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Cross-Species Amplification of Shorea Microsatellite DNA Markers in Parashorea Malaanonan (Dipterocarpaceae)

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Cross-Species Amplification of Shorea Microsatellite DNA Markers in Parashorea Malaanonan (Dipterocarpaceae) Philippine Journal of Science 138 (1): 23-28, June 2009 ISSN 0031 - 7683 Cross-Species Amplification of Shorea Microsatellite DNA Markers in Parashorea malaanonan (Dipterocarpaceae) Myralyn A. Abasolo1, Edwino S. Fernando2*, Teresita H. Borromeo3, and Desiree M. Hautea3 1Institute of Renewable Natural Resources, College of Forestry and Natural Resources, 2Department of Forest Biological Sciences, College of Forestry and Natural Resources, 3Crop Science Cluster, Institute of Plant Breeding, College of Agriculture, University of the Philippines, Los Baños, College 4031 Laguna, Philippines Sixteen (16) microsatellite DNA markers previously developed for two Bornean species of Shorea (Dipterocarpaceae), viz. 5 from Shorea curtisii (Shc) and 11 from Shorea leprosula (Sle), were tested on Philippine Parashorea malaanonan for cross-species amplification. Initially, these loci were tested in Parashorea malaanonan from the Makiling Forest Reserve on Luzon Island. All five Shc loci had amplification products, three (3) were polymorphic and two (2) were non- polymorphic. Of the 11 Sle loci, five (5) were polymorphic, three (3) were non-polymorphic, two (2) did not have amplification products, and one (1) had multiple bands. Four loci were further tested on individuals of Parashorea malaanonan from a plantation on Leyte Island. High levels of genetic variability were observed. Cross-transferability was 75%. Expected heterozygosity (He) ranged from 0.39 to 0.82. Results indicate potential use of these markers derived from Shorea in studies of genetic diversity assessments in Parashorea malaanonan. Key Words: cross transferability, Dipterocarpaceae, genetic diversity, Parasahorea malaanonan, Shorea, SSR INTRODUCTION assessing population variation in Stemonoporus oblongifolius Thw. by Murawski and Bawa (1994), Parashorea malaanonan (Blanco) Merr. is one of Lee et al. (2000a) in Shorea leprosula Miq., and Lee about 45 species of the timber family Dipterocarpaceae et al. (2000b) in Dryobalanops aromatica Gaertn.f. thus far recorded from the Philippines (Ashton 1982; Cao et al. (2006) used amplified fragment length Newman et al. 1996). It is known from at least 10 of polymorphism (AFLP) to determine genetic diversity the larger islands in the Philippine archipelago and also and differentiation among Indonesian populations from the northeast of Borneo (Newman et al. 1996; of Shorea leprosula and Shorea parvifolia Dyer. Ashton 2004). The species is common in lowland Microsatellite DNA markers have previously been forests and occurs up to 1300 m alt. One recent study used by Konuma et al. (2000) and Nagamitsu et al. has characterized the mating system and assessed (2001) in analyses of dipterocarp breeding systems the genetic diversity of this species using isozymes and genetic diversity assessments. (Gamboa-Lapitan and Hyun 2005). In order to aid in the assessment of genetic diversity in In most dipterocarps, genetic variation within species Parashorea malaanonan we investigated the potential is largely unknown. Isozymes have been used in use of microsatellite DNA markers. These markers, *Corresponding author: [email protected] 23 Philippine Journal of Science Abasolo et al.: Cross-Amplification of Vol. 138 No. 1, June 2009 Shorea SSRs in Parashorea also known as simple sequence repeats (SSRs), have DNA extraction was done from frozen (-80˚ C) mature the potential to resolve genetic relationships at all levels leaf material using the protocol of Tsumura et al. (1996) of population structure (Jarne and Lagoda 1996). The with modifications to improve the quality and quantity development of new microsatellite markers, however, is of the DNA obtained. Using this protocol, a viscous and often time-consuming and expensive owing to the need brownish pellet was obtained indicating that polyphenols for DNA sequence data of these short tandem repeats and tannins precipitated with the DNA. Sodium bisulfite and their flanking regions from the genome of the target was used in place of beta mercaptoethanol as antioxidant. species. One alternative approach is to exploit the Incubation period was extended to 1 hour at a higher available information by cross-species amplifications temperature of 65° C to allow for the efficient breakage among a range of phylogenetically related species. There of the cells considering that mature leaves already is increasing number of studies on plants that report some developed rigid cell walls. Both RNAse A and proteinase cross-species amplification of SSR loci, with occasional K treatments were extended to 1 hour each to allow for informative transferability of loci to divergent taxa (e.g. sufficient degradation of RNA and other proteins. Dayanandan et al. 1997; Zucchi et al. 2002; Aldrich et al. 2003; Yasodha et al. 2005). Phenol:chloroform:octanol (25:24:1) was effective in removing impurities of DNA extracted