J Forensic Sci, September 2015, Vol. 60, No. 5 doi: 10.1111/1556-4029.12789 Available online at: onlinelibrary.wiley.com
TECHNICAL NOTE CRIMINALISTICS
Hitomi S. Kikkawa,1 Ph.D.; Makoto Tahara,2 Ph.D.; and Ritsuko Sugita,1 Ph.D.
Forensic DNA Analysis of Wheat Flour as Commonly Used in White Powder Cases*
ABSTRACT: In the wake of terrorist attacks using anthrax and ricin, white powder is often encountered in cases of malicious mischief and terrorist threats. Wheat flour is a common white powder encountered in such criminal investigations. We used DNA analysis to investigate wheat flour samples for identification and discrimination as trace evidence. Species identification of commercially available wheat flour was carried out by sequencing a partial region of the ribulose bisphosphate carboxylase large subunit gene (rbcL). Samples were discriminated using short tandem repeat (STR) analysis. The rbcL sequences of all wheat flour samples were identical and showed a high level of similarity to known wheat (Triticum aestivum L.) sequences. Furthermore, flours had characteristic patterns in STR analyses, with specific cultivars showing distinctive patterns. These results suggested that the identification of wheat flour species is possible using rbcL sequencing, and that STR analysis is useful for discriminating between samples.
KEYWORDS: forensic science, criminalistics, white powder material, DNA barcoding, ribulose bisphosphate carboxylase large subunit gene, DNA typing, short tandem repeats
White powder is often encountered in possible terrorism cases in the aftermath of the 2001 anthrax attacks via the United States postal system, as well as the related terrorism attacks using ricin in 2008 and 2013, also in the United States (1–5). Identification of the raw materials in white powder samples recovered from a crime scene can provide valuable information in criminal investigations (6,7). If materials contained in such samples can be linked to a suspect, the findings can be used as evidence to associate the suspect with the site (8). Commonly used white substances such as talcum powder, cement, dust, sugar, and starch are often involved in anthrax-related incidents (9). Wheat flour is the most common white powder sold globally, and five million kilograms is distributed annually in Japan (10). The possibility of wheat flour being used in criminal acts is, therefore, quite high. Forensic botanical samples are often identified based on morphological observations (11,12), but wheat flour has a similar appearance to other powdery materials, making accurate species identification based on such characteristics more difficult. White powders derived from cereals and beans are analyzed by several methods, including iodostarch reaction, Fourier transform infrared spectroscopy, inductively coupled plasma mass spectrometry, and scanning electron microscopy (13–15). Although these methods are effective, cross-reactivity among white powders is often observed (13). If the forensic sample is suspected to include
1
National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa 277-0882, Japan. 2 Graduate School of Environmental and life Science Okayama University, 1-1-1 Tsushimanaka, Kita-ku, Okayama 700-8530, Japan. *Presented at the 18th Annual Meeting of the Japanese Association of Forensic Science and Technology, November 15–16, 2012, in Tokyo, Japan. Received 24 Nov. 2013; and in revised form 31 May 2014; accepted 16 Aug. 2014.
1316
unknown particles from plants, DNA analysis might be useful to identify the plant species and discriminate the possible product sources within the sample. However, an official DNA analysis procedure for botanical samples in forensic examination has not yet been established. In a plant forensic examination, two steps might be required: (1) identification of species in the sample using species-specific markers; (2) discrimination of the possible sources within samples using polymorphic markers that can distinguish cultivars within a species (16,17). Direct sequencing of specific loci, known as DNA barcoding, has been used successfully for plant species identification (18– 22). For a forensic examination, the procedure needs to be standardized, taking into account limitations in analysis time and amounts of sample. Additionally, the method must be broadly applicable for botanical materials, as forensic laboratories often deal with unknown plant material. DNA barcoding is advantageous for forensic applications because the species of the plant material in the questioned sample can be identified by a single test run. In addition, an official recommendation for plant barcoding was recently established (19), and large databases such as GenBank (23) and the Barcode of Life data systems (BOLD) (24) are readily accessible. Discrimination of possible sources among samples should be carried out only after the species has been identified, as polymorphic markers are highly species specific, and most are only capable of distinguishing differences within a limited set of closely related species. Molecular markers such as microsatellites or single nucleotide polymorphisms ideally provide resolution at the genotype level (25). Recently, short tandem repeat (STR) markers have been developed for Japanese domestic wheat to analyze flour products such as udon noodles and determine the source cultivars (26). However, it remains unclear as to whether DNA analysis can be applied to commercially distributed wheat flour samples. © 2015 American Academy of Forensic Sciences
KIKKAWA ET AL.
