Development of Gene-based Identification Markers for Phalaenopsis ‘KS Little Gem’ Based on Comparative Genome Analysis

다국어 초록 (Multilingual Abstract)

Gene-based markers are useful tools for genetic background surveys and comparative genome analysis dueto their ease of application across closely related species. We developed gene-based DNA markers for Phalaenopsis‘KS Little Gem’, a new Phalaenopsis variety with excellent ornamental traits, based on a genome-wide comparisonof chloroplast and nuclear sequences with those of orchids as well as monocot species, including rice and banana, toestablish a molecular basis for species identification. Sequence comparisons of chloroplast DNA from P. ‘KS LittleGem’ with those of P. aphrodite and P. equestris identified six variable genic regions of the chloroplast genome inwhich an intron of rpl16 was the most polymorphic between related orchid species. To develop conserved orthologset (COS) markers, we compared transcriptome unigenes of P. ‘KS Little Gem’ with the P. equestris, rice, andbanana genomes and identified 582 Phalaenopsis COS candidates with at least two exons. PCR application usingprimer sets targeting six variable regions of the chloroplast genome and introns of 45 randomly selected COScandidate genes showed 92-98% amplification in three Phalaenopsis species. Among the candidate genes, wedeveloped rpl16 and three COS genes as diagnostic cross-species markers for P. ‘KS Little Gem’ and diverse,related orchids. The gene-based molecular markers developed in this study will play an important role in speciesidentification to facilitate the protection of P. ‘KS Little Gem’ variety rights, as well as breeding and genetic studiesof Phalaenopsis orchids.

참고문헌 (Reference)

1 Chapman M, "Universal markers for comparative mapping and phylogenetic analysis in the Asteraceae (Compositae)" 115 : 747-755, 2007

2 International Trad Centre, "Trade statistics for international business development"

3 Atwood J, Jr, "The size of Orchidaceae and the systematic distribution of epiphytic orchids" 9 : 171-186, 1986

4 Lin B-Y, "The mitochondrial DNA markers for distinguishing Phalaenopsis species and revealing maternal phylogeny" 60 : 68-78, 2015

5 International Rice Genome Sequencing Project, "The map-based sequence of the rice genome" 436 : 793-800, 2005

6 Cai J, "The genome sequence of the orchid Phalaenopsis equestris" 47 : 65-72, 2014

7 Kim G-B, "The complete chloroplast genome of Phalaenopsis "Tiny Star"" 27 : 1300-1302, 2016

8 D'Hont A, "The banana (Musa acuminata) genome and the evolution of monocotyledonous plants" 488 : 213-217, 2012

9 Schnable P, "The B73 maize genome : complexity, diversity, and dynamics" 326 : 1112-1115, 2009

10 Luo R, "SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler" 1 : 18-, 2012

11 Bushakra J, "Rosaceae conserved orthologous set(RosCOS)markers as a tool to assess genome synteny between Malus and Fragaria" 8 : 643-658, 2011

12 Tsai C, "Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA" 256 : 1-16, 2006

13 Fulton T, "Identification, analysis, and utilization of conserved ortholog set markers for comparative genomics in higher plants" 14 : 1457-1467, 2002

14 Liewlaksaneeyanawin C, "Identification of COS markers in the Pinaceae" 5 : 247-255, 2008

15 Kelchner S, "Group II introns as phylogenetic tools : structure, function, and evolutionary constraints" 89 : 1651-1669, 2002

16 Quraishi U, "Genomics in cereals: from genome-wide conserved orthologous set (COS) sequences to candidate genes for trait dissection" 9 : 473-484, 2009

17 Altschul S, "Gapped BLAST and PSI-BLAST : a new generation of protein database search programs" 25 : 3389-3402, 1997

18 Grabherr M, "Full-length transcriptome assembly from RNA-seq data without a reference genome" 29 : 644-652, 2011

19 Lin J-Y, "Evaluation of chloroplast DNA markers for distinguishing Phalaenopsis species" 192 : 302-310, 2015

20 Cabrera A, "Development and bin mapping of a Rosaceae Conserved Ortholog Set(COS)of markers" 10 : 562-, 2009

21 Krutovsky K, "Comparative mapping in the Pinaceae" 168 : 447-461, 2004

22 Jeong Y-M, "Comparative analysis of the radish genome based on a Conserved Ortholog Set(COS)of Brassica" 127 : 1975-1989, 2014

23 Wu F, "Combining bioinformatics and phylogenetics to identify large sets of single-copy orthologous genes (COSII) for comparative, evolutionary and systematic studies: a test case in the euasterid plant clade" 174 : 1407-1420, 2006

24 Larkin M, "Clustal W and Clustal X version 2.0" 23 : 2947-2948, 2007

25 Hollingsworth, P. M, "Choosing and using a plant DNA barcode" 6 (6): e19254-, 2011

26 De Riek J, "Are molecular markers strengthening plant variety registration and protection?" 552 : 215-223, 2001

27 Timms L, "Analyses of synteny between Arabidopsis thaliana and species in the Asteraceae reveal a complex network of small syntenic segments and major chromosomal rearrangements" 173 : 2227-2235, 2006

28 Padolina J, "A phylogeny of Phalaenopsis using multiple chloroplast markers" 26 : 23-27, 2005