基于不同长度的COI和COII基因片段分子鉴定嗜尸性双翅目昆虫
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摘要
1季节性演替实验鉴定法医学重要节肢动物
背景
法医昆虫学(forensic entomology)研究内容主要是对在尸体内、尸体表面或尸体附近采集到的昆虫标本,进行检验、鉴定、分类后,明确昆虫种类或确定某一昆虫生长发育状态,结合昆虫在尸体上的生态群落演替,帮助法医完成死亡时间(postmortem interval, PMI)的推断。昆虫在尸体上是按一定的次序出现并繁殖。目前,在埃及有关不同腐败阶段的动物区系演替实验和他们在法律上应用报道还很少。而且,也没有埃及东北部有关法医重要意义昆虫的数据报道。
目的
1.第一次在埃及东北部(32015'E,30°36'N)调查法医重要性意义的昆虫。
2.调查冬夏季节动物尸体上的主要昆虫类群,并分析尸体分解速度的季节性差异。
3.观察最早到达尸体的双翅目种类及嗜尸性昆虫的演替规律。
方法：
1.连续2年(2011/2012)的冬夏季节用动物尸体进行实验。
2.在每次观察中,检查尸体的腐败阶段,采集不同发育阶段的有代表性的样本(死的和活着的样本),并记录温度和相对湿度等环境变量。
3.请分类专家依据本地物种的形态学特征对物种行形态学鉴定。
结果
1.夏季在动物尸体上取得了3目7科10种的物种,而冬季在动物尸体上获得了4目8科12种的物种。虽然在冬季可以收集到更丰富的样本物种,但在夏季每个物种的样本数量更多。
2.一般情况下,蝇科和厕蝇科在夏季研究中占主导地位而麻蝇科在冬季占主导地位。
3.冬季动物尸体的分解的速度明显慢于在夏季的速度。
4.相比夏季,在冬季双翅目到达尸体明显延迟。夏季最先到达尸体的类群是家蝇,而在冬季最先到达的白头裸金蝇。
5.通过分析所收集到的样本,构建了夏季和冬季的物种的共生矩阵表。
结论
本研究结果提示开展地域性昆虫演替规律调查研究是十分重要的,在不同的区域范围内确定最有可能出现的昆虫种类对于法医学案例也至关重要。演替实验也是评估分子标记鉴定能力的一个基本步骤。
2细胞色素氧化酶基因Ⅰ (COⅠ):利用短片段和长片断鉴定嗜尸性双翅目昆虫
背景
在法医学领域,正确的鉴定是一个关键性的问题,因为有缺陷的法医学证据可能导致审判不公。在以被准确评估后的遗传标记为指导的鉴定工具包中,以分子为基础的方法被认为是一个重要工具。本文第一次使用线粒体细胞色素氧化酶基因Ⅰ (COⅠ)的272-bp和1173-bp两个片断对来自埃及的嗜尸性昆虫进行评估。并且,由于现有中国的相关数据全部来自对短片段的研究,本文也第一次使用COI的长片断对中国的嗜尸性昆虫进行评估。因此,这些研究对于确定以上片断的适用性是非常有意义的。从两个地区收集到的样本对于使用这些分子标记鉴定物种和种群具有重要意义。
目的
-确定COI长、短两个片断对于鉴定嗜尸性双翅目昆虫的鉴别效力,并且通过以下三个方面评估和比较这两个片断：
1、种内及种间差异
2、所研究物种的单系分离
3、通过邻接法建立于与分类学一致的系统发育树
-确定这些片断在种群水平上鉴定同种的鉴别效力。
方法
1、形态学鉴定来自18个物种的50个样本。
2、DNA提取。
3、使用两个片断的引物进行PCR扩增,之后产物直接用于测序。
4、使用Kimura双参数距离模型(K2P)对核苷酸序列的分歧进行计算,通过邻接法(NJ)建立系统发育树。
结果
1、在种内和种间差异中两个片断显示了类似的结果。
2、长片断能够以高的自举值完整地区分单系物种,明显优于短片段。
3、使用长片断对这些物种建立的NJ系统发育树与分类学结果较为一致,远远优于短片段。
结论
1、COI长片段的鉴定结果更为可靠和安全,因为它能够优化分歧,特别是对那些具有低水平分歧的物种。
2、长片断另外一个优点是除了能够得到与形态学分类相一致的结果之外,还特别适用于鉴定已建立本地数据库中的新物种。
3、在种群水平上COI基因不能区分同种昆虫。
3细胞色素氧化酶基因Ⅱ：利用短片段鉴定嗜尸性双翅目昆虫
背景
由于相对较高的变异度,细胞色素氧化酶基因Ⅱ可以成功用于进化学研究、群体遗传学和分类学。然而,仅有少数文献报道使用该基因进行嗜尸性蝇类鉴定。在将任何遗传学标记用于实际案例之前,需要对这些标记进行评估。为了作为可用的法医学工具,这些标记必须含有有效的变异区能在种及种群水平上产生唯一的标识。由于短片段具有简单、低成本和很好的应用前景,最近有文献报道使用一个189-bp COⅡ片段鉴定来自中国的3种麻蝇。目前,在埃及没有使用该片断发表的数据。因此,本研究利用该片断鉴定来自两个不同地区的三科昆虫(家蝇科、麻蝇科和蝇科)14种蝇类,进而评估该片断的实用性。
目的
通过以下方法评估长度小于200-bp小片段的使用性：
1、测量种内及种间差异。
2、在科水平上证实所研究物种的单系分离及正确归类。
方法
1、形态学鉴定来自14个物种的53个样本。
2、DNA提取。
3、使用该片断的引物进行PCR扩增,之后产物直接用于测序。
4、使用Kimura双参数距离模型(K2P)对核苷酸序列的分歧进行计
算,通过邻接法(NJ)建立系统发育树。
结果
1、同种蝇类无种内差异,种间差异为2%～25%。
2、除了H. melanura和秋家蝇M. autumnalis,所有样品都正确的归类。
结论
尽管分子生物学对于鉴定嗜尸性昆虫是一种有效的方法,然而我们建议小于小于200-bp小片段用于鉴定仅仅是对传统方法的补充。
4章基于COI和COⅡ基因利用PCR-RFLP技术鉴定嗜尸性麻蝇
