Spatio-temporal change detection of land use and land cover in Malakand Division Khyber Pakhtunkhwa, Pakistan, using remote sensing and geographic information system.

The land use land cover (LULC) change due to the rapidly growing population is a common feature of the urban area. The rapidly growing population in Malakand Division is a greater threat to the LULC of the area due to its negative impact on environment and ecology. This research aims to detect the variations in LULC from 1991 to 2017 in the Malakand Division, Khyber Pakhtunkhwa (KP) province of Pakistan. The study relies on secondary dataset downloaded from the US Geological Survey (1991, 2001, 2011, and 2017 imageries) and the United Nations Office for the Coordination of Humanitarian Affairs (UN OCHA) website. Maximum likelihood technique under supervised image classification was opted to analyze the LULC changes in between 1991 and 2017. The results were based on six major land use classes including agriculture built-up area, vegetation cover, water bodies, snow cover, and barren land. The results from 1991 to 2017 show a substantial reduction in snow cover and barren land which is consequence of climate change. A known change has been recorded in built-up area which shows an increase from 1.02 to 6.2% with a change of 5.18% of the total land. The vegetation cover water bodies were also showing increase in area. The vegetation cover increased from 28.89 to 44.67% while barren land decreased from 45.68 to 40.29% of the total area. Furthermore, the built-up area increased from 1.02 to 6.2%, whereas water covers increased from 0.63% (1991) to 0.86% (2017) of the total area. The study concludes that there is an immense need for planning to preserve the natural habitat for sustainable development in the area.

Growth performance, haematological assessment and chemical composition of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) fed different levels of Aloe vera extract as feed additives in a closed aquaculture system.

A 105-day experimental trial was conducted to assess different levels of dietary Aleo vera extract supplementation on water quality parameters, proximate composition, growth performance and haematological parameters of fry Oreochromis niloticus. Four different percentages of dietary leaf extract powder of Aleo vera (ALE) with a basal feed, designated as, i.e., T0 (Control group; without ALE), T1 (1% ALE), T2 (2% ALE), and T3 (3% ALE). Fish fry was reared in concrete tanks (7.0 m, 1.6 m, 1.0: L, W, H; water volume 11.2 m3/tank), with an average initial weight 4.04 ± 0.03 g/ fry, and each treatment was triplicated. Fry was randomly distributed at a stocking rate of 450 individuals/ tanks. The water quality parameters revealed that temperature, pH, salinity, dissolved oxygen (DO) and nitrates were found in a promising range as given by FAO/WHO limits. However, the record values obtained for Electric Conductivity (EC), Total dissolved solids (TDS), and alkalinities were not found in all tanks' suitable range according to FAO/WHO limits. The results revealed a significant impact of different percentages of dietary ALE supplementation on fry's body composition and haematological parameters. Moreover, the final body weight, final body length, average daily weight gain (g), net weight gain (g) and specific growth rate (%) were significantly higher (p < 0.05) in T1 and T2 compared with T0 and T3 treatments. The poorest feed conversion ratio was recorded in the T2 group compared with other treatments. Thus, the current study provides information about the nutritional quality of Nile tilapia culturing in Pakistan.

Effect of acute exposure of triazophos on histological structure and apoptosis of the brain and liver of zebrafish (Danio rerio).

