Development of microsatellite markers for Dendrobium orchids

Authors

  • Thanita Boonsrangsom Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, 10900 Thailand
  • Pradit Pongtongkam Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, 10900 Thailand
  • Sumon Masuthon Department of Botany, Faculty of Science, Kasetsart University, Bangkok, 10900 Thailand
  • Surin Peyachoknagul Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, 10900 Thailand

Abstract

Microsatellite markers for Dendrobium orchids were developed using SSR enrichment procedure. Two genomic libraries were constructed from the DNA digested with either MseI or TaqI. They were screened for the presence of microsatellite sequences with 6 types of biotinylated oligonucleotide probes ((CA)15, (GA)15, (ACC)10, (CCT)10, (GAT)10 and (ATCT)7) for DNA digested with MseI and with 4 types of biotinylated oligonucleotide probes ((CA)15, (GA)15, (ACC)10 and (CCT)10)) for those digested with TaqI. The positive clones were reconfirmed by dot blot hybridization showing 26% and 90% of the respective two groups. The total of 195 positive clones were sequenced, of which 62.8% and 88.8% contained SSR motifs, respectively. Different types of repeat motif were found comprising of GA/TC (50.0%), CCT/AGG (22.8%), 15 types of compound repeat (20.4%), GGGTTTAn/TAAACCCn (5.6%), CA/TG (0.6%) and GAT/ATC (0.6%). Seventy-three clones were chosen for primer design. Eight primer pairs could be used to amplify the products giving the expected sizes and detect genetic polymorphism in the population with the allele numbers ranging from 4 to 7 (averaged 5.25 alleles per locus), observed heterozygosity (Ho) of 0.0612 to 1.0000 (averaged 0.7398), expected heterozygosity (He) of 0.0788 to 0.7323 (averaged 0.5871) and effective number of allele (ne) of 1.0855 to 3.7355 (averaged 2.7850). The genetic relationships of 49 Dendrobium hybrids were analyzed using NTSYS-pc version 2.1m program. The results showed that all Dendrobium samples could be identified but they were not clearly separated into distinct clusters. However, the abundance of microsatellite in Dendrobium orchids and the high information content of the developed markers will be useful for genetic diversity analysis and cultivar discrimination.

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Published

2012-05-25

Issue

Vol. 1 No. 1 (2008): Jan-Jun 2008

Section

Research Articles