条斑紫菜(Porphyra yezoensis)dbEST中筛选微卫星位点及引物种间转移扩增

采用电子查询的方法，借助Blast软件从条斑紫菜表达序列标签数据库的20979条序列中筛选微卫星位点，共发现非冗余微卫星位点211个，占整个条斑紫菜EST数据库的1．01％。其中双碱基重复微卫星位点共有35个，占查找微卫星序列总数的16．6％；三碱基微卫星有176个，占83．4％。双碱基重复微卫星中AG／CT形式最多，而三碱基中GGC／GCC最多。从筛选出的微卫星位点中选取序列设计合成引物，用在坛紫菜上进行引物种间转移扩增研究。在总共15对引物中有13对引物在两个种间都有扩增产物。结果表明，微卫星位点在这两种紫菜之间具有很高的同源性。获得的微卫星引物可用来进行种群遗传多样性研究、谱系鉴定和遗传图谱的构建。而微卫星标记的共显性可用来进行紫菜二倍体丝状体纯合性检验，为纯系紫菜品系的鉴定提供分子生物学鉴定方法。

ISOLATION OF MICROSATELLITE LOCI FROM dbEST OF ALGAE PORPHYRA YEZOENSIS AND PRIMER AMPLIFICATION OF INTERSPECIES TRANSFER

To establish the molecular basis for the pure line conchocelis tests of algae Porphyra, marking microsatellite of the species was performed. However, the biggest obstacle is to obtain the sequence information for designing primer on flanking regions. Comparing high cost for isolation microsatellite loci in genomic DNA, screening microsatellite loci from public database is very easy and less expensive. In recent years, with increasing databases of expressed sequence tags, microsatellite loci derived from EST have been reported for many species. Microsatellites derived from EST databases are equivalent to those from cDNA libraries, as an EST is a sequenced cDNA. Thus, microsatellite lengths, mixtures of repeat types, and polymorphic nature of microsatellites from cDNA and ESTs are identical. In this research, microsatellite loci were screened from EST database of Porphyra yezoensis by on-line software Blasting. Total 714 loci were isolated from 20979 sequences in the database and 211 loci (about 1.01% of the sequences in database) were un-redundant ones. Among these un-redundant microsatellite loci, 35 loci (16.6%) were di-nucleotide that repeated no less than 7 times. AG/TC motif was dominant. 176 loci (83.4%) had tri-nucleotide core unit with dominant GGC/GCC motif that repeated no less than 5 times. GGC/GCC motif also the most abundant, amount to 1/3 of the total loci, and AGC/GCT, AG/CT and TGC/GCA were less abundant loci. Besides, the microsatellite loci were screened by software, so the microsatellites in perfect form were the most common (168; 79.6%) ones, followed by the imperfect form (34; 16.1%) and compound form (9; 4.3%). These results were similar to the published papers regarding EST-SSRs. From those 211 loci, 15 sequences were selected for designing microsatellite primers on the flanking regions using software Primer Premier 5.0. The criteria of the design were as followed: primer length within (20±2)bp, GC content between 50% and 75%, melting temperature between 50°C and 65°C, expected product size between 100bp and 300bp and no secondary structure. Among these selected sequences, 14 contained tri-nucleotide repeats and 1 was bi-nucleotide repeat. With those 15 primer pairs the transferring amplifications of interspecies were carried on in 3 wild and cultivated populations of P. haitanensis Meanwhile, a wild population of P. yezoensis was checked as the positive control. The amplification results showed that 13 primers produced bands in both P. yezoensis and P. haitanensis, implying high conservation of these microsatellite primers in P. yezoensis and P. haitanensis. So, the microsatellite primers derived from P. yezoensis can be used in genetic study of other species in Porphyra. Besides, co-dominance and high polymorphism make the microsatellite marker very useful in DNA fingerprint mapping and identification of homozygote diploid.