Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:923±926 (2000)

Microsatellite markers for individual treegenotyping: application in forest crimeprosecutionsEleanor White,* Jerome Hunter, Cory Dubetz, Renee Brost, Alexandra Bratton,Sandra Edes and Robert SahotaPacific Forestry Centre, Canadian Forest Service, 506 West Burnside Road, Victoria, British Columbia, Canada V8Z 1M5

(Rec

* CoCanaE-maContCont

# HPubl

Abstract: Microsatellite markers similar to those used in human forensic investigations are being

developed for western red cedar (Thuja plicata). They will be used to match DNA from illegally

harvested tree stumps to suspected stolen wood. A method to extract DNA from wood samples is

described, an example of a primer pair suitable for individual tree genotyping is given, and its

application in forensic analysis is described.

# Her Majesty the Queen in Right of Canada, 2000

Keywords: DNA; microsatellite; forensic; Thuja plicata

1 INTRODUCTIONTheft of standing timber is a growing problem in

British Columbia (BC) forests. Tree theft costs the

province of BC as much as $20 million per year in lost

royalties alone. Illegal tree harvesting destroys habitat

and reduces diversity within parks and protected areas.

Thieves often `high grade', removing the most valu-

able trees. When theft occurs in an area where

harvesting authority has been issued, costs result since

forest management plans have to be changed. Red

cedar (Thuja plicata) is particularly targeted by thieves

because of its high value.

The expansive forested areas of BC (93 million

hectares, with more than 160000km of forest roads

and one of the longest forested coastlines in the world),

are dif®cult to patrol. In the absence of witnesses, most

of the evidence in cases of tree theft is circumstantial.

To improve the evidence available and curb illegal tree

harvest, the Canadian Forest Service, the BC Ministry

of Forests' Compliance and Enforcement Branch, and

the Royal Canadian Mounted Police are developing

microsatellite markers for western red cedar similar to

those used in human forensic investigations. They will

be used to match DNA from illegally harvested tree

stumps to suspected stolen wood.

Here, a method for extracting and purifying DNA

from red cedar wood is described, the results of

screening a genomic library for microsatellite or simple

sequence repeats (SSRs) are given, and an example of

a microsatellite suitable for forensic analysis is

described.

eived 26 May 2000; accepted 6 June 2000)

rrespondence to: Eleanor White, Pacific Forestry Centre, Canadian Fda V8Z 1M5il: ewhite@PFC.Forestry.CAract/grant sponsor: BC Ministry of Forestsract/grant sponsor: Canadian Biotech Strategy Genomics Fund

er Majesty the Queen in Right of Canada, 2000ished for Society of Chemical Industry by John Wiley & Sons, L

2 EXPERIMENTAL2.1 DNA extractionDiscs of wood ca 3cm thick were clamped onto a drill

press so that the edge of a clean 2.5cm (1inch) drill

just cleared the edge of the block of wood. The wood

was drilled and wood shavings were collected at the

open edge of the hole, by aspiration into ®lter sacks

made of clean interfacing fabric fastened over the end

of a shop vacuum hose. Twenty to thirty grains of

shavings per sample were collected and immediately

wrapped in aluminium foil and stored in liquid

nitrogen.

Shavings were ground in liquid nitrogen to a ®ne

powder in a Retsch 9001 ball mill. Frozen powder was

transferred to extracting solution pre-heated to 65°Cin disposable screw-cap centrifuge tubes, 10±15g of

powder per 50cm3 solution. The extracting solution

was 2X cetyltrimethylammonium bromide (CTAB) of

Rogers and Bendich,1 with 1.5% insoluble PVP

instead of 1% PVP 40000. Tubes were incubated at

65°C for 12±1h with occasional mixing by inversion.

The mixture was squeezed through one layer of nylon

cloth, the ®ltrate was extracted with an equal volume

of chloroform/isoamyl alcohol 24/1, the layers were

separated by centrifugation, and DNA was precipi-

tated from the supernatant with 2/3 volume cold

isopropanol.2

The DNA pellet was dissolved in the minimum

amount of TE and run on a 0.6% agarose gel in TBE

using a sample well 3±11cm long, depending on

sample size, obtained by taping the teeth of the well-

orest Service, 506 West Burnside Road, Victoria, British Columbia,

td. 923

E White et al

forming comb.3 Size standards were run in wells

beside the sample slot. After electrophoresis, a band of

agarose containing large molecular weight DNA was

excised from the gel. To avoid UV damage to the

DNA, the band was located and quickly marked with a

scalpel under UV, and then excised under visible light.

