Culture Of Brown Macroalgae Gametophytes And Analysis Of Laminaria Self-breeding Lines By AFLP

Brown macroalgae such as Undaria pinnatifida and Laminaria japonica gametophytes are not only the material sources of seaweed aquiculture,breeding and seedling, but also the enter-piont for seaweed genetics research.So how to abtain the gametophytes which can satisfy different demands becomes the focus. Effects of daylength, irradiance and temprature on the vegetative growth, maturation and sporophyte production of U. pinnatifida gametophytes were examined using a factorial experimental design in culture. The results suggest that many entironmental factors regulate the growth of gametophytes. It increases with the increasing of daylength, irradiance and temprature. But different factors have different results, they are diversity .The optimal conditions for gametophytes culture in our lab are 22±1℃, 1500lx and 14:10h(L:D)daylength. At the same time, the maximum fertility occurs at 18±1℃,14:10h(L:D)and 2000lx, respectively.During the culture of gametophytes, diatom pollution is a knotty problem. They block the growth of the gametophytes, even can induce the death .They can live in large-scale temperatures, the ability of adaption is very strong and reproduce too fast. Gametophytes are always surrounded by them and a majority of nutrition and light are contested by them.So they restrict the natural growth of gametophytes , and even induce the death. The study screening gametophytes'endurance concentration range of germanium dioxide (GeO2),and comparing the growth changes of diatom. It is determined that a concentration of GeO2 which is the restrainal preparation of gametophytes between 1 to 5mg per litre in seawater is feasible and 1mg/L was the best. At this time the gametophytes can have a natural nutrition growth and the pollution of diatom can fall to the lowest level.Basing on the gametophytes culture and hybridize breeding technics, we cultivate the major aquacultural Laminaria lines in china. In the study of Laminaria genetic breeding, only according to the character identify of samples is not enough, it is necessary for us to use biochemistry and molecular marker methods to analyse genetic distance of them. We applied the amplified fragment length polymorphism (AFLP) markers to contrast the genetic identity and relationship of 100 self-breeding lines, in parallel with wild self-breeding lines and cultivar mix-breeding lines as a control.A total of 434 bands are detected in ten lines using ten selective primer pairs and the number of polymorphic bands are 141.The polymorphic ratio distribution is from 20% to 40% .The average amplified bands, polymorphic bands and polymorphic ratio is 43.4,14.4 and 32.5%,respectively. Results from electrophoresis patterns of the amplified fragment length polymorphism (AFLP) products support that YANZA, YANTAI901 and QINGDAO lines have the highest polymorphic ratio, their diversity bands are obvious than other lines. These conclusions can offer new ideas for Laminaria breeding and breed improvement.In this paper, modified SDS method, CTAB method, and Trizol and RNAiso Reagent kits were applied to extract the total RNA from Undaria pinnatifida gametophyte. We compared the quality and purity of obtained RNAs and then checked validities of the RNAs by RT-PCR for future using in the experiments. The results showed that the Trizol and RNAiso Reagent kits were not suitable to seaweed RNA isolation because there is no clear bend be detected on the gel. The RNA isolated by the CTAB method showed smear bands and also was contaminated by genomic DNA. However, the modified SDS method could give birth to good quality RNAs although there was a little genomic DNA contamination.The OD260/OD280 ratio of the RNA was between 1.8-2.0. These RNAs could be used for RT-PCR. So it is a very good method for extracting total RNAs from gametophyte of Undaria pinnatifida and some other seaweeds.