**3. Results**

112 Biodiversity Conservation and Utilization in a Diverse World

The amplification reaction used for amplification of the D-loop fragment was also used (with little modifications in temperature cycling) in the other experiments according to the

The amplification reaction was carried out in a 25 μl reaction mixture consisting of 1.25 unit Taq polymerase (DyNAzyme), 1X enzyme buffer (1X is 10 mM Tris-HCl, pH 8.8 at 25 0C, 1.5 mM MgCl2, 50 mM KCl and 0.1% Triton X-100) supplied by the manufacture, 1 μM of each forward and reverse primer, 0.2 mM dNTPs and 100 ng of DNA. The reaction mixture was overlaid with sterile mineral oil and was run in an MJ research PTC-100 Thermocycler. The temperature cycling was as follows: 30 cycles of 45 seconds at 94°C; 1 minute at 58°C and 1 minute at 71°C, followed by a final extension at 71°C for 5 minutes. All PCR amplifications included a negative control reaction which lacked template DNA. No product was seen in any negative control. Small quantities of the reaction products (5 μl each) were used for electrophoresis with an appropriate size marker on 1.5% agarose in 1X-Tris acetate buffer

After electrophoresis the gels were stained with ethidium bromide and were examined with UV lamp at a wave length 312 nm to verify amplification of the chosen specific fragment. The PCR products were purified using QIAquick PCR purification kit (Qiagen, Inc.) and the resulting purified products were used in the subsequent sequencing reactions. Sequencing was performed on an Applied Biosystems 310 genetic analyzer (Applied Biosystem) using Big Dye terminator cycle sequencing ready reaction mixture according to manufacturer's

Pairwise sequence alignments were carried out using NCBI-BLASTN 2.2.5 version & PSI BLAST. Multiple sequence alignments were done using the MUSCLE 3.6 software and CLUSTALW (1.82). Analysis, manipulation, conservation plots, positional entropy plot and conserved region analysis was done using the BIOEDIT package. Variable sites were

Phylogenetic model selection was done using the FINDMODEL server available from the HCV LANL database at (http://hcv.lanl.gov/ /content/hcv-db/findmodel/). A Bayesian phylogenetic tree was constructed by Markov chain Monte Carlo (MCMC) method as implemented in the MR BAYES 3.1 package using the Hasegawa-Kishino-Yano plus Gamma model HKY+G substitution model with an invariant four category gamma distribution among sites. A 50% consensus tree was generated and the analysis was repeated two times. Maximum parsimony tree was conducted using MEGA version 4, with 1000 bootstraps for

extracted from the multiple sequence alignment using the MEGA 3.1 package **[12]**.

**2.3. The amplification reaction** 

conditions of each experiment.

instructions (Applied Biosystems).

**2.5. Phylogenetic analysis** 

reliability.

**2.4. Sequence analysis and multiple sequence alignment** 

(TAE).

Our experience in the field of molecular identification or DNA barcoding through a series of published research papers are represented in this section Results with some illustrated figures and tables are represented here but the complete information could be obtained through obtaining the complete published papers from the publication section.

Shows the Positional entropy plot of the D-loop for the buffalo, and cow sequences The Bayesian phylogenetic trees of cow and buffalo sequences were constructed using MRBAYES software (Figure 2) and Maximum parsimony tree using the Kimura twoparameter model and the closest neighbor interchange method of the MEGA 3.1 software package (Figure 3). Table 1. Shows the Substitution events detected in complete D-loop sequences from multiple sequence alignments between cows and buffaloes.

**Figure 1.** Positional Entropy Plot of the D loop for the Buffalo, and cow sequences. The X axis is the nucleotide position, while the Y axis is the entropy (lack of information) at that position. The central region is mainly conserved, and the beginning and end regions are highly variable (shaded areas).

Biological Identifications Through DNA Barcodes 115

**Figure 3.** Maximum parsimony tree constructed using the Kimura two-parameter model and the closest neighbor interchange method of the MEGA 3.1 software package. The numbers show the percentage of

bootstrap confidence.

