ScienceAsia 31 (2005): 145-149 DNA Fingerprint Database of Some Economically Important Thai Plants: Litchi chinensis Sonn, Dimocarpus longan Lour, and Peuraria spp. Rungrach Wangspa a , Robert W. Cutler b,* , Supranee Sitthiprom a , Ruttaporn Chundet a , Nadtaya Dumampai c , and Somboon Anuntalabhochai a a Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50202, Thailand. b Department of Biology, Bard College, Annandale-on-Hudson, New York 12504, USA. c Trang Horticultural Research Center Siako, Trang 92150, Thailand. * Corresponding author, E-mail: cutler@bard.edu Received 26 July 2004 Accepted 12 Jan 2005 ABSTRACT: The high annealing temperature random amplified polymorphic DNA (HAT-RAPD) method can be used to generate highly polymorphic data from PCR amplification of DNA samples to determine relatedness in plant cultivars. Using the HAT-RAPD method, we created a fingerprint database of tropical plants for fourteen subspecies of Dimocarpus longan Lour., twelve subspecies of Litchi chinensis Sonn., and seven distinct varieties of the genus Peuraria, two of which were previously uncharacterized. Out of a total of 22 distinct primer sets, a subset of primers with reproducible DNA band patterns was characterized for each species. From this data, we developed a web-accessible database which both graphically and quantitatively depicts the existent bands and species phylogenies using character state data generated from the banding patterns. Using historical and geographic data for these plant species and subspecies, the generated phylogenies support the currently accepted species relationships for D. longan and L. chinensis and characterize the unidentified varieties of Peuraria. The HAT-RAPD experimental method combined with band pattern recognition is a cost effective and easily characterized methodology that can be used to identify plant varieties, as well as to advance the knowledge of biodiversity in previously uncharacterized species. KEYWORDS: HAT-RAPD, DNA fingerprinting, Online Database, Bioinformatics. INTRODUCTION Since Thailand’s economy is based largely on agricultural production, many plant varieties have been generated by means of selective breeding to improve crop yield or to overcome growth limitations and diseases. This gave rise to an assortment of hybrids and divergent strains in some economically important species. For example, there are over 50 varieties of Litchi chinensis Sonn. and some 45 distinct varieties of Dimocarpus longan Lour. found in orchards throughout the country. Based on physical appearance alone, uncertain or even misleading identification of an individual plant is certain. An easy and reliable means to distinguish one variety from another is needed. Molecular techniques such as RAPD with arbitrary primers, 9,13,15,16 restriction fragment length polymorphism, 5,8,17 amplified fragment length polymorphism, 3,12 variable number of tandem repeats, 10 and sequence-tagged simple sequence repeats are powerful techniques for species and variety identification. The RAPD technique can detect DNA segments present in individual samples to infer genetic relationships between different populations. However, a serious shortcoming of RAPD is inconsistency of banding patterns. 6 Our group has further developed a technique called HAT-RAPD. The technique, instead of using lower annealing temperatures, utilizes temperatures between 40-46 o C to increase the specificity of the annealing conditions. It was successfully shown that, coupled with several random decamer primers, repeatable highly polymorphic bands can be generated. 1 Independent of this work, optimization of RAPD analysis by elevating annealing temperature, which then yielded reproducible DNA profiles, has also been reported. 2 By using the presence or absence of conserved bands in the amplified products (which we designate as a character state measure for the Phylip DISCRETE software package 7 ), varieties and hybrids of L. chinensis Sonn. 1 and D. longan Lour., 14 were effectively and reliably identified. Thus, the HAT-RAPD profiles can further be adapted for plant identification. The purposes of this work are to: (1) develop an algorithm to identify both conserved and polymorphic bands generated with each primer, (2) assemble HAT- RAPD marker polymorphic data into a database for