Triazophos (TAP) has become a part of widespread pollutant of the aquatic environment due to its residue. Current study was designed to investigate the toxic effect of TAP at different doses (0.06, 0.3 and 1.5 mg/L) to the model organism of zebrafish (Danio rerio) by using multi-endpoint analysis in a 96 h acute exposure test. The direct observation that histological and ultrastructural alteration of zebrafish brain and liver were carried out via paraffin section in hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM), respectively. In addition, a series of methods were applied for exploring the physiological parameters related to cellular apoptosis. Results indicated that vacuolar structure after 96 h treatment with TAP were appeared in the molecular and granular layers of cerebellum. A large number of nuclear retraction, tissues vacuolation and cytoplasmic loss were observed in liver at histological level. From the fine structural level, the mitochondrial vacuolation and membrane damage of brain cells were found and the cristae of mitochondria disintegrated partly in hepatocytes. Onset of such histological structure alterations were one of the most intuitive reflection to TAP exposure, which needs to analyze biochemical alterations for further study. The mitochondrial membrane potential (MMP) showed a downward trend in the brain and liver of zebrafish. Simultaneously, the activity of caspase-3 and caspase-9 increased after 96 h exposure with a concentration-dependent manner, which could be served as a suitable indicator of cellular apoptosis. Furthermore, apoptosis-related genes (Apaf-1, p53, Bax, Bcl-2, caspase-3 and caspase-9) transcription showed different alterations in response to the TAP treatment. These results indicated that TAP exposure led to apoptosis in zebrafish brain and liver and it was speculated that the apoptosis may occur through mitochondrial pathway. The present study demonstrated that the exposure of zebrafish to the insecticide TAP led to observe its effects at both histological structure and apoptosis level in liver and brain.

Comparative expression analysis of let-7 microRNAs during ovary development in Megalobrama amblycephala.

As a critical regulator of gene expression, let-7 family miRNAs have been reported to be involved in multiple physiological processes. In this study, in order to elucidate the putative regulatory effect of let-7 miRNAs during fish gonadal development and to identify which member is crucial for this regulation, the expression of ten members including let-7a/b/c/d/e/f/g/h/i/j were quantified in ovary, pituitary, and brain tissues during the different ovarian developmental stages of blunt snout bream Megalobrama amblycephala. According to the data from analysis of expression patterns, let-7a showed the highest expression value in almost all the tested ovaries, pituitaries, and brains, with let-7b and let-7d moderately expressed, following by other let-7 miRNAs. In terms of the differential expression levels of ten let-7 miRNAs at each developmental stage, the results showed that let-7a/b/d/f/h expression gradually increased during the ovary development from stage I to V and dropped significantly at the phase VI in ovary tissues. However, the expression of let-7a/b/e/f in pituitary increased during the ovary development from stage I to IV and declined at stage V. Among all the let-7 miRNAs, let-7a/b/d had the highest expression and their expression patterns were consistent with the gonad development of M. amblycephala. Furthermore, the mostly predicted target genes of let-7 miRNAs are significantly enriched in signaling pathways closely related to gonadal development through KEGG enrichment analysis. These results indicate that let-7 miRNA members, especially let-7a/b/d, may play important roles in the regulation of ovary development in M. amblycephala through negatively regulating expression of their target genes.

The association between the perception of crime and walking in gated and non-gated neighbourhoods of Asian developing countries.

It has been reported in the literature that the perception of crime at the neighbourhood level inversely affects the walking behaviour of individuals. On the other hand, the gated neighbourhoods are considered safe from crime, however, there is a lack of research on the association of the perception of crime and walking in gated neighbourhoods. Therefore, the objectives of the study reported in this paper were to investigate the association between the perception of crime and walking in gated and non-gated neighbourhoods. A questionnaire was used to collect the data on walking and the perception of crime in 16 neighbourhoods of Karachi Pakistan, 8 out of which were gated. Independent sample t-test and gamma tests were used for the data analysis. The results show that although there is a lower perception of crime in the gated neighbourhoods, yet the inhabitants of gated neighbourhoods engage in less physical activity comprising of walking. In spite of a greater perception of crime in non-gated neighbourhoods, higher values of walking were reported by the residents of those neighbourhoods. Therefore, it has been concluded that there does not exist a definite relationship between the perception of crime and walking behaviour at the neighbourhood level and the perceived safety from crime claimed by the proponents of the gated neighbourhoods does not encourage walking among the residents.

Molecular response and association analysis of Megalobrama amblycephala fih-1 with hypoxia.