**Figure 2.** Bayesian phylogenetic tree of the cow and buffalo sequences using the MRBAYES software.

**Figure 2.** Bayesian phylogenetic tree of the cow and buffalo sequences using the MRBAYES software.

**Figure 3.** Maximum parsimony tree constructed using the Kimura two-parameter model and the closest neighbor interchange method of the MEGA 3.1 software package. The numbers show the percentage of bootstrap confidence.

116 Biodiversity Conservation and Utilization in a Diverse World


Biological Identifications Through DNA Barcodes 117

**Figure 5.** Phylogenetic tree constructed between the Egyptian and GenBank database chicken samples.

Sample DQ629875 was used as out-group for its high diversity.

**Table 1.** Substitution events detected in complete D-loop sequences from multiple sequence alignment between Cows (35 animals) and buffaloes (53 animals).

Shows the PCR amplification of chicken mitochondrial D loop fragments while the phylogenetic tree constructed between the Egyptian and GenBank database chicken samples is represented in Figure 5. The Polymorphic sites and their positions are shown in Table 2.

**Figure 4.** PCR amplification of chicken mitochondrial D loop fragment. The PCR reactions were run on 1% agarose gel, stained with ethidium bromide and examined with UV. Samples from 1 to 4 for Dandarawi breed and samples from 5 to 8 for Fayoumi breed. The *Hae* III digest of Ф X174 DNA was used as ladder (1353, 1078, 872, and 603 base pairs).


Total number of indels (insertions/

between Cows (35 animals) and buffaloes (53 animals).

used as ladder (1353, 1078, 872, and 603 base pairs).

Animals Cows (35) Buffaloes (53) Cows &

Substitutions and genetic distances Value S E Value S E Value S E

Genetic distance between the two groups 0.156 0.016 Overall (all animals) distance 0.06 0.006

**Table 1.** Substitution events detected in complete D-loop sequences from multiple sequence alignment

Shows the PCR amplification of chicken mitochondrial D loop fragments while the phylogenetic tree constructed between the Egyptian and GenBank database chicken samples is represented in Figure 5. The Polymorphic sites and their positions are shown in Table 2.

**Figure 4.** PCR amplification of chicken mitochondrial D loop fragment. The PCR reactions were run on 1% agarose gel, stained with ethidium bromide and examined with UV. Samples from 1 to 4 for Dandarawi breed and samples from 5 to 8 for Fayoumi breed. The *Hae* III digest of Ф X174 DNA was

Total numbers of transitions 44 20 100 Total numbers of transversions 4 1 61

deletions) 1 7 15

Total number of substitutions 49 28 176 R ratio (transversions/transitions) 0.09 0.05 0.61

Genetic distance within group 0.023 0.003 0.007 0.002

buffaloes (88)

**Figure 5.** Phylogenetic tree constructed between the Egyptian and GenBank database chicken samples. Sample DQ629875 was used as out-group for its high diversity.


Biological Identifications Through DNA Barcodes 119

**Figure 7.** Agarose gel representing the PCR-amplified product of ITS. M: 50 bp DNA size marker, lane

5'CAATCTAATAAGTTTCAGACTTTCGGGGCTTTGGGTATAATTTATGCTATATTGTCTATTGGG ATTTTAGGGTTTATTGTATGAGCTCATCATATATTTACAGTAGGAATAGATGTTGATTCTCGAG CTTATTTTACCGCTGCTACGATAATTATTGCGGTACCTACAGGTATTAAGGTTTTTTCTTGATT ATCTACAATGTATGGGTCAGTAGTAAAGTGAGGAGTAGTTGTTTTGTGAAATTTTGGTTTTATT