This report describes the application of DNA analysis to wheat flour samples as white powder materials for forensic identification and discrimination. Species identification of commercially obtained wheat flour samples was carried out by partial sequencing of an ribulose bisphosphate carboxylase large subunit gene (rbcL) region, which was used as a marker for DNA barcoding (19,21). These wheat flour samples were then discriminated by four STR markers that were developed for cultivar identification.
.
FORENSIC DNA ANALYSIS OF WHEAT FLOUR TABLE 1––Wheat flour samples analyzed.
Sample Number
Brand Name (The Best Before Date)
Manufacturer
Description Strong flour made from kitanokaori (Japanese domestic cultivar) Strong flour made from kitanokaori (Japanese domestic cultivar) Strong flour made from haruyutaka (Japanese domestic cultivar) Strong flour made from yukichikara (Japanese domestic cultivar) Strong flour made from haruyokoi (Japanese domestic cultivar) Strong flour Strong flour Strong flour Soft flour Soft flour Soft flour Soft flour Soft flour Soft flour Soft flour made from Japanese domestic cultivar Soft flour Soft flour Soft flour Soft flour Strong flour Strong flour Produced from crops grown in a Hokkaido prefecture Medium flour produced from crops grown in a Nagano prefecture Soft flour produced from crops grown in a Nagano prefecture Soft flour produced from crops grown in Japan Strong flour made from Japanese domestic cultivar
1
A1
A
2
B1
B
3
C1
C
4
C2
C
5
C3
C
6 7 8 9 10 11 12 13 14 15
D1 (2003) D1 (2009) D1 (2012) D2 (2006) D2 (2009) D3 E1 (2003) E1 (2008) E1 (2009) E2
D D D D D D E E E E
16 17 18 19 20 21 22
F1 F2 (2012) F2 (2012) F3 G1 H1 H2
F* F* F* F* G H H
23
I1
I
24
I2
I
25
I3
I
26
J1
J*
Materials and Methods Samples Twenty-six wheat flours commercially available in Japan, including 11 that were harvested domestically, were sampled (Table 1). Samples were stored at room temperature until experimental analysis. Total genomic DNA was extracted from samples weighing 100 mg using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions and was stored at 20°C until analysis. The DNA concentration of each sample was measured using a U-0080D photodiode array spectrophotometer (Hitachi High-Technologies, Tokyo, Japan). PCR Amplification of rbcL A partial rbcL sequence was amplified by PCR using the primer set rbcLa_F and rbcLa_R (Table 2) (19,27). Amplification was carried out using a MyCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA) in a total volume of 10 lL. Each reaction contained 200 lM of dNTPs, 0.01% BSA, 0.2 lM of each primer, 0.1 U of ExTaq DNA polymerase (Takara, Otsu, Japan), 19 ExTaq reaction buffer, and 10 ng of DNA. The PCR conditions were as follows: 95°C for 4 min, 35 cycles of 94°C for 30 sec, 55°C for 60 sec, and 72°C for 60 sec, with a final extension at 72°C for 10 min. The amplified products were detected on a 2% agarose gel and purified with the QuickStep2 PCR Purification Kit (Edge BioSystems, Gaithersburg, MD). DNA Sequencing of rbcL Cycle sequencing reactions were carried out using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Foster City, CA) with a MyCycler in a total volume of 20 lL. Direct sequencing of the PCR products was performed using both forward and reverse primers. The cycle sequencing products were analyzed with POP-6 Performance Optimized Polymer (Life Technologies) and detected by a 310 Genetic Analyzer (Life Technologies). The partial rbcL sequence was determined for each sample. Homology searches were performed for similar sequences stored in the GenBank, European Molecular Biology Laboratory (EMBL), and DNA Data Bank of Japan (DDBJ) databases using the Basic Local Alignment Search Tool (BLAST) accessible through the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
1317
*Distributor. The manufacturer is unknown.
