CAJ | 학술논문

通过磁珠富集的方法分离池蝶蚌（Hyriopsis Schlegelii）的微卫星分子标记。将池蝶蚌基因组 DNA酶切，连接Brown接头，采用PCR技术富集，与探针杂交后杂交复合物结合到包被有链霉亲和素的磁珠上，再将这些片段洗脱，PCR扩增，克隆构建含有微卫星片段的“基因组文库”，所得到的阳性克隆进行测序。从所获得的212个阳性克隆中选取110个进行测序，其中54．5％含有微卫星序列，80％的重复序列重复数在10以上，微卫星序列中21．4％为完美型。除探针中使用的 AC、AG等重复单元外，还观察到CT、GA、ATG等重复序列。设计获得60对微卫星引物，合成其中的30对经PCR筛选，结果有10对引物扩增出多态性条带。该实验表明：磁珠富集法是获得池蝶蚌微卫星分子标记的一种有效的方法，同时，制备出的微卫星标记可为今后研究池蝶蚌分子遗传多样性提供有用的遗传标记。
통과자주부집적방법분리지접방（Hyriopsis Schlegelii）적미위성분자표기。장지접방기인조 DNA매절，련접Brown접두，채용PCR기술부집，여탐침잡교후잡교복합물결합도포피유련매친화소적자주상，재장저사편단세탈，PCR확증，극륭구건함유미위성편단적“기인조문고”，소득도적양성극륭진행측서。종소획득적212개양성극륭중선취110개진행측서，기중54．5％함유미위성서렬，80％적중복서렬중복수재10이상，미위성서렬중21．4％위완미형。제탐침중사용적 AC、AG등중복단원외，환관찰도CT、GA、ATG등중복서렬。설계획득60대미위성인물，합성기중적30대경PCR사선，결과유10대인물확증출다태성조대。해실험표명：자주부집법시획득지접방미위성분자표기적일충유효적방법，동시，제비출적미위성표기가위금후연구지접방분자유전다양성제공유용적유전표기。
Microsatellites molecular markers of Hyriopsis Schlegelii were obtained by using magnetic beads enrichment.The genomic DNA was firstly cut by Sau3AI,Then,the purified fragment was linked to Brown adapter.These fragments were hybridized with a biotin-labeled SRS (simple repeat sequenced)probe (AC) 12.The hybrid mixture was incubated with magnetic beads coated with streptavidin.After washing to re-move the non-SSR fragments,the eluted single-stranded DNA contained the selected microsatellites DNA. The target DNA were amplified by using primers according to the linkers.The PCR product was cloned in-to the pMD19-T vector and transformed into DH5α.In this study,110 of 212 positive clones were se-quenced and 60 clones were identified as microsatellites sequences.In microsatellites sequences,the repeat-ed number of 80% sequences were above 10 repeats and 21.4% sequences were perfect.In addition,30 pairs of primers were designed according to microsatellites sequences and 10 pairs were used to amplify spe-cial fragments.These results showed that magnetic beads enrichment was an efficient method to acquire microsatellites.In a word,the microsatellites which were obtained can offer useful genetic markers for stud-ying the diversity of Hyriopsis Schlegelii in the future.