**Figure 8.** Sequence of the mitochondrial gene fragment coding for cytochrome oxidase subunit I(COI)

5'GNCACCTGATTTAGATCACGTCATAATGGTTTGTTTTGGTTACTCTTGTGACCAAGAGAATT TATACCATTGCATGGCGATACCGACGAGGGCTGCAGCAAAGGTAAATACGTATGATACGGTT TATATACTCGAAACAAATGTAGCACAGATACAGTAAGTGTCCGTGCTGAAATTTTCATTCAA AAACACAATGCTCACAAAATTTCACAACTCACATCAATTTCCACAAATTACTATGTTTTTCAT CGATTTAAGGACTAAGTGATCCCCCATATTGAGTCTTGGTTTTTTTCTTCTCGATAGCACAACT TACTTCCAAAGGAAGTGAAAAGGTTTGTCGGAATGGTTACCGACTTACTGTCGCATACGCCTT CCCCGTAACCAAAAGGGACCGGTTAAACACCCACCAATTCGAAGCGGTTGTCGCCCAAAAA GGGACCGGTTTGGAAGACCGTTATCCACCGTTAAAACCTAAATTACAGGTTTGTTTTTTTCAT

TTTTTATTCAGGTTAGGTGGTTTAACCGGGGTGGTTTTGTCAAA3'

**Figure 9.** Sequence of the internal transcribed spacers (ITS) region in the predacious mite*, Amblyseius* 

1: ITS fragment and lane 2: blank.

in the predacious mite, *Amblyseius swirskii.*

TGGTCCTTTTTCGAAAAAACACAN3

*swirskii.* N: unknown base.

**Table 2.** The Polymorphic sites and their positions. The nucleotide positions were given with respect to the Egyptian nucleotide numbers in GenBank database. The left column shows the breeds with their accession numbers.

The following Figures: show polyacrylamide gel representing the PCR-amplified fragment of CO1 and its sequence (Figures 6 & 8) while the PCR-amplified fragment of ITS region and its sequence were presented in Figures 7 & 9.

**Figure 6.** 10% polyacrylamide gel representing the PCR-amplified product of CO1. M: 50 bp DNA size marker, lane 1: CO1 fragment and lane 2: blank (PCR cocktail without DNA).

its sequence were presented in Figures 7 & 9.

accession numbers.

Breed & Accession number Variable sites and their positions

23 35 276 457 464 483

EgyDand1 EF586881 T A C A G T

EgyDand2 EF586882 T A C C T T EgyDand3 EU352856 T A C A G A

EgyFay1 EF586879 T A A A G A EgyFay2 EF586880 T A C C T A

DQ629875 A \* C A G A Database public sequence T A C A G A

**Table 2.** The Polymorphic sites and their positions. The nucleotide positions were given with respect to the Egyptian nucleotide numbers in GenBank database. The left column shows the breeds with their

The following Figures: show polyacrylamide gel representing the PCR-amplified fragment of CO1 and its sequence (Figures 6 & 8) while the PCR-amplified fragment of ITS region and

**Figure 6.** 10% polyacrylamide gel representing the PCR-amplified product of CO1. M: 50 bp DNA size

marker, lane 1: CO1 fragment and lane 2: blank (PCR cocktail without DNA).

**Figure 7.** Agarose gel representing the PCR-amplified product of ITS. M: 50 bp DNA size marker, lane 1: ITS fragment and lane 2: blank.

5'CAATCTAATAAGTTTCAGACTTTCGGGGCTTTGGGTATAATTTATGCTATATTGTCTATTGGG ATTTTAGGGTTTATTGTATGAGCTCATCATATATTTACAGTAGGAATAGATGTTGATTCTCGAG CTTATTTTACCGCTGCTACGATAATTATTGCGGTACCTACAGGTATTAAGGTTTTTTCTTGATT ATCTACAATGTATGGGTCAGTAGTAAAGTGAGGAGTAGTTGTTTTGTGAAATTTTGGTTTTATT TTTTTATTCAGGTTAGGTGGTTTAACCGGGGTGGTTTTGTCAAA3'