DNA was removed from the band of agarose by

displacement electrophoresis (isotachophoresis) by

the method of OÈ verstedt et al,4 essentially as

described by Hammann and Tabler.5 Five 100-mm3

fractions were collected off the column, with most of

the DNA generally in the third and fourth fractions. If

necessary, DNA was pooled and concentrated by

ultra®ltration according to the manufacturer's direc-

tions (Amicon's Microcon 100).

2.2 Construction and screening of genomic DNAlibrariesTwo genomic libraries were constructed by standard

methods;3 for one library, genomic DNA digested with

Sau 3A1 was ligated into the Bam HI site of the

plasmid pTZ18R (Pharmacia), while for the other, RsaI digested DNA was ligated into the Sma I site of the

same vector. The second library had the advantage

that non-insert-containing circularized vector in the

ligation reaction could be linearized by Sma I digestion

before transformation of competent cells, thereby

greatly reducing background colonies.6 Insert-con-

taining colonies were stored glycerized at ÿ80°C prior

to screening.3

For colony screening, nylon membranes were

prepared by lysing recombinant colonies grown

directly on the membrane, removing protein, and

UV ®xing DNA. Membranes were hybridised with

digoxygenin-labelled (GA)10 and (CA)10. Hybridisa-

tion was detected with alkaline phosphatase-linked

anti-digoxygenin and CSPD, on Kodak X-Omat AR

®lm, according to manufacturer's protocols.7

Figure 1. DNA extracted from red cedar heartwood. Lane 1, Lambda HindIII size standards; lanes 2 –4, DNA from growth rings 170–190, 195–205and 210–250 years old; lane 5, size standard.

2.3 PCRPlasmid DNA from strongly hybridizing colonies was

sequenced with an ABI Prism model 377 version 3.0

auto-sequencer at the University of Victoria. Primers

¯anking repeated sequences were designed using

`Primer 3' software.8

PCR ampli®cation of genomic DNA, extracted from

red cedar trees covering the species range, was carried

out using the primer pair 91-G. The reaction mix

contained 10mmol dmÿ3 Tris±HCl, 1.5mmol dmÿ3

MgCl2, 50mmol dmÿ3 KCl, pH 8.3, ca 10±20ng

template DNA, 0.2mmol dmÿ3 each dNTP,

1mmol dmÿ3 each primer, and 2U Taq polymerase

in a 50mm3 reaction. A hot-start, touchdown protocol

was used, with a 2min initial denaturation of the

template in the reaction buffer, after which the dNTPs

and enzyme were added at 80°C and ampli®cation

cycles started. Two cycles were carried out at 94°C for

45s, 56.5°C for 45s, 72°C for 45s, followed by 10

cycles with the annealing temperature decreased by

924

0.5°C every two cycles, followed by 18 cycles at 94°Cfor 45s, 53.5°C for 45s, 72°C for 45s.

3 RESULTS3.1 DNA extraction from woodResults of extraction of DNA from heartwood of red

cedar are shown in Fig 1. The yield declined as

successively older growth rings were extracted, and the

degree of degradation increased (non-gel puri®ed

samples, not shown). However, suf®cient DNA for

PCR could readily be obtained from growth rings 210±

250 years old two weeks after the wood was cut. Initial

extracts contained very high levels of phenolics and

other contaminants. These were removed in several

steps of the extraction protocol, ®rst by bonding to

insoluble PVP in the initial extraction solution, then by

extraction of the ®ltrate with CHCl3, by isopropanol

precipitation of the DNA, by agarose gel electrophor-

esis of the DNA in which high molecular weight DNA

had much lower mobility than phenolics, and ®nally by

isotachophoresis. Degraded DNA was removed from

samples during agarose gel electrophoresis; only the

high molecular weight fraction was excised from the

gel.

3.2 Screening genomic librariesApproximately 1400 clones from the Sau 3AI/ Bam Hl

and 1900 clones from the Rsa I/Sam I library were

screened and 53 putative positive clones were identi-

®ed. Sequence data have been obtained for 17 of these.

