为给梨品种S基因芯片的制备及梨品种自交不亲和性S-RNase基因和S基因型的检测奠定基础,结合已有的对梨自交不亲和S-RNase基因的研究成果及Genbank中梨S-RNase基因信息,利用生物学比对软件Genedoce对梨S-RNase等位基因序列进行比对,得到等位基因HV区的特异序列;用oligo6.71、Premier Premier 5.0和Clustal.W等生物学软件设计特异性高、解链温度Tm值接近、长度均一的oligo探针,获得了24条32 mer的oligo探针。
Based on the c DNA sequences from hyper variable(HV) regions of identified 52 S-alleles in Oriental pear cultivars, S-RNase c DNA probes were designed, and a c DNA microarray for S-RNase detections was established. Each microarray contained 240 sites from 55 c DNA probes, including all specific c DNA sequences from the HV regions of the S-alleles. Using the c DNA of pistils of tested pear cultivars as template and Cy3 fluorescently labeling primers by PCR amplification, microarray hybridization detected the S-genotype of each pear cultivar. The genotypes inferred from the c DNA microarray hybridization signals of pear cultivars such as ‘Lijiang Huangsuanli', ‘Xiuyu', ‘Midu Yuli', ‘Baimianli', and ‘Deshengxiang' were similar to the known genotypes of all tested cultivars. The S-RNase c DNA microarrays and the oligonucleotide gene chips were then used to conduct parallel testing of 24 P. pyrifolia cultivars with unknown S-genotypes. In conclusion, the construction of c DNA microarrays has further improved the pear S-RNase detection platform.