**Figure 8.** Sequence of the mitochondrial gene fragment coding for cytochrome oxidase subunit I(COI) in the predacious mite, *Amblyseius swirskii.*

5'GNCACCTGATTTAGATCACGTCATAATGGTTTGTTTTGGTTACTCTTGTGACCAAGAGAATT TATACCATTGCATGGCGATACCGACGAGGGCTGCAGCAAAGGTAAATACGTATGATACGGTT TATATACTCGAAACAAATGTAGCACAGATACAGTAAGTGTCCGTGCTGAAATTTTCATTCAA AAACACAATGCTCACAAAATTTCACAACTCACATCAATTTCCACAAATTACTATGTTTTTCAT CGATTTAAGGACTAAGTGATCCCCCATATTGAGTCTTGGTTTTTTTCTTCTCGATAGCACAACT TACTTCCAAAGGAAGTGAAAAGGTTTGTCGGAATGGTTACCGACTTACTGTCGCATACGCCTT CCCCGTAACCAAAAGGGACCGGTTAAACACCCACCAATTCGAAGCGGTTGTCGCCCAAAAA GGGACCGGTTTGGAAGACCGTTATCCACCGTTAAAACCTAAATTACAGGTTTGTTTTTTTCAT TGGTCCTTTTTCGAAAAAACACAN3

**Figure 9.** Sequence of the internal transcribed spacers (ITS) region in the predacious mite*, Amblyseius swirskii.* N: unknown base.

Sequencing of a fragment of ITS region (ITS1, ITS2, 5.8S) indicated almost complete identity of the Egyptian samples with *Neoseiulus swirskii,* accession number EU 310505 (= *A. swirskii*). Regardless the locality within Egypt, taxa were less identical when compared to the related species *N. Cucumeris (*Oud*.) N. andersoni* (Chant), and *N. fallacis* (Gar-man).

Biological Identifications Through DNA Barcodes 121

nucleotide Base position

Thymine position 172

Guanine position 158 Cytosine position 267 Adenine position 293

Thymine positions 32 and 267 Guanine position 293 Cytosine position 72

Guanine positions 193, 266 and 273

Thymine positions 26, 36, 186 & 190 Cytosine positions 253, 294 and 295.

Guanine positions 172, 231 and 300

Adenine 27 and 174

Adenine position 190 Thymine position 271 Cytosine position 349 insertion of Adenine position 164-165 deletion of Adenine position 259

Guanine position 299 Thymine positions 260, 261 and 317 Cytosine positions 225 and 259 deletion of Adenine deletion at position 273

**Table 4.** Nucleotide variation in a specific 12S rRNA gene fragment of four studied Bovidae species. Nucleotide positions correspond to Egyptian buffaloes GenBank accession numbers (FJ828575-FJ828583).

Considering multiple alignment results between homologous 16S rRNA sequences obtained from GenBank database with the reference sequence, it was shown that, the entire 16S rRNA fragment (422 bp. in size) contains more than 57 variable sites (from base no. 21 to base no. 323) inside the two conserved regions. The bases outside this variable region are completely conserved in the four species (Figure 10 and Table 5). From these variable sites, 25 specific nucleotides were chosen (which gave clear significant results in both types of alignment comparisons (two and multiple alignment sequences programs) as a reference for identification of unknown species (from base no. 21 to base no. 308). It was also shown that the size of the amplified fragments were less by one nucleotide (421 bp) in case of goat and

Detection of specific variable sites between Egyptian buffalo 16S rRNA gene fragment and the other studied three species is shown to be a good marker for identification of the four studied species. The detected variable sites can be classified as represented in both Fig. 10 and Table 5.

Guanine at positions 110, 132 and 196

Cytosine positions 71, 269, 271 and 348.

