棉属D基因组棉种着丝粒FISH标记的筛选初报

为了筛选着丝粒探针, 在棉花染色体荧光原位杂交中标记染色体着丝粒区域, 以便于构建棉花粗线期染色体细胞遗传学图谱. 在棉花遗传连锁图上选择尽可能接近着丝粒区的单拷贝的分子标记, 以海岛棉pima 90-53的BAC文库为素材, 采用两维筛库法从该文库中筛选BAC克隆, 然后用BAC-FISH技术进行染色体定位检测. 用10号染色体长臂末端的SSR引物BNL3563筛选到一个BAC克隆150D24, 该克隆在四倍体陆地棉和海岛棉部分染色体着丝粒区有杂交信号, 但信号强度不高. 以二倍体D基因组为靶DNA进行FISH时, 染色体着丝粒区有明显信号, 但是以二倍体A, C, E等基因组棉种染色体为靶DNA进行FISH时, 染色体着丝粒区未发现杂交信号. BAC克隆150D24可能含有D组特有卫星重复序列, 可用作棉属D基因组棉种(包括四倍体棉种D亚组)的着丝粒FISH探针.

Screen of FISH marker of chromosomes at Gossypium D genome species

Appropriate centromere probes for fluorescence in situ hybridization (FISH) of cotton were screened and high density cytogenetic maps based on pachytene-FISH in D genome cotton were developed. A BAC library of Gossypium barbadense var. pima 90-53 was screened using two-dimensional PCR with molecular markers close to the centromere region and 12 BAC clones were identified by BAC-FISH. BAC clone 150D24 screened with SSR marker BNL3563, located at the end of long arm of Chromosome 10 was identified. Signals for BAC clone 150D24 were located in centromere regions of some shorter chromosomes in tetraploid cotton G. hirsutum (AD)1 and G. barbadense (AD)2. FISH conducted on diploid cotton demonstrated that there were clear signals in the centromere region of D genome diploid cotton species when BAC clone 150D24 was used as a probe. No signals were evident in A, C or E genome diploid cotton. This suggests that 150D24 contains repeat sequences, which are specific to D genome species centromere region; therefore, 150D24 can be used as a specific FISH marker for centromere regions in D genome diploid cotton, including the D sub-genome.