from mature Ujino et al. (1998) developed eight microsatellite loci leaves that were not degraded by the enzymes RNAse and for Shorea curtisii Dyer ex King, five of which were proteinase. Phenol and chloroform are organic solvents transferable to other Bornean species belonging to the and they effectively trapped hydrophobic substances such following genera of the family Dipterocarpaceae, viz. as polysaccharides and other PCR-inhibiting substances, Anisoptera, Hopea, Neobalanocarpus, Parashorea, leaving the hydrophilic DNA in the aqueous phase. Vatica and Upuna. Lee et al. (2004) also characterized Chloroform:octanol (24:1) purification removed the 20 microsatellite loci for Shorea leprosula and 16 of these cloudiness of the solution brought about by PVP (Porebski loci were cross-transferable to Shorea parvifolia. et al. 1997). Precipitation of the DNA by absolute ethanol and sodium acetate increased its yield. In this study we investigated the transferability of these two sets of microsatellite loci characterized for two The amount of DNA extracted was determined by Bornean species of Shorea to Parashorea malaanonan comparing it with a standard uncut lambda (λ) phage from the Philippines. DNA marker. Two μL of resuspended DNA was mixed with 18 μL of 1x blue juice (0.04% bromphenol blue, 6.67% sucrose in water). The mixture was loaded on a 1% agarose gel run in 1x TBE buffer (90 mM Tris borate, MATERIALS AND METHODS 2mM EDTA) at 100V for 30 minutes. DNA was visualized by staining with ethidium bromide (10mg/μL) and viewed Leaf samples and DNA extraction under ultraviolet light (410-720 nm) using Biorad Gel We obtained leaf material from four individuals of Documentation System (Biorad Laboratories, Segrate Parashorea malaanonan from separate populations and Milan, Italy). DNA working stock (5ng/μL) was prepared different elevations in the Mt. Makiling Forest Reserve from the visual quantification of DNA per sample. on Luzon Island, Philippines for the initial screening of cross-amplification. The collected leaves were tagged, PCR and Electrophoresis placed in resealable plastic bags and stored in a styrofoam Sixteen (16) microsatellite DNA markers or simple container with ice while in the field, and immediately sequence repeats (SSRs) developed for two Bornean transferred to a biofreezer in the laboratory until DNA species of Shorea (Dipterocarpaceae), viz. Shorea curtisii extraction. Since the main objective of this initial and Shorea leprosula, were used in this study (Table 1). study was to assess the transferability of SSRs rather Five of the SSRs were previously developed for Shorea than polymorphism, the number of individuals tested curtisii (Shc) (Ujino et al. 1998) and 11 for Shorea is considered satisfactory. In addition, leaf material of leprosula (Sle) (Lee et al. 2004). These primers were this species was also obtained from nine individuals chosen based on the high (>0.60) expected heterozygosity in a tree plantation in the Cienda–San Vicente Forests (He). The oligonucleotide primers used in this study were near Baybay on Leyte Island. This was done because synthesized by Invitrogen Philippines. These sixteen of the potential presence of unique alleles in distant (16) loci were screened using individuals of Parashorea populations, especially in an archipelagic setting such malaanonan from four different sites within the Mt. as the Philippines. Makiling Forest Reserve. Annealing temperatures were adjusted to obtain the best amplification product as applied 24 Philippine Journal of Science Abasolo et al.: Cross-Amplification of Vol. 138 No. 1, June 2009 Shorea SSRs in Parashorea Table 1. Amplification of Shorea curtisii (Ujino et al. 1998) and Shorea leprosula (Lee et al. 2004) SSR loci in Parashorea malaanonan using four individuals from the Mt. Makiling Forest Reserve, Philippines Source species Target species Locus SSR motif Size range No. of Size range AT b (˚C) Ac AT b (˚C) Ac TRd (bp)a trees (bp)a Shorea curtisii Parashorea malaanonan Shc01 (CT)8 (CA)10 CT (CA)4 CTCA 152 56 20 20 138 52 1 +* Shc04 (CT)16 149 52 12 20 103-139 52 7 + (CT) CA (CT) CACCCC (CTCA) CT Shc07 8 5 3 169 54 11 20 82-112 52 2 + (CA)10 Shc09 (CT)12 197 54 9 20 178 50 1 +* Shc11 (CT)4TT(CT)5 166 54 4 20 170-183 48 5 + Shorea parvifolia Parashorea malaanonan Sle074a (CT)11 110-130 52 11 20 181-240 52 5 + Sle079a (CT)11 155-198 52 20 n/a 56 - Sle111a (GA)14 138-154 52 12 20 146 52 1 +* Sle118 (GA)16 145-176 52 20 n/a 56 - Sle216 T12 (CT)6 93-112 50 7 99-135 58 2 + Sle280 (CT)7 107-137 52 11 109 52 1 +* Sle290 A21 N5 (GAA)6 (GA)18 195-211 52 14 136-147 52 2 + Sle303a (GA)12 145-169 55 11 151 55 1 +* Sle392 (GA)11 161-231 55 15 n/a 60 - Sle562 (GT)8 154-180 52 12 154-155 52 4 + Sle566 (GA)13 59-104 52 15 ≤ 67-106 52 5 + aSize of allele in base pairs (bp); bAT - annealing temperature (in ˚C); cA - the number of alleles; dTR – type of result: (-) a smear, multiple banding pattern or no fragment was detected; (+) successful amplification products of one or two bands of expected size close to the original species for which the primer was designed (source species); * polymorphism not detected.
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