背景
利用麻蝇进行死亡时间推断是十分有限的,因为麻蝇的形态学鉴定十分困难,无论是在幼虫、蛹还是成虫阶段,它们都具有相似的特征。几乎所有可用的PCR-RFLP数据都来自丽蝇科昆虫。并且,对使用PCR-RFLP技术进行物种鉴定仍存在争议。目前,尚无来自中国或埃及有关被检验的物种和分子标记的数据。
目的
1、评估部分COI-COII基因的使用性；基于直接测序鉴定四种常见的嗜尸性麻蝇。
2、评估该PCR-RFLP技术的实用性；基于COI-COII片断,PCR-RFLP作为一种简单、快速和低成本的技术鉴定四种常见的嗜尸性麻蝇。
方法
1、形态学鉴定4个麻蝇物种。
2、DNA提取。
3、使用该片断的引物进行PCR扩增,之后产物直接用于测序。
4、使用限制性内切酶Hinfl和MfeI对PCR产物进行酶切,使用电泳确定条带图谱。
5、使用Kimura双参数距离模型(K2P)对核苷酸序列的分歧进行计算,通过邻接法(NJ)建立系统发育树。
结果
1、所有被检测样品都正确的归类。
2、联合使用限制性内切酶Hinfl和MfeI产生了不同的限制性片断长度多态性(RFLP)图谱,并且对3种同地区的麻蝇同样有效。
结论
本研究证实了基于部分COI-COII基因的PCR-RFLP技术可以正确的鉴定嗜尸性麻蝇。
1Seasonal succession studies for identification of forensically important arthropods
Background
Forensic entomology, the study of insects pertaining to legal investigations, is primarily concerned with estimations of post mortem interval (PMI). Insect colonization of carrion has been demonstrated to occur in a predictable sequence. There are few available Egyptian studies on the faunal succession related to stages of decomposition and their application in legal medicine. However, there are no published data on the forensically important insects in northeastern Egypt.
Objectives
1. To identify forensically important species in northeastern Egypt (32°15'E and30°36'N) for the first time.
2. To document the arthropod succession pattern (record their presentation at different stages of decomposition).
3. To assess rate of decomposition in the2studied seasons.
4. To document the early arriving of Diptera (time of arriving and first colonizer).
5. To identify the dominant arthropods in each season.
Methods
1. The2seasonal experiments had been conducted in2consecutive years (2011/2012) by using exposed animal carcasses.
2. At each visit, examination of carcasses had been performed to assess the stage of decomposition. Representative samples (dead and still alive) of different life stages have been collected. Environmental variables such as ambient temperature and relative humidity were recorded during each sampling.
3. Morphological identification has been conducted by taxonomy experts with using local published keys.
Results
1. Carcasses yielded3-4orders representing7-9families with11-12collected species in summer and winter experiments respectively. Interestingly, although more species have been collected in winter study but the abundance of each species was much more in summer study.
