| Gracilaria lemaneiformis is one of important agarophytic species of red alga and important cultivable species in the genus. G. lemaneiformis is also an ideal material for studies of genetics and molecular biology. The cDNA libraries of G. lemaneiformis have been constructed, and analyses of expressed sequence tags (EST) and inter-simple sequence repeat (TSSR) have also been carried out.In the gametophytic cDNA library construction, total RNA was extracted by IJNIQ-10 Trizol Total RNA Preparation Kit, and cDNAs were synthesized and amplified by cDNA Synthesis Kit and PCR cDNA Library Kit. Finally, the ligated cDNAs with pMD 18-T vector were transducted into E. coll JM109. The constructed cDNA library was used to the latter analysis of EST.In order to study the differential expressed genes between different phases and sexes of G. lemaneiformis, a cDNA library from its tetrasporophyte was constructed. RNeasy Plant Mini Kit was used to isolate total RNA. SMART cDNA Library Construction Kit was used to synthesis of cDNA and construction of cDNA library, while packing system was purchased from Promega corporation. Results showed that the library contained 1.28×106 independent clones, the titer of the amplified library was 1.98×10 9 pfu/ml and 85.0% clones were recombinants. Almost all the inserts were between 500 bp and 1500 bp.EST technique has proved to be a useful means of identifying novel genes, producing molecular markers and characterizing genes expressed in an organism. The cDNA library from gametophyte of G. lemaneiformis was used to generate 180 ESTs (151 non-redundant groups). Thirty groups were found matches to known nucleic acid sequences. The submitted 136-group ESTs were used to search for similarities at the databases of EST from red algae, and 22 groups were found similarities. Of the 73 clones homologous to known protein sequences, majority of them were related to energy metabolism, transcription/translation and photosynthesis. Those related to growth/development, signal transduction, stress/defense response were 6, 4 and 3 groups, respectively. Of the submitted ESTs, 58.1% could find homology either at nucleic acid level or at amino acid level, and 41.9% were novel sequences.ISSR technique has been widely applied to various fields, such as genetic diversity, phylogenesis and identification of variants. In this study, it was used to investigate the genetic variation of genus Gracilaria. Seven strains of algae werenalyzed, including 4 strains of G. lemaneifarmis, of which 3 came from Qingdao (China), Venezuela and South Africa, respectively, and the fourth was selected strain from Qingdao sample, and three agarophytic species of G. tennistipitata var. liui, G. vermiculophylla, G. blodgettii. A total of 151 bands were amplified with six primers, which could produce clear bands, and 117 (77.48%) revealed polymorphism. Analyses of data showed that G. lemaneiformi from Qingdao and its selected strain displayed the highest genetic similarity (S = 0.939) of all the samples. Among the three strains of G. lemaneiformis originated from different habitats, samples of Qingdao and South Africa had the nearest genetic distance (S = 0.643), while similarity coefficient of Venezuela sample with them was 0.167 and 0.192, respectively. So conclusion could be drawn that Venezuela sample shouldn't be ascribed to species of G. lemaneiformis. The similarity coefficients of G. lemaneiformis (from Qingdao or South Africa), G. tenuistipitata var. liui, G. vermiculophylla and G. blodgettii ranged from 0 to 0.143, much lower than common genetic similarities in other species.ISSR analysis was also used to study wild type and five mutants of G. lemaneiformis, including yellow mutant (yelOO) and four green mutants of grl 80, grl 84, grl 86 and grl 88. Results showed that there was almost no difference between the six strains except for intensity of individual bands.Finally, tetrasporophyte, female gametophyte and male gametophyte of G. lemaneiformis were analyzed with six ISSR primers. Of the two primers co...
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