用磁珠富集法从AFLP片段中分离鹅微卫星DNA标记

Geese Microsatellite Enrichment from AFLP Fragments by Magnetic Beads

用磁珠富集法从AFLP片段中分离微卫星DNA标记,可以避免基因组文库的构建和筛选这一繁重的工作,既节省时间又节约费用.利用少量的内切酶组合建立一系列AFLP DNA,然后用生物素标记的简单重复序列(SSR)作探针与其杂交,杂交复合物固定到包被有链亲和素的磁珠上,经过一系列洗涤过程,含有SSR的AFLP片段被吸附到磁珠表面.这些片段经洗脱下来后,先用对应的AFLP引物扩增,再进行克隆和测序,根据SSR两端的保守序列设计引物,经过多态性分析后,便可得到微卫星DNA标记.用这种方法已成功地从6个中国地方品种鹅中分离到30个微卫星DNA标记.其中有11个引物表现为多态性好,特异性强且相互之间并不连锁.各品种平均多态信息含量(PIC)在0.513～0.546之间.说明这11个微卫星均为高度多态,可作为有效的遗传标记用于鹅品种间遗传多样性和系统发生关系的分析,并为建立鹅遗传图谱、重要经济性状的基因定位及标记辅助选择奠定基础.

Microsatellite enrichment from AFLP fragments by magnetic beads can obviate the tedious work of library construction which save not only time but also expense. The genomic DNA was converted into pre-amplified AFLP fragments by using a few restriction enzymes combination and hybridized with biotin-labeled SSR probes. Then the hybrid mixture was used to incubated with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-strand DNA which was cloned and sequenced was largely enriched for microsatellites. Primers can then be designed according to the sequence flanking the repeat motifs and used for polymorphism analysis. Thirty primers had been developed from six China indigenous geese breeds successfully. Eleven of them were highly polymorphic better specific and did not link each other. The mean Polymorplism Information Content （PIC） of six geese breeds was also all over 0.5. The highest was the Gushi goose （0.546）, and the lowest was the Yan goose （0.513）. The resuits showed that the 11 rnicrosatellites had higher PIC. genetic diversity, construction of saturated maps, and in These 11 microsatellites could be used in the analysis of some cases, in marker-assited selection.