2. To analyze succession patterns of collected species, occurrence matrices were constructed in the summer and winter studies.
3. As expected, the rate of decomposition in winter was significantly slower than in summer study.
4. There was significance delay in early arriving of Diptera in winter than in summer. The initial colonizers in summer were Musca domestica and Sarcophaga argyrostoma while the initial colonizer in winter was Chrysomya albiceps.
5. In general, Muscidae and Fanniidae were the dominant in summer study and Sarcophagidae was the dominant in winter.
Conclusion
The results of present study affirms that local surveys are critical in identifying what species are present in various areas, and therefore, what species can be expected to be most important in a medico-legal case. It also constitutes a mandatory and primary step to assess the utility of any molecular markers in identification.
2Cytochrome oxidase I gene:utility of short versus long fragments in identification of forensically significant Diptera
Background
Correct identification is an important issue from a forensic perspective because flawed forensic evidence may lead to miscarriages of justice. Molecular based method is considered as an important tool in identification toolkit guided with accurate evaluation of the chosen genetic marker. This is the first time to assess and provide molecular data of forensically important species from Egypt either by using short "272-bp"or long "1173-bp" fragment of the mitochondrial cytochrome oxidase I (COI) gene. Moreover, it is considered also the first time to assess and provide data about long fragment of COI from China because up to our knowledge up to date all available studies from china extracted from short fragment. Thus, assessment will be valuable to determine the suitability of these fragments. The collection of some species from2geographical regions is important to evaluate the dual role of these markers in identification of species and population.
Objectives
-To determine the power of short and long COI fragment in discrimination of Diptera of forensic relevance then compare between
2investigated fragments by assessing:
1. Intra and interspecific variations.
2. Monophyletic separation of investigated species.
3. NJ tree has been constructed consistent with taxonomic classification.
-To determine the ability of these fragment to discriminate within same species on population level.
Methods
1. Morphological identification (50specimens belonging to18species).
2. DNA extraction was performed for all examined specimens.
3. The2investigated markers were amplified by polymerase chain reaction (PCR) followed by direct sequencing.
4. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.
Results
1. Both tested fragment displayed overlapping between intra and interspecific variations.
2. Long marker outperforms short one in completeness of monophyletic separation with high bootstrap support.
3. NJ tree based on long fragment clustered species more in accordance with their taxonomic classification than in short fragment.
Conclusion
1. Identification based on long COI fragment is more reliable and safer because it has the ability to optimize divergence particularly for species characterized by low levels of divergence.
2. The other advantage of using long fragment is obtaining phylogenetic data that in accordance with that based on morphological taxonomy. That will help in correct identification even new cryptic species when local databases had been constructed.
3. COI gene displayed inability to discriminate within same species on population level.
3Cytochrome oxidase Ⅱ gene:identification of forensically significant Diptera based on short fragment
Background
Although it has been reported that cytochrome oxidase Ⅱ (COII) gene is useful in evolution studies, population genetics and systematics due to the relatively high degree of variation in the region, but there are few publications about identification of forensically relevant flies based on COII alone. Before introducing any genetic marker to practice on real cases, we need to assess that marker. In order to be useful as a forensic tool, it must contain enough variation to generate unique identifiers. Because of the value of short DNA fragment as being simple and cheap as well as promising result of recent study which assessed189-bp COII in discrimination between3Sarcophagidae species in China. There is no data up to date about that fragment from Egypt. Based on these circumstances, the present study assessed the utility of189-bp COII fragment in identification of14species; belonging to Calliphoridae, Sarcophagidae and Muscidae, originating from2different geographical areas.
Objectives
To evaluate the utility of too small fragment (<200-bp in length) by assessmg:
1. Intra and interspecific variations.
2. Monophyletic separation of investigated species and correct assignment on family level.
Methods
1. Morphological identification (53specimens belonging to14species).
2. DNA extraction was performed for all examined specimens.
3. The investigated marker was amplified by polymerase chain reaction (PCR) followed by direct sequencing.
4. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.
Results
1. No intraspecific variation within same species (0%), while interspecific variation ranged from2%to25%.
2. All specimens were properly assigned into correct species and families apart from H. melanura and M. autumnalis.
Conclusion
Although molecular methods are very useful to identify forensically important insect, we propose to use this fragment (too small fragment<200-bp) only in addition to the conventional methods.
