Internal transcribed spacer-based CAPS marker development for Lilium hansonii identification from wild Lilium native to Korea

Abstract

We have developed a cleaved amplified polymorphic sequence (CAPS) marker with the aim of identifying the best species among the different wild Lilium species native to Korea for breeding purpose. The CAPS marker is based on single nucleotide polymorphisms (SNPs) in the nucleotide sequences of the internal transcribed spacer genes, which are widely used in the breeding of angiosperms. We cloned and sequenced the ITS regions of nuclear ribosomal genes from 11 Lilium species and deduced the phylogenetic relationships among them. Phylogenetic analysis based on the maximum likelihood method showed that Lilium species formed a monophyletic clade and were divided into two groups that linked to the two sections, Martagon and Sinomartagon, out of seven sections of Lilium. The lengths of the ITS1 and ITS2 regions in the wild native Lilium species were distinct, and nucleotide polymorphisms were distinguished in these regions. A conserved region in the sequence was found from position 330？340 in all the species. We analyzed the SNP sites and found a suitable restriction endonuclease, Eco52I, having six base pair recognition site, to be suitable for the development of the CAPS marker. Among the 11 Lilium species, only the amplicon from the ITS region of L. hansonii contained the restriction site and produced two distinct bands of around 623 and 268 bp upon digestion with Eco52I. The genotyping of the 11 wild Lilium species by the developed marker can be recommended for breeding programs as it provides an indirect selection of plants and can effectively differentiate wild native lilies. The method using CAPS marker is simple, quick, and highly reliable for identifying the best species for efficient breeding of Lilium