No. of

6 + 2

6 + 1

SNPs Specificity Representing

<sup>8</sup>all buffaloes (haplotypes 1 & 2)

3 buffalo haplotype 1 only

4 buffalo haplotype 2 only

12 cattle (*Bos taurus*)

indels sheep (*Ovis aries)*

deletion goat (*Capra hircus)*

two nucleotides (420 bp) in case of both cattle and sheep.

According to the molecular analysis (Table 3), samples are grouped into three groups.


**Table 3.** The variable sites (a = Adenine, c = Cytosine, g = Guanine, t = Thymine, - = deletion and n = Not detected) detected in a fragment of nuclear ITS region of six samples of *A. swirskii* collected from citrus and grapes in the Nile delta of Egypt.

The results of 12S rRNA showed that, two haplotypes of 12S rRNA sequences were identified from the multiple alignment results between the nine tested Egyptian buffalo sequences and other examples of homologous buffalo sequences selected from GenBank database. Two buffalo haplotypes were revealed, of which haplotype 1 which include Egyptian buffaloes and haplotype 2 which include Chinese swamp buffalo; breed: Haikou (accession AY702618), Mediterranean (accession AY488491) and *Bubalus bubalis* (accession AF231028). The detected SNPs can be classified as shown in table 4.

Eleven SNPs were detected which can be used to discriminate between subfamily Bovinae, represented by buffalo and cattle and the subfamily Caprinae represented by sheep and goat.


Nucleotide number (Accession No. EU924213)

Sample Accession

Sample 3 EU924213

Sample 1 EU924212

Sample 5 EU924216

goat.

Number

citrus and grapes in the Nile delta of Egypt.

Sequencing of a fragment of ITS region (ITS1, ITS2, 5.8S) indicated almost complete identity of the Egyptian samples with *Neoseiulus swirskii,* accession number EU 310505 (= *A. swirskii*). Regardless the locality within Egypt, taxa were less identical when compared to the related

According to the molecular analysis (Table 3), samples are grouped into three groups.

2 3 3 3 3 5 8 8 1 4 5

Sample 4 EU924214 c c c a c c a t a c c a g t c a t a c c - a g - a g

Sample 2 EU924215 a a t a t c a t a - c a g t c a t a c c - a g - a g

*N. swirskii* EU310505 a a t - t g t - - - t - t t t t - t - t t t t t a t

Sample 6 EU924217 n n n - t g t - - - t a g a t a - c - t t a t - g t

The results of 12S rRNA showed that, two haplotypes of 12S rRNA sequences were identified from the multiple alignment results between the nine tested Egyptian buffalo sequences and other examples of homologous buffalo sequences selected from GenBank database. Two buffalo haplotypes were revealed, of which haplotype 1 which include Egyptian buffaloes and haplotype 2 which include Chinese swamp buffalo; breed: Haikou (accession AY702618), Mediterranean (accession AY488491) and *Bubalus bubalis* (accession

Eleven SNPs were detected which can be used to discriminate between subfamily Bovinae, represented by buffalo and cattle and the subfamily Caprinae represented by sheep and

**Table 3.** The variable sites (a = Adenine, c = Cytosine, g = Guanine, t = Thymine, - = deletion and n = Not detected) detected in a fragment of nuclear ITS region of six samples of *A. swirskii* collected from

1 4 6 1 4 7

Group ITS1 5.8S rRNA ITS2

1 4 8 1 4 9 1 6 3 1 8 2 2 1 6 2 2 3 2 4 9

c c c a c c a t a c c a g t c a t a c c - a g - a g

a a t - t g t - - - t - t t t t - t - t t t t t a -

n a t - t g t - - - t a g a t t - c c t t a t g g t

2 6 2 2 7 1 2 7 4 2 8 7 3 0 0 3 0 4

3 1 4 3 1 5

3 2 3 3 3 2

species *N. Cucumeris (*Oud*.) N. andersoni* (Chant), and *N. fallacis* (Gar-man).

Phylogenetic

Group 1

Group 3

Group 2

AF231028). The detected SNPs can be classified as shown in table 4.