The procedure for STR profiling was the same as in a previous report (26). PCR was performed in a final volume of 10 lL, which contained 200 lM of dNTPs, 0.2 lM of each primer, 19 AmpliTaqGold buffer, 0.2 U of AmpliTaqGold DNA polymerase (Life Technologies), and 15 ng of DNA. STR regions were amplified using the following parameters: initial denaturation at 94°C for 9 min, 30 cycles of 94°C for 30 sec, 60°C for 1 min, and 72°C for 1 min, followed by a final extension step of 7 min at 72°C. The amplification reactions were conducted using a MyCycler.
PCR Amplification for STR Genotyping The same extracted DNA as was used for DNA barcoding was also used in STR profiling. Table 2 provides information regarding the primers used for amplification of each STR site.
Capillary Electrophoresis and Data Analysis Fragment analysis was performed using a 310 Genetic Analyzer (Life Technologies). Capillary electrophoresis separation
1318
JOURNAL OF FORENSIC SCIENCES TABLE 2––Primers used to amplify rbcL and STR loci.
rbcL STR
Primer
Sequence
rbcLa_F rbcLa_R TaSE3-F TaSE3-R TaSE37-F TaSE37-R TaSe63-F TaSe63-R TaSe96-F TaSe96-R
50 -gattcgcagatcctccagacgtagagc-30 50 -gattcgcagatcctccagacgtagagc-30 50 -caccgatcgatcaacaagtcaaaa-30 50 -NED-catcatcatcggttcttgga-30 50 -atccgctacggaagaaataccaca-30 50 -VIC-gttgctggcctgccatgttta-30 50 -6FAM-cgtgtgctctcgcagtttcatagt-30 50 -cctcgccttctaattaagctccgt-30 50 -PET-tgggacaagtccctaggtaagacg-30 50 -gtagtccgcccagcctctactttt-30
TABLE 3––Search results for rbcL sequences in the GenBank, EMBL, DDBJ, and PDB databases. Locus rbcL TaSE3 TaSE37
n X i ¼1
n X i ¼1
P2i
P2i
n1 X n X i ¼1 j ¼ iþ1
2P2i P2j
where n is the total number of alleles detected for a locus and Pi is the frequency of the i th allele, respectively (30,31). Power of discrimination (PD) of each locus was determined according to the equation: PD ¼ 1
m X
Pi 2
i ¼1
where m is the total number of genotypes detected for a locus and Pi is the frequency of the ith genotype (32). Power of discrimination of the combination of all loci (APD) was calculated by the formula: APD ¼ 1
k Y
aestivum aestivum aestivum aestivum aestivum
Homology (%)
HQ590312.1 JN133499.1 EU492898.1 AB042240.3 D00206.1
100 100 100 100 100
TaSE96
and PIC ¼ 1
Triticum Triticum Triticum Triticum Triticum
Accession Number
TaSE63
was carried out using the POP-6 polymer (Life Technologies), and the sizes of amplified bands were calculated based on GeneScan 600 LIZ size standard (Life Technologies). Output data were analyzed using GeneMapper ID version 3.2.1 software (Life Technologies). The fluorescent signals that were reached above 1/10 of the main peak height were used for further analysis (28). Genotypic data analyses were performed using GENEPOP v.4.2 (29). Expected heterozygosity (He) and the polymorphic information content (PIC) were calculated for each locus using the formulae: He ¼ 1
Sequence Matched
ð1 PDi Þ
i ¼1
where k is the total number of loci (33). Cluster analysis was performed using the unweighted pairgroup method (UPGMA) with Jaccard similarity coefficients using BioNumerics software (Appliedmath, Kortrijk, Belgium).