Hypoxia is one of the most important environmental factors which affect fish growth, development and survival, but regulation mechanisms of hypoxia in fish remain unclear. Therefore, to further understand molecular functions of factor inhibiting HIF-1 (Fih-1), an essential hypoxia sensor, the full-length cDNA of fih-1 was cloned from Megalobrama amblycephala, a hypoxia-sensitive cyprinid fish. The deduced amino acid sequence showed high homology with that of other vertebrates, and all structural and functional domains were highly conserved. The mRNA level in different tissues and developmental stages indicated that M. amblycephala fih-1 expression was higher in liver and muscle, followed by gill, intestine and spleen. During embryogenesis, the fih-1 mRNA was highly expressed in the early embryonic development, then decreased to a very low level, and maintained a relative high level of expression after hatching. In most tissues, the fih-1 mRNA was down-regulated at 2 h but up-regulated at 4 h after hypoxia treatment. In addition, the promoter sequence of M. amblycephala fih-1 was obtained using thermal asymmetric interlaced PCR. Three single nucleotide polymorphism (SNP) sites were found in the cDNA and promoter sequences, and identified significant association with hypoxia trait by correlation analysis in hypoxia-sensitive group and hypoxia-tolerant group. These results demonstrated that M. amblycephala fih-1 plays important roles in embryo development and hypoxia response, which will contribute to systematic understanding of the molecular mechanisms of fish in response to hypoxia, and provide help for fish genetic breeding with hypoxia-tolerant strains or breeds.

Alternative splicing transcription of Megalobrama amblycephala HIF prolyl hydroxylase PHD3 and up-regulation of PHD3 by HIF-1α.

PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3. Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala.

Molecular characterization and expression analysis of a complement component C3 in large yellow croaker (Larimichthys crocea).

The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. Complement component 3 is a key molecule in the complement system, whose activation is essential for all the important functions performed by this system. In this study, the complete C3 cDNA sequence was isolated from the large yellow croaker (Larimichthys crocea), which was high similarity to other complement C3. We reported the primary sequence, tissue expression profile, polypeptide domain architecture and phylogenetic analysis of L. crocea complement component C3 (L.c-C3) gene. Its open reading frame (ORF) is 4962 bp and encodes for 1653 amino acids with a putative signal peptide of 23 amino acid residues. The deduced amino acid sequence showed that L.c-C3 has conserved residues and domains known to be crucial for C3 function. Phylogenetic analysis showed that L. crocea was closely related to Miichthys miiuy. The mRNA expressions of L.c-C3 was detectable at different tissues. L.c-C3 was expressed in a wide range of adult tissues, it showed highest expression in the liver. But the different developmental stages from fertilized egg to newborn larvae of the large yellow croaker the highest expression levels of L.c-C3 gene were not found. Bacterial challenge experiments showed that the levels of L.c-C3 mRNA expression were up-regulated in the liver, spleen and brain of adult large yellow croaker respectively. The results showed that L.c-C3 mRNA expression in the large yellow croaker is influenced by bacterial stress and L.c-C3 might play an important role in immunity mechanisms. This study will further increase our understanding of the function of L.c-C3 and molecular mechanism of innate immunity in teleosts.

Molecular cloning and expression analysis of immunoglobulin M heavy chain gene of blunt snout bream (Megalobrama amblycephala).

Immunoglobulins (Igs), which bind antigens with high specificity, are essential molecules in adaptive immune system of jawed vertebrates. In this study, cDNA encoding the secreted form of the immunoglobulin heavy chain of IgM (sIgM) was cloned from the mesonephros of blunt snout bream (Megalabrama amblycephala) using RT-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of sIgM heavy chain gene has 1961 nucleotides encoding a putative protein of 569 amino acids, constant region shares high amino acid identity with that of Ctenopharyngodon idella (80%), Carassius auratus langsdorfii (65%) and Danio rerio (59%). Multiple protein sequence alignment revealed that blunt snout bream sIgM was clustered with the homologues of cyprinid fish and constructed one clade. Using quantitative real-time PCR (qRT-PCR) analysis, the level of sIgM mRNA was determined, with a V-shape change pattern: decreased initially from unfertilized egg stage to 4 cells stage and increased from 16 cells stage to prelarva. This sharp drop indicates that sIgM mRNA is maternally transferred, and was continuously degraded until 16 cells stage. The drastic rising in sIgM level from blastula stage to prelarva might be attributed to embryonic stem cell differentiation procedure. Compared with juvenile fish, the expression of sIgM was significantly higher in pronephros, liver, spleen, gill and muscle of adult fish. After the injection of Aeromonas hydrophila, the expression pattern of sIgM was found first down-regulated at 4 h, then up-regulated and reached the peak at 7 d and 21 d in mesonephros, spleen, liver and gill, respectively.

