RAPD markers and compared each other.
The major findings were listed as the following:
Instantaneous growth rate of Laminaria gametophyte clones at static cultivation was obviously lower than that at suspension cultivation. Therefore, static cultivation was not appropriate for the large scale vegetative amplification of Laminaria gametophyte clones. However, at a low initial density, the medium containing 6.0ppm N and 1.0ppm P and the replacing frequency of one time each week, static cultivation met the need of laboratory use.
At static cultivation, temperature affected the vegetative amplification of Laminaria gametophyte clones obviously. The optimum temperature for the amplification of Laminaria gametophyte clones ranged from 10 to 15℃. It was found that 25℃was lethal to Laminaria gametophyte clones.
Laminaria gametophyte clones showed the highest growth rate under an illumination intensity of 4500lx. However, the decrease of cytochrome was observed at such intensity, indicating that Laminaria gametophyte clones were not tolerant to high illumination intensity. It was suggested that the illumination intensity of 2500lx should be used in large scale amplification of Laminaria gametophyte clones, the optimal illumination time is 24h/d.
Nineteen random primers were obtained after primary selection. Among 252 loci, 228 were polymorphic. The ratio of polymorphic loci was 90.5%. Some loci showed obvious differentiation between clones with different species origins and genders. The phylogenetic tree constructed based on the genetic distances depicted that Laminaria gametophyte clones isolated from different species and those with different genders can be easily discriminated. RAPD marker was proved to be effective for determining the genetic diversity of Laminaria gametophyte clones.
Nei’s gene diversity of L .longissima gametophyte clones and Laminaria japonica gametophyte clones were relatively high. There was obvious genetic differentiation between the clones isolated from two species. The variation levels between male and female gametophyte clones within a species and among the whole collection of clones were different obviously. The differentiation between different genders of the gametophyte clones from L. longissima was obviously higher than that of L. japonica. For Laminaria of the same species, female gametophytes have significantly higher genetic diversity than their male analogs.
引文
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