Results and Discussion Identification of Raw Materials Using rbcL Sequence of Wheat Flour While most wheat flour products distributed in Japan are made from imported wheat, some flours are made from Japanese
domestic cultivars. In this study, various wheat flour products were examined (Table 1). To identify the species of the source materials, DNA was extracted from commercial flours and rbcL partial regions (553 base pairs [bp]) were sequenced. The concentration of DNA recovered from the extracts ranged from 24.8 ng/lL (No. 25) to 80.9 ng/lL (No. 14). The rbcL region was successfully amplified from all samples and sequenced using both rbcL primers. All sequences were identical among the samples, in spite of the possibility that wheat flour samples contain multiple cultivars. The database search results for the rbcL region are shown in Table 3. Sample sequences matched the wheat (Triticum aestivum L.) type strain sequence, which was the only species that showed 100% similarity. This result indicated that unknown white powder could be identified as wheat flour by determining the rbcL sequence. Previous studies have confirmed sample species from their morphological features (16,18,34), but forensic samples, such as wheat flour, often lack such morphological characteristics. We hypothesized that it might be possible to identify such samples using DNA analysis. Thus, we attempted to determine wheat species in the samples using DNA barcoding and successfully identified all samples (Table 3). Official analytical methods for species identification of wheat are established to detect wheat as an allergenic ingredient in processed foods, but these methods can only determine whether or not a sample contains wheat. Forensic analysis requires rapid testing, so it is desirable to obtain as much information as possible in a single procedure (27). In this study, species (or related species) of the unknown samples could be identified by DNA barcoding, suggesting that it is suitable for forensic analysis (27). Discrimination of Wheat Flour Samples using STR Markers As all samples analyzed in this study were confirmed as wheat flour by rbcL sequencing, possible combinations of wheat cultivars included in each sample were investigated by STR marker analysis. Ten STR markers for Japanese domestic wheat strains have been reported (26), and six of the 10 markers can discriminate both imported and Japanese cultivars. Preliminary examination of these six STR markers showed that markers TaSE3, TaSE37, TaSE63, and TaSE96 were sufficient to discriminate different flour products (data not shown). Therefore, in the current study, we analyzed these four STR sites for all of the flour samples. To examine the accuracy and repeatability of wheat cultivar genotyping using the STR markers, we analyzed the five products listed as having been made from one cultivar using the four STR markers. As expected, the samples showed a single identical product at all four STR sites, and the pattern of product sizes for the markers was consistent with published data (26). The observed peak sizes were slightly different from those reported; this was most likely due to the influence of the dye mass
KIKKAWA ET AL.
.
FORENSIC DNA ANALYSIS OF WHEAT FLOUR
1319
TABLE 4––Characteristics of the STR markers used in this study.
Loci TaSE3 TaSE37 TaSE63 TaSE96 Combination of 4 loci
Allele Size Range (bp)
Number of Alleles
Number of Rare Alleles (
TECHNICAL NOTE CRIMINALISTICS
Hitomi S. Kikkawa,1 Ph.D.; Makoto Tahara,2 Ph.D.; and Ritsuko Sugita,1 Ph.D.
Forensic DNA Analysis of Wheat Flour as Commonly Used in White Powder Cases*
ABSTRACT: In the wake of terrorist attacks using anthrax and ricin, white powder is often encountered in cases of malicious mischief and terrorist threats. Wheat flour is a common white powder encountered in such criminal investigations. We used DNA analysis to investigate wheat flour samples for identification and discrimination as trace evidence. Species identification of commercially available wheat flour was carried out by sequencing a partial region of the ribulose bisphosphate carboxylase large subunit gene (rbcL). Samples were discriminated using short tandem repeat (STR) analysis. The rbcL sequences of all wheat flour samples were identical and showed a high level of similarity to known wheat (Triticum aestivum L.) sequences. Furthermore, flours had characteristic patterns in STR analyses, with specific cultivars showing distinctive patterns. These results suggested that the identification of wheat flour species is possible using rbcL sequencing, and that STR analysis is useful for discriminating between samples.
KEYWORDS: forensic science, criminalistics, white powder material, DNA barcoding, ribulose bisphosphate carboxylase large subunit gene, DNA typing, short tandem repeats
White powder is often encountered in possible terrorism cases in the aftermath of the 2001 anthrax attacks via the United States postal system, as well as the related terrorism attacks using ricin in 2008 and 2013, also in the United States (1–5). Identification of the raw materials in white powder samples recovered from a crime scene can provide valuable information in criminal investigations (6,7). If materials contained in such samples can be linked to a suspect, the findings can be used as evidence to associate the suspect with the site (8). Commonly used white substances such as talcum powder, cement, dust, sugar, and starch are often involved in anthrax-related incidents (9). Wheat flour is the most common white powder sold globally, and five million kilograms is distributed annually in Japan (10). The possibility of wheat flour being used in criminal acts is, therefore, quite high. Forensic botanical samples are often identified based on morphological observations (11,12), but wheat flour has a similar appearance to other powdery materials, making accurate species identification based on such characteristics more difficult. White powders derived from cereals and beans are analyzed by several methods, including iodostarch reaction, Fourier transform infrared spectroscopy, inductively coupled plasma mass spectrometry, and scanning electron microscopy (13–15). Although these methods are effective, cross-reactivity among white powders is often observed (13). If the forensic sample is suspected to include
1
National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa 277-0882, Japan. 2 Graduate School of Environmental and life Science Okayama University, 1-1-1 Tsushimanaka, Kita-ku, Okayama 700-8530, Japan. *Presented at the 18th Annual Meeting of the Japanese Association of Forensic Science and Technology, November 15–16, 2012, in Tokyo, Japan. Received 24 Nov. 2013; and in revised form 31 May 2014; accepted 16 Aug. 2014.
