关键词: 柑橘, 表达序列标签, 单核苷酸多态性, 酶切扩增多态性序列, 等位基因特异PCR

Abstract: The objective of this study was to develop CAPS markers and AS-PCR primers that can be used as molecular genetic markers in cultivar identification and genetic diversity studies. With the QualitySNP software package，1 521 putative single nucleotide polymorphism（SNP）sites were identified among the 93 955 sweet orange [Citrus sinensis（L.）Osbeck] expressed sequence tags（ESTs）downloaded from the GenBank. Furthermore，those ESTs containing the putative SNP sites were analysed with WatCut program and 125 putative Cleaved Amplified Polymorphic Sequences（CAPS）sites were obtained. Forty ESTs with CAPS sites were randomly chosen to design primer pairs，with which PCR amplifications were performed and then the amplification products were digested with restriction enzymes. Meanwhile，25 sets of AS-PCR primers were designed according to other 25 putative SNP that change the deduced amino acid type. Afterwards，6 citrus cultivars with different genotypes were used for the validation of each SNP site. As a result，26 CAPS sites with cleaved amplified polymorphism among 12 citrus cultivars were identified，all of them can be used as CAPS markers. Furthermore，fifteen sets of Allele-specific PCR primers with good reproducibility and polymorphism were obtained，which can be applied for further SNP genotyping in citrus. This study indicated that it is feasible to develop CAPS markers and AS-PCR primers based on public citrus EST sequences.

Key words: citrus, expressed sequence tags（EST）, single nucleotide polymorphism（SNP）, cleaved amplified polymorphic sequences（CAPS）, allele specific PCR