Of these, 15 contained microsatellite repeats. Two

pairs were copies of the same clone, and two clones did

not have suf®cient ¯anking sequence around the

repeat to design PCR primers.

J Chem Technol Biotechnol 75:923±926 (2000)

Figure 2. Amplification products for marker region RS 9 11 G from different red cedar trees. (a) Lanes 2–5 and 7–10, pairs of amplification products from four treeDNAs, template at 1/10 and 1/100 dilution; remaining lanes, 10bp standard. (b) Lanes 1–2, 4–7 and 9–10, amplification products from eight different trees;remaining lanes, 10bp standard.

Table 1. Frequency of RS9 11 G alleles in red cedar

Allele

Frequency

(%)

1 8.8

Microsatellite markers

3.3 PCRResults of PCR ampli®cation using primers designed

for clone RS 9 11G are shown in Fig 2. Ampli®cation

products from four trees (genotypes) are illustrated in

Fig 2(a), with duplicate ampli®cations from the same

DNA template at 1/0 or 1/100 dilution. Figure 2(b)

shows ampli®cation products from eight more geno-

types. Table 1 shows the frequency of different alleles

in 102 trees originating across the range of western red

cedar. Alleles which have been sequenced differ by

multiples of (CT)2. For example, Fig 3 shows the

sequence alignment of alleles 2 and 3, which have

(CT)13 and (CT)15 respectively, among other differ-

ences.

2 18.6

3 18.6

4 1.0

5 1.5

6 2.0

7 5.9

8 3.9

9 3.4

10 5.4

11 4.9

12 6.4

13 5.4

14 9.3

15 2.5

16 2.5

4 DISCUSSIONDNA evidence is a matter of probabilities. In the

absence of mutation, when two samples do not have

the same DNA markers they could not have originated

from the same individual ±the evidence excluding a

suspect sample is absolute. However if two samples

have the same markers the question becomes: what is

the signi®cance of the match? The strength of DNA

evidence is that the probability of a random match will

be very low if suf®cient markers are analysed. The

courts have accepted the basic genetic concept that the

J Chem Technol Biotechnol 75:923±926 (2000)

probability of a particular genotype is the product of

the probabilities at independent loci.9

Using this `product rule', the probability of a red

cedar tree having the genotype with the two least

frequent alleles in Table 1 is 1/100�1.5/100=1.5/

10000. Not surprisingly, after surveying 102 trees, we

925

Figure 3. Alignment of DNA sequence for amplified DNA from two different alleles.

E White et al

have not found any with this genotype. Similarly, the

probability of a genotype with the two most frequent

alleles is 18.6/100�18.6/100=346/10000. We have

found three trees with this genotype, an indication that

the frequency of genotypes will be those expected of a

population that is in Hardy±Weinberg equilibrium.

These results demonstrate the feasibility of using

DNA evidence in cases of tree theft, to match

suspected stolen wood to stumps of illegally harvested

trees. Suf®cient good quality DNA can be extracted

from wood, even from heartwood with high levels of

enzyme inhibiting compounds. Since sample size is

usually not an issue in tree theft cases, the lower limits

for sample size have not been tested, but they are lower

than the 10g described here. Some cases of tree theft

involve trees which have been uprooted from steep

banks into the ocean by pumping water around their

roots to create a landslide, allowing the trees to be

claimed as salvage. In these cases, suf®cient DNA

could be extracted from small roots remaining on the

bank to compare to suspect wood.

More microsatellite containing clones were identi-

®ed in genomic libraries screened with the GA10 than

with the CA10 probe, similar to results with Norway

spruce.10 More microsatellites have been identi®ed

from libraries enriched for repeat sequences. Primers

are currently being designed and tested for these

potential markers. More single locus polymorphic

markers such as RS 9 11G will be identi®ed and

validated, until a suf®cient number are available to

forensic analysis agencies to provide the low probabil-

ities of a random match that have come to be expected

for DNA evidence.

926

ACKNOWLEDGEMENTSThe authors are grateful to the BC Ministry of Forests

and the Canadian Biotech Strategy Genomics Fund

for ®nancial support.

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J Chem Technol Biotechnol 75:923±926 (2000)