1316
unknown particles from plants, DNA analysis might be useful to identify the plant species and discriminate the possible product sources within the sample. However, an official DNA analysis procedure for botanical samples in forensic examination has not yet been established. In a plant forensic examination, two steps might be required: (1) identification of species in the sample using species-specific markers; (2) discrimination of the possible sources within samples using polymorphic markers that can distinguish cultivars within a species (16,17). Direct sequencing of specific loci, known as DNA barcoding, has been used successfully for plant species identification (18– 22). For a forensic examination, the procedure needs to be standardized, taking into account limitations in analysis time and amounts of sample. Additionally, the method must be broadly applicable for botanical materials, as forensic laboratories often deal with unknown plant material. DNA barcoding is advantageous for forensic applications because the species of the plant material in the questioned sample can be identified by a single test run. In addition, an official recommendation for plant barcoding was recently established (19), and large databases such as GenBank (23) and the Barcode of Life data systems (BOLD) (24) are readily accessible. Discrimination of possible sources among samples should be carried out only after the species has been identified, as polymorphic markers are highly species specific, and most are only capable of distinguishing differences within a limited set of closely related species. Molecular markers such as microsatellites or single nucleotide polymorphisms ideally provide resolution at the genotype level (25). Recently, short tandem repeat (STR) markers have been developed for Japanese domestic wheat to analyze flour products such as udon noodles and determine the source cultivars (26). However, it remains unclear as to whether DNA analysis can be applied to commercially distributed wheat flour samples. © 2015 American Academy of Forensic Sciences
KIKKAWA ET AL.
This report describes the application of DNA analysis to wheat flour samples as white powder materials for forensic identification and discrimination. Species identification of commercially obtained wheat flour samples was carried out by partial sequencing of an ribulose bisphosphate carboxylase large subunit gene (rbcL) region, which was used as a marker for DNA barcoding (19,21). These wheat flour samples were then discriminated by four STR markers that were developed for cultivar identification.
.
FORENSIC DNA ANALYSIS OF WHEAT FLOUR TABLE 1––Wheat flour samples analyzed.
Sample Number
Brand Name (The Best Before Date)
Manufacturer
Description Strong flour made from kitanokaori (Japanese domestic cultivar) Strong flour made from kitanokaori (Japanese domestic cultivar) Strong flour made from haruyutaka (Japanese domestic cultivar) Strong flour made from yukichikara (Japanese domestic cultivar) Strong flour made from haruyokoi (Japanese domestic cultivar) Strong flour Strong flour Strong flour Soft flour Soft flour Soft flour Soft flour Soft flour Soft flour Soft flour made from Japanese domestic cultivar Soft flour Soft flour Soft flour Soft flour Strong flour Strong flour Produced from crops grown in a Hokkaido prefecture Medium flour produced from crops grown in a Nagano prefecture Soft flour produced from crops grown in a Nagano prefecture Soft flour produced from crops grown in Japan Strong flour made from Japanese domestic cultivar
