Development of Microsatellite Markers for Vanda Orchid

Abstract

The aim of this research was to develop microsatellite (SSR) markers for the Vanda orchid from the enriched library using dinucleotide repeats [(CA)₁₅ and (GA)₁₅] and trinucleotide repeats [(ACC)₁₀ and (CCT)₁₀] as probes. Positive clones were selected using dot blot hybridization. The results showed that 82.45% of dinucleotide-enriched libraries but only 9.91% of trinucleotide-enriched libraries gave positive signals. After sequencing, 83.12% of the positive clones contained microsatellite repeats. The four most frequently found sequences were the compound repeats of (GA)_n(GT)_n (45.19%), (GA)_n (22.59%), (CA)_n (15.93%) and (CCT)_n (9.26%). Fifty-six pairs of primers were designed and nine primer pairs could amplify the DNA giving the expected PCR product with polymorphism. There was a range from 3 to 9 alleles per locus and the expected heterozygosity (H_e) range was 0.3150-0.7438. Based on the nine loci of these microsatellite markers, the probability of identity (PI) of any two Vanda and related orchid cultivars having the same genotype was approximately 1 in 1,000,000. Therefore, these markers could be used for identification of the Vanda orchid samples studied. After analyzing the genetic relationships of 33 Vanda and related orchid cultivars using NTSYS-pc 2.1m, the result indicated that the Vanda and related orchid cultivars could be divided into four groups. The first three groups were strap-leaved Vanda, while the fourth one was terete Vanda, which was clearly clustered separately from the other Vanda groups. This study showed the isolation efficiency of the enrichment procedure, the abundance of microsatellites in Vanda orchids and their potential use for the individual identification of Vanda and related orchid samples.