Thesis for Degree of Doctor of Philosophy

Genetic Marker Development by Constructing DNA and cDNA Library Systems and Phylogenic Analysis on Korean Orchid, Cymbidium

DNA cDNA library

The Graduate School of Kongju National University Department of Plant Resources, Crop Science and Biotechnology

MOE KYAW THU

2012.05

Thesis for Degree of Doctor of Philosophy

Genetic Marker Development by Constructing DNA and cDNA Library Systems and Phylogenic Analysis on Korean Orchid, Cymbidium

DNA cDNA library

Professor Yong-Jin Park

This paper is submitted for the requirement of Ph.D degree

2012.05

The Graduate School of Kongju National University Plant Resources Department, Crop Science and Biotechnology MOE KYAW THU

Fulfillment of Requirement for the Degree of Doctor of Philosophy, Department of Plant Resources,

2012.05

Chairman Examiner Examiner Examiner Examiner

Kim Seoung Mim ( Koo, Han Mo Park, Ha Seung Shin, Hak Ki Park, Yong Jin ( ( ( (

) ) ) ) )

The Graduate School of Kongju National University

ABSTRACT Genetic Marker Development by Constructing DNA and cDNA Library Systems and Phylogenic Analysis on Korean Orchid, Cymbidium
Moe Kyaw Thu

Department of Plant Resources Graduate School of Kongju National University, Yesan, Korea
(Supervised by Professor Yong-Jin Park)

The Oriental genus Cymbidium contains some of the most important and popular species of ornamental orchids in Korea. The present study reports the
development of 14 novel simple sequence repeat (SSR) markers through the construction of an enriched library, and 52 EST-SSRs through cDNA library of C. goeringii. Although more motifs (403) were identified and more primers (206) were designed in the genomic library compared to the cDNA library, a larger number of successful primers were obtained from the cDNA library (59.9%) than from genomic DNA library (51.1%). However, higher PIC and gene diversity were identified in genomic SSRs. The average allele number per locus was also higher in genomic SSRs (7.3) than EST-SSRs (5.2), among the 24 evaluated Cymbidium accessions. AT/TA was comparatively high in EST-SSRs, while this motif was not as common in genomic SSRs. The CTT/AAG/TCT/AGA/TTC/GAA and TGC/GCA/GCT/AGC/CTG/CAG motifs were the most abundant tri-nucleotide sequences in EST-SSRs, while GTT/AAC/TGT/ACA/TTG/CAA was the most frequent in genomic SSRs. The number of repeats ranged from 3 to 12 in EST-SSRs. Currently, developed polymorphic SSR markers will be useful for germplasm assessments, core set construction, evaluation of genetic diversity, and marker assisted selection (MAS) based Cymbidium breeding. Genetic characterization of Cymbidium is vital for its conservation and

management, as well as for understanding the genetic relationships among accessions. So, we evaluated phylogenetic relationship of 100 Cymbidium accessions collected from Korea. Among 100 tested accessions, 226 alleles were detected with an average of 16.1 alleles per simple sequence repeat locus; the number of alleles ranged from 28 for the KNU-CC-32 locus to 7 for KNU-CC-25. The allele size ranged from 103 to 380 bp. Thirteen loci were deviated from HardyWeinberg equilibrium, and showed highly significant linkage disequilibrium (P < 0.01). These results indicate that influencing disturbance in the Cymbidium population, such as natural selection and/or human intervention (i.e., plant breeding), are taking place among the species in Korea. The values for heterozygosity ranged from 0.000 to 0.969, with a mean value of 0.402. The average gene diversity and polymorphism information content values were 0.679 and 0.656, respectively, and ranged from 0.223 (KNU-CC-52) to 0.936 (KNU-CC-32) and from 0.219 (KNU-CC-52) to 0.933 (KNU-CC-32), respectively. All Cymbidium accessions were put into three main groups, and no evidence of mixed population ancestry was observed among the three populations identified. Cymbidium sinensis is not as well distributed and abundant as C. goeringii in Korea. Cymbidium goeringii is endemic in Korea. The genetic diversity in Cymbidium is not related to geographical area, which indicates that the species are randomly distributed in Korea. Our finding helps explain the genetic relationships and the population structure of Cymbidium species in Korea

A thesis submitted to the committee of Graduate School, Kongju National University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Conferred in August 2012.