1
A1
A
2
B1
B
3
C1
C
4
C2
C
5
C3
C
6 7 8 9 10 11 12 13 14 15
D1 (2003) D1 (2009) D1 (2012) D2 (2006) D2 (2009) D3 E1 (2003) E1 (2008) E1 (2009) E2
D D D D D D E E E E
16 17 18 19 20 21 22
F1 F2 (2012) F2 (2012) F3 G1 H1 H2
F* F* F* F* G H H
23
I1
I
24
I2
I
25
I3
I
26
J1
J*
Materials and Methods Samples Twenty-six wheat flours commercially available in Japan, including 11 that were harvested domestically, were sampled (Table 1). Samples were stored at room temperature until experimental analysis. Total genomic DNA was extracted from samples weighing 100 mg using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions and was stored at 20°C until analysis. The DNA concentration of each sample was measured using a U-0080D photodiode array spectrophotometer (Hitachi High-Technologies, Tokyo, Japan). PCR Amplification of rbcL A partial rbcL sequence was amplified by PCR using the primer set rbcLa_F and rbcLa_R (Table 2) (19,27). Amplification was carried out using a MyCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA) in a total volume of 10 lL. Each reaction contained 200 lM of dNTPs, 0.01% BSA, 0.2 lM of each primer, 0.1 U of ExTaq DNA polymerase (Takara, Otsu, Japan), 19 ExTaq reaction buffer, and 10 ng of DNA. The PCR conditions were as follows: 95°C for 4 min, 35 cycles of 94°C for 30 sec, 55°C for 60 sec, and 72°C for 60 sec, with a final extension at 72°C for 10 min. The amplified products were detected on a 2% agarose gel and purified with the QuickStep2 PCR Purification Kit (Edge BioSystems, Gaithersburg, MD). DNA Sequencing of rbcL Cycle sequencing reactions were carried out using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Foster City, CA) with a MyCycler in a total volume of 20 lL. Direct sequencing of the PCR products was performed using both forward and reverse primers. The cycle sequencing products were analyzed with POP-6 Performance Optimized Polymer (Life Technologies) and detected by a 310 Genetic Analyzer (Life Technologies). The partial rbcL sequence was determined for each sample. Homology searches were performed for similar sequences stored in the GenBank, European Molecular Biology Laboratory (EMBL), and DNA Data Bank of Japan (DDBJ) databases using the Basic Local Alignment Search Tool (BLAST) accessible through the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
1317
*Distributor. The manufacturer is unknown.
The procedure for STR profiling was the same as in a previous report (26). PCR was performed in a final volume of 10 lL, which contained 200 lM of dNTPs, 0.2 lM of each primer, 19 AmpliTaqGold buffer, 0.2 U of AmpliTaqGold DNA polymerase (Life Technologies), and 15 ng of DNA. STR regions were amplified using the following parameters: initial denaturation at 94°C for 9 min, 30 cycles of 94°C for 30 sec, 60°C for 1 min, and 72°C for 1 min, followed by a final extension step of 7 min at 72°C. The amplification reactions were conducted using a MyCycler.
PCR Amplification for STR Genotyping The same extracted DNA as was used for DNA barcoding was also used in STR profiling. Table 2 provides information regarding the primers used for amplification of each STR site.
Capillary Electrophoresis and Data Analysis Fragment analysis was performed using a 310 Genetic Analyzer (Life Technologies). Capillary electrophoresis separation
1318
JOURNAL OF FORENSIC SCIENCES TABLE 2––Primers used to amplify rbcL and STR loci.
