一个可区分银鲫F系和A+系BAC序列的特征、染色体定位和SCAR标记筛选

摘要

为了揭示F系和A+系的遗传差异以及解析F系的遗传特征,研究基于前期基因组Survey分析发现一个特异于银鲫F系大小为782 bp片段的基础,首先采用混合池筛选方法从银鲫F系BAC文库中筛选出一个包含该特异片段的BAC克隆(F782-BAC).鸟枪法测序结果表明,F782-BAC序列全长大小为172625 bp,富含转座子序列,包含1个具备完整结构的Tc l-like转座子,以及不具备完整结构的transposase-like、PIF/Harbinger、target-primed reversed transcription(TPRT)和transpon X-element等6个转座子.用地高辛标记F782-BAC质粒得到的DNA探针进行了银鲫F和A+系有丝分裂中期相的染色体荧光原位杂交,结果表明不论是在F系还是A+系的有丝分裂中期相中,3个阳性荧光信号皆清晰地定位于3条大小相同的近端着丝粒染色体短臂的近着丝粒位置.进一步开展了银鲫A+系相应同源的F782-BAC序列的差异鉴定和序列分析,揭示A+系缺失了大小为3395 bp的一段序列,且特异于银鲫F系大小为782 bp的片段正好位于其中,并由此筛选出可作为区分银鲫F系和A+系的SCAR标记.研究为银鲫的多倍化起源提供了进一步的染色体定位证据,鉴定的SCAR标记可为银鲫分子标记辅助育种提供新的遗传标记.%Gibel carp (Carassius gibelio Bloch) has been widely used for basic and applied studies on developmental genetics and genetic engineering because of the polyploid background and multiple reproduction modes.In this study,we isolated a positive BAC clone with the fragment (F782-BAC) from a BAC library of F strain based on a 782 bp fragment (F782) specific to F strain revealed by comparative genome surveys between F and A+ strains.Shotgun sequencing revealed the complete sequence of the F782-BAC that is composed of 172625 bp containing a complete Tc1-like transposon and six incomplete transposons such as transposase-like transposon,PIF/Harbinger transposon,targetprimed reversed transcription (TPRT) transposon,and transpon X-element transposon.Using the DIG-labeled F782-BAC sequence as a probe,three positive green fluorescence signals were clearly detected on short arms of three homologous acrocentric chromosomes in mitotic metaphases of both F and A+ strains.Moreover,differential screening and sequence analysis of the homologus F782-BAC sequence were performed on the A+ strain,and a deleted fragment of 3395 bp was identified in the corresponding F782-BAC sequence of A+ strain,in which includes the F strain-specific fragment F782.Furthermore,we developed valuable SCAR markers for discriminating strain F from strain A+ in gibel carp.Therefore,the current data provide genetic evidence for polyploidy origin in gibel carp and the identified SCAR markers for the marker-assisted selective breeding in gibel carp.