**Table 4.** Nucleotide variation in a specific 12S rRNA gene fragment of four studied Bovidae species. Nucleotide positions correspond to Egyptian buffaloes GenBank accession numbers (FJ828575-FJ828583).

Considering multiple alignment results between homologous 16S rRNA sequences obtained from GenBank database with the reference sequence, it was shown that, the entire 16S rRNA fragment (422 bp. in size) contains more than 57 variable sites (from base no. 21 to base no. 323) inside the two conserved regions. The bases outside this variable region are completely conserved in the four species (Figure 10 and Table 5). From these variable sites, 25 specific nucleotides were chosen (which gave clear significant results in both types of alignment comparisons (two and multiple alignment sequences programs) as a reference for identification of unknown species (from base no. 21 to base no. 308). It was also shown that the size of the amplified fragments were less by one nucleotide (421 bp) in case of goat and two nucleotides (420 bp) in case of both cattle and sheep.

Detection of specific variable sites between Egyptian buffalo 16S rRNA gene fragment and the other studied three species is shown to be a good marker for identification of the four studied species. The detected variable sites can be classified as represented in both Fig. 10 and Table 5.


Biological Identifications Through DNA Barcodes 123

Guanine (36, 189, and 297)

Thymine (190 and 221)

Guanine (252 and 254) Thymine (227) Cytosine (50).

Thymine (102, 129 and 249)

Cytosine (298 and 308)

Adenine (298) Cytosine (102, 129 and 249) Thymine (308)

Guanine (119, 171, and 251) Adenine (122) Thymine (167) Cytosine (295 and 301)

Thymine (29) Cytosine (21 and 182)

Specificity Representing nucleotides Base position

Three Cattle (*Bos taurus*) Guanine (55)

**Table 5.** Nucleotide variation in a specific 16S rRNA gene fragment of four studied Bovidae species. Nucleotide Positions correspond to Egyptian buffaloes GenBank accession numbers (FJ748599–FJ748607)

The ability of molecular trees to encompass both short and long periods of time is based on the observation that different genes evolve at different rates. The DNA specifying ribosomal RNA (rRNA) changes relatively slowly, so comparisons of DNA sequences in these genes are useful for investigating relationships between taxa that diverged hundreds of millions of years ago. Studies of the genes for rRNA have shown, for example, that fungi are more closely related to animals than to green plants—something that certainly could not have

In contrast, the DNA in mitochondria (mtDNA) evolves relatively rapidly and can be used

The methodology used in DNA barcoding has been straightforward. Sequences of the barcoding region are obtained from various individuals. The resulting sequence data are then used to construct a phylogenetic tree using a distance-based 'neighbour-joining' method. In such a tree, similar, putatively related individuals are clustered together. The term 'DNA barcode' seems to imply that each species is characterized by a unique sequence,

**4.1. DNA barcoding, genome evolution & phylogenetic trees** 

No. of Variable sites (SNPs)

Five

Five

**4. Discussion** 

Six River buffaloes

Seven Sheep (*Ovis aries*)

Four Goat (*Capra hircus*)

Group one (river buffaloes and cattle, Subfamily Bovinae)

Group two (sheep and goat, Subfamily Caprinae).

been deduced from morphological comparisons alone.

to investigate more recent evolutionary events.

**Figure 10.** Multiple sequence alignments result showing the total variable sites between river buffalo (BBU), Cattle (Bost, Bosi and JBC), Sheep (Ovis) and goat (Capra) in the specific 16S rRNA fragment. Sequences of the 16S rRNA fragment of Egyptian buffaloes (FJ748599–FJ748607). Differing nucleotides are noted (T, A, G and C)


**Table 5.** Nucleotide variation in a specific 16S rRNA gene fragment of four studied Bovidae species. Nucleotide Positions correspond to Egyptian buffaloes GenBank accession numbers (FJ748599–FJ748607)