rbcL STR
Primer
Sequence
rbcLa_F rbcLa_R TaSE3-F TaSE3-R TaSE37-F TaSE37-R TaSe63-F TaSe63-R TaSe96-F TaSe96-R
50 -gattcgcagatcctccagacgtagagc-30 50 -gattcgcagatcctccagacgtagagc-30 50 -caccgatcgatcaacaagtcaaaa-30 50 -NED-catcatcatcggttcttgga-30 50 -atccgctacggaagaaataccaca-30 50 -VIC-gttgctggcctgccatgttta-30 50 -6FAM-cgtgtgctctcgcagtttcatagt-30 50 -cctcgccttctaattaagctccgt-30 50 -PET-tgggacaagtccctaggtaagacg-30 50 -gtagtccgcccagcctctactttt-30
TABLE 3––Search results for rbcL sequences in the GenBank, EMBL, DDBJ, and PDB databases. Locus rbcL TaSE3 TaSE37
n X i ¼1
n X i ¼1
P2i
P2i
n1 X n X i ¼1 j ¼ iþ1
2P2i P2j
where n is the total number of alleles detected for a locus and Pi is the frequency of the i th allele, respectively (30,31). Power of discrimination (PD) of each locus was determined according to the equation: PD ¼ 1
m X
Pi 2
i ¼1
where m is the total number of genotypes detected for a locus and Pi is the frequency of the ith genotype (32). Power of discrimination of the combination of all loci (APD) was calculated by the formula: APD ¼ 1
k Y
aestivum aestivum aestivum aestivum aestivum
Homology (%)
HQ590312.1 JN133499.1 EU492898.1 AB042240.3 D00206.1
100 100 100 100 100
TaSE96
and PIC ¼ 1
Triticum Triticum Triticum Triticum Triticum
Accession Number
TaSE63
was carried out using the POP-6 polymer (Life Technologies), and the sizes of amplified bands were calculated based on GeneScan 600 LIZ size standard (Life Technologies). Output data were analyzed using GeneMapper ID version 3.2.1 software (Life Technologies). The fluorescent signals that were reached above 1/10 of the main peak height were used for further analysis (28). Genotypic data analyses were performed using GENEPOP v.4.2 (29). Expected heterozygosity (He) and the polymorphic information content (PIC) were calculated for each locus using the formulae: He ¼ 1
Sequence Matched
ð1 PDi Þ
i ¼1
where k is the total number of loci (33). Cluster analysis was performed using the unweighted pairgroup method (UPGMA) with Jaccard similarity coefficients using BioNumerics software (Appliedmath, Kortrijk, Belgium).
Results and Discussion Identification of Raw Materials Using rbcL Sequence of Wheat Flour While most wheat flour products distributed in Japan are made from imported wheat, some flours are made from Japanese
domestic cultivars. In this study, various wheat flour products were examined (Table 1). To identify the species of the source materials, DNA was extracted from commercial flours and rbcL partial regions (553 base pairs [bp]) were sequenced. The concentration of DNA recovered from the extracts ranged from 24.8 ng/lL (No. 25) to 80.9 ng/lL (No. 14). The rbcL region was successfully amplified from all samples and sequenced using both rbcL primers. All sequences were identical among the samples, in spite of the possibility that wheat flour samples contain multiple cultivars. The database search results for the rbcL region are shown in Table 3. Sample sequences matched the wheat (Triticum aestivum L.) type strain sequence, which was the only species that showed 100% similarity. This result indicated that unknown white powder could be identified as wheat flour by determining the rbcL sequence. Previous studies have confirmed sample species from their morphological features (16,18,34), but forensic samples, such as wheat flour, often lack such morphological characteristics. We hypothesized that it might be possible to identify such samples using DNA analysis. Thus, we attempted to determine wheat species in the samples using DNA barcoding and successfully identified all samples (Table 3). Official analytical methods for species identification of wheat are established to detect wheat as an allergenic ingredient in processed foods, but these methods can only determine whether or not a sample contains wheat. Forensic analysis requires rapid testing, so it is desirable to obtain as much information as possible in a single procedure (27). In this study, species (or related species) of the unknown samples could be identified by DNA barcoding, suggesting that it is suitable for forensic analysis (27). Discrimination of Wheat Flour Samples using STR Markers As all samples analyzed in this study were confirmed as wheat flour by rbcL sequencing, possible combinations of wheat cultivars included in each sample were investigated by STR marker analysis. Ten STR markers for Japanese domestic wheat strains have been reported (26), and six of the 10 markers can discriminate both imported and Japanese cultivars. Preliminary examination of these six STR markers showed that markers TaSE3, TaSE37, TaSE63, and TaSE96 were sufficient to discriminate different flour products (data not shown). Therefore, in the current study, we analyzed these four STR sites for all of the flour samples. To examine the accuracy and repeatability of wheat cultivar genotyping using the STR markers, we analyzed the five products listed as having been made from one cultivar using the four STR markers. As expected, the samples showed a single identical product at all four STR sites, and the pattern of product sizes for the markers was consistent with published data (26). The observed peak sizes were slightly different from those reported; this was most likely due to the influence of the dye mass
KIKKAWA ET AL.
.
FORENSIC DNA ANALYSIS OF WHEAT FLOUR
1319
TABLE 4––Characteristics of the STR markers used in this study.
Loci TaSE3 TaSE37 TaSE63 TaSE96 Combination of 4 loci
Allele Size Range (bp)
Number of Alleles
Number of Rare Alleles (