Characterization of muscle morphology and satellite cells, and expression of muscle-related genes in skeletal muscle of juvenile and adult Megalobrama amblycephala.

Hyperplasia and hypertrophy are two distinct processes of skeletal muscle growth regulated by four myogenic regulatory factors (MRFs, contains MyoD, Myf5, Mrf4 and myogenin (MyoG)) and myostatin (MSTN). In this study, characterization of muscle morphology and satellite cells in juvenile (1-year-old) and adult (2-year-old) Megalobrama amblycephala was described. Compared with 1-year-old M. amblycephala, the diameter scope of epaxial, horizontal septum and hypaxial muscle fibers including red and white muscle in 2-year-old fish exhibited broader with dramatic reduction in frequency distribution of <20 µm diameter, nevertheless observable increase in frequency distribution of >50 µm diameter. Intermyofibrillar (IM) nuclei were also found except numerous subsarcolemmal (SS) nuclei in 2-year-old fish, whereas only SS nuclei were observed in 1-year-old fish. Immunofluorescence results showed that more satellite cells existed in red muscle than white muscle in 1-year-old fish, rather than 2-year-old fish. Moreover, we observed predominant increase in the mRNA levels of MyoD, Myf5, Mrf4, and MSTN during muscle development of fish in 2-year-old fish except MyoG.

Anti-stress properties and two HSP70s mRNA expressions of blunt snout bream (Megalobrama amblycephala) fed with all-plant-based diet.

The influence of all-plant-based diet on fingerling blunt snout breams (Megalobrama amblycephala) was tested by examining growth performance, anti-stress properties and related gene expression. Healthy fish were randomly divided into triplicate groups per dietary treatment and fed with different formulated diets. The results showed that both weight gain, specific growth rate and protein efficiency ratio of all-plant-based diet group were significant higher than those of the control (p < 0.05). In contrast, FCR of all-plant-based diet group was significantly lower than that of the control (p < 0.05). Therefore, all-plant-based diets could not affect the growth performance of blunt snout breams. Compared to the control group, the lysozyme levels in serum and mucus, and glutamic-oxaloacetic transaminase activities in serum and liver decreased significantly (p < 0.05). In contrast, the glutamic-pyruvic transaminase activities in serum and liver increased significantly (p < 0.05). For blunt snout breams fed with all-plant-based diets, the superoxide dismutase activities in mucus, serum and liver as well as catalase activity in serum and liver were decreased significantly (p < 0.05) comparing with that of the control group. But malondialdehyde contents were higher (p < 0.05) in serum and liver than that of control group. The expression of HSC70 mRNA increased significantly (p < 0.05) in blunt snout breams fed with all-plant-based diet, whereas the HSP70 mRNA expression decreased significantly (p < 0.05) when compared with control group. In conclusion, all these results indicated that the application of all-plant-based diet could decrease the anti-stress properties (non-specific immunity, stress resistance and antioxidant ability) and HSP70 mRNA expression in blunt snout breams fingerling. Although all-plant-based diets could not affect the growth performance of blunt snout breams, the application of all-plant-based diet should be discreet in the production practice.

PPARγ, an important gene related to lipid metabolism and immunity in Megalobrama amblycephala: cloning, characterization and transcription analysis by GeNorm.

In order to be able to modulate and improve the function of PPARγ and decrease further some metabolic diseases of M. amblycephala, we have cloned and identified the full-length cDNA of PPARγ in M. amblycephala and examined its transcription patterns at different embryo developmental stages and in different tissues of adult and immature fish. We also accurately normalized seven reference genes by GeNorm and calculated their gene transcription normalization factors. The full-length of PPARγ was 1968 bp, consisting of 218 bp 5'-untranslated region, 1,533 bp open reading frame encoding 510 amino acids residues and 217 bp 3'-untranslated region. M. amblycephala PPARγ peptide was predicted to consist of 4 conserved domains, i.e. N-terminal domain, DNA-binding domain, ligand binding domain and flexible hinge region. PPARγ mRNAs were detected in all studied tissues of adult and immature fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, PPARγ transcription in liver was highest, followed by gills and it was lowest in female gonads. Moreover, the differences among liver, gill, intestine/brain, spleen/white muscle, kidney and female gonads were greatly significant (p<0.01). The transcription of PPARγ in male gonads was significantly higher than in female gonads (p<0.01). In immature fish, the transcription of PPARγ was highest in intestines followed by adipose tissue, and it was lowest in hearts and white muscles. A great difference was observed (p<0.01) in the transcription of PPARγ among adipose tissue, intestines, liver and heart/white muscles. At different embryo developmental stages, PPARγ transcription in unfertilized spermatozoa was greatly higher than in unfertilized ovum (p<0.01) and it was highest among different embryo developmental stages. The transcription of PPARγ increased gradually during 2 cells stage and 32 cells stage and then decreased until gastrula stage at which it was lowest. The transcription of PPARγ increased again on first day after hatching. There was a significant difference (p<0.01) in the transcription of PPARγ between 2 cells stage and 32 cells stage and it was same between 32 cells stage and gastrula stage. These results revealed that transcription of PPARγ showed a tissue-dependent regulation and a developmental-stage-dependent regulation that are valuable and helpful to improve the function of PPARγ and to decrease some metabolic diseases in the culture of M. amblycephala.

Flow cytometric determination of genome size for eight commercially important fish species in China.

The genome size (C value) of eight commercially important fish species in China was measured using flow cytometry. Chicken (Gallus domesticus) erythrocytes were used as reference cells. When using propidium iodide (PI) as the fluorescent dye, genome sizes were 1.09 ± 0.08, 2.75 ± 0.12, 1.05 ± 0.05, 1.35 ± 0.11, 0.99 ± 0.05, 0.90 ± 0.08, 0.90 ± 0.07, and 0.88 ± 0.07 pg for Japanese eel (Anguilla japonica), mullet (Myxocyprinus asiaticus), yellowcheek carp (Elopichthys bambusa), blunt snout bream (Megalobrama amblycephala), yellow catfish (Pelteobagrus fulvidraco), ricefield eel (Monopterus albus), mandarin fish (Siniperca chuatsi), and snakehead (Ophicephalus argus), respectively. However, genome sizes were 1.25 ± 0.00, 3.08 ± 0.02, 1.25 ± 0.00, 1.57 ± 0.01, 0.96 ± 0.01, 1.00 ± 0.01, 0.91 ± 0.01, and 0.89 ± 0.01 pg for these fishes, respectively, when 4', 6-diamidino-2-phenylindole (DAPI) was used as the fluorescent dye. Regardless of the dye used, the more evolutionarily advanced species had a smaller genome size than those with a lower evolutionary status. For each species, we also measured the size of erythrocytes and their nucleus and evaluated the relationships between erythrocyte size, nucleus size, chromosome number, and genome size. Genome size was positively correlated with erythrocyte nucleus size and chromosome number when using PI as the fluorescent dye, but it was only correlated with erythrocyte nucleus size when DAPI was used.

Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits, HIF-1α/2α/3α of the hypoxia-sensitive freshwater species, Chinese sucker.

Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in oxygen tension in the cellular environment. In this study, we identified full-length cDNAs of HIF-1α, HIF-2α and HIF-3α in an endangered hypoxia-sensitive fish species, Chinese sucker. The HIF-1α/2α/3α cDNAs are 3890, 3230 and 3374 bp in length, encoding 780, 782 and 632 amino acid residues, respectively. The real-time PCR results suggested that HIF-1α and HIF-3α mRNAs were highly expressed in liver and gonads, followed by spleen and muscle. Moreover, HIF-1α and HIF-3α transcription factors revealed similar developmental expression patterns, with the lowest expression at 48h post-fertilization, and reaching the highest expression level at 360 h post-fertilization. Short-term hypoxia exposure (2.2, 2.8 and 3.2mg/L dissolved oxygen for 24h) increased mRNA levels of HIF-1α and HIF-3α. HIF-2α mRNA showed similar expression patterns with that of HIF-1α and HIF-3α, however, its expression was extremely low in the spatio-temporal expression patterns and hypoxia treatment. This is the first report describing the potential to identify hypoxia-sensitive/tolerant fishes according to the number of the serine residues of fish oxygen-dependent degradation (ODD) domain. It was suggested that Cyprinomorpha fishes, with less than 40 serine residues in fish ODD domain were hypoxia-sensitive fishes and more than 40 serine residues in this domain were hypoxia-tolerant fishes.

Expression characterization and the promoter activity analysis of zebrafish hdac4.

Histone deacetylase 4 (HDAC4) is an important modifier enzyme for chromatin remodeling and plays an essential role in regulating gene expression. Spatio-temporal expression spectrum revealed that zebrafish hdac4 mRNA, ubiquitously distributed in various tissues, were significantly higher at 36 hpf (hours post-fertilization) and 6 dpf (days post-fertilization) than other periods. Trichostatin A (TSA) inhibited the development of zebrafish embryos and transcription of hdac4 and mef2a (myocyte enhancer factor-2A). Moreover, five vectors containing different promoter regions of hdac4 were constructed in order to analyze promoter activity. The vector containing the region from -125 to +160 exhibited maximum luciferase activity that was approximately 30.3-fold and 58.9-fold higher than the control in two kinds of cells, respectively. By comparing the luciferase activities between the region from -302 to +30 and -698 to +30, it was suggested that the region between -698 and -302 might contain mild negative regulatory elements.

Cloning, identification and accurate normalization expression analysis of PPARα gene by GeNorm in Megalobrama amblycephala.

Megalobrama amblycephala suffers from serious liver diseases recently and PPARα gene has been reported to play an important role in the immune system of animal liver. On the basis of these facts, we have cloned and identified full-length cDNA of PPARα and examined its expression patterns at different embryo developmental stages and in different tissues of adult and young fish in order to improve liver disease immunity of M. amblycephala. We also accurately normalized seven reference genes by GeNorm and calculated their gene expression normalization factors. The total length of PPARα cDNA was 2021 bp, comprising of 214-bp 5'-untranslated region; 1404-bp open reading frame (encoding 467-amino acids); and 403-bp 3'-untranslated region. PPARα peptide was predicted to consist of 4 domains, i.e. A/B, C, D, and E/F. PPARα mRNAs were detected in different tissues of adult and young fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, the expression of PPARα in white muscles was highest followed by liver and it was lowest in gonads. Its expression in male gonads was significantly higher than female gonads. In young fish, the expression of PPARα was highest in brain, followed by intestines and it was lowest in spleen. At different embryo developmental stages, the expression of PPARα was highest at 2 cells stage and it was lowest at gastrula stage, but it increased on first day after hatching. In unfertilized spermatozoa, the expression of PPARα was higher than unfertilized ovum.

DNA extraction from crayfish exoskeleton.

Crayfish exoskeleton (CE) samples are generally less invasive and easy to be collected. However, it is difficult to extract DNA from them. This study was intended to investigate CE as a DNA source and design an easy and efficient DNA extraction protocol for polymerase chain reactions. Specific primer pair (PPO-F, PPO-R) was used to amplify extracted DNA from CE, and compared to crayfish tail muscle DNA sample. Moreover, seven microsatellites markers were used to amplify the CE DNA samples set. Since the extracted DNA from CE is suitable for gene amplification, the results present usefulness of CE as an easy and convenient DNA source for PCR-based population genetic research.