4Applicability of PCR-RFLP in identification of forensically important Sarcophagidae (Diptera) based on COI and COII genes
Background
The use of Sarcophagidae (Diptera) for PMI estimation is limited since morphological determination is often hampered due to similar characteristics in the larval, pupal and even adult stage. Almost all available PCR-RFLP data extracted from Calliphoridae. Furthermore, there is a debate about the usefulness of PCR-RFLP technique in identification. Therefore, we aimed to assess the utility PCR-RFLP; as simple and inexpensive technique, based on partial COI-COII genes in identification of4common forensically important Sarcophagidae (Flesh flies) in Egypt and China.
Objectives
1. To assess the utility of partial COI-COII genes; based on direct sequencing, in identification of4common forensically important Sarcophagidae (Flesh flies).
2. To assess the utility of PCR-RFLP; as simple, fast and inexpensive technique based on partial COI-COII genes in identification of4common forensically important Sarcophagidae.
Methods
1. Morphological identification of4species belong to Sarcophagidae.
2. DNA extraction was performed for all examined specimens.
3. The investigated marker was amplified by polymerase chain reaction (PCR) followed by direct sequencing.
4. Both restriction enzymes Hinfl and MfeI has been used on amplified PCR products then electrophoresis to determine the banding profile.
5. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.
Results
1. All examined specimens were assigned to the correct species.
2. Combinations of the restriction enzymes Hinfl and MfeI provide4different restriction fragment length polymorphism (RFLP) profiles even among3sympatric species belong to Sarcophaga genus.
Conclusion
This study demonstrates that PCR-RFLP based on partial mt COI-COII genes was accurate to identify forensically important flesh fly species.
背景
法医昆虫学(forensic entomology)研究内容主要是对在尸体内、尸体表面或尸体附近采集到的昆虫标本,进行检验、鉴定、分类后,明确昆虫种类或确定某一昆虫生长发育状态,结合昆虫在尸体上的生态群落演替,帮助法医完成死亡时间(postmortem interval, PMI)的推断。昆虫在尸体上是按一定的次序出现并繁殖。目前,在埃及有关不同腐败阶段的动物区系演替实验和他们在法律上应用报道还很少。而且,也没有埃及东北部有关法医重要意义昆虫的数据报道。
目的
1.第一次在埃及东北部(32015'E,30°36'N)调查法医重要性意义的昆虫。
2.调查冬夏季节动物尸体上的主要昆虫类群,并分析尸体分解速度的季节性差异。
3.观察最早到达尸体的双翅目种类及嗜尸性昆虫的演替规律。
方法：
1.连续2年(2011/2012)的冬夏季节用动物尸体进行实验。
2.在每次观察中,检查尸体的腐败阶段,采集不同发育阶段的有代表性的样本(死的和活着的样本),并记录温度和相对湿度等环境变量。
3.请分类专家依据本地物种的形态学特征对物种行形态学鉴定。
结果
1.夏季在动物尸体上取得了3目7科10种的物种,而冬季在动物尸体上获得了4目8科12种的物种。虽然在冬季可以收集到更丰富的样本物种,但在夏季每个物种的样本数量更多。
2.一般情况下,蝇科和厕蝇科在夏季研究中占主导地位而麻蝇科在冬季占主导地位。
3.冬季动物尸体的分解的速度明显慢于在夏季的速度。
4.相比夏季,在冬季双翅目到达尸体明显延迟。夏季最先到达尸体的类群是家蝇,而在冬季最先到达的白头裸金蝇。
5.通过分析所收集到的样本,构建了夏季和冬季的物种的共生矩阵表。
结论
本研究结果提示开展地域性昆虫演替规律调查研究是十分重要的,在不同的区域范围内确定最有可能出现的昆虫种类对于法医学案例也至关重要。演替实验也是评估分子标记鉴定能力的一个基本步骤。
2细胞色素氧化酶基因Ⅰ (COⅠ):利用短片段和长片断鉴定嗜尸性双翅目昆虫
背景
在法医学领域,正确的鉴定是一个关键性的问题,因为有缺陷的法医学证据可能导致审判不公。在以被准确评估后的遗传标记为指导的鉴定工具包中,以分子为基础的方法被认为是一个重要工具。本文第一次使用线粒体细胞色素氧化酶基因Ⅰ (COⅠ)的272-bp和1173-bp两个片断对来自埃及的嗜尸性昆虫进行评估。并且,由于现有中国的相关数据全部来自对短片段的研究,本文也第一次使用COI的长片断对中国的嗜尸性昆虫进行评估。因此,这些研究对于确定以上片断的适用性是非常有意义的。从两个地区收集到的样本对于使用这些分子标记鉴定物种和种群具有重要意义。
目的
-确定COI长、短两个片断对于鉴定嗜尸性双翅目昆虫的鉴别效力,并且通过以下三个方面评估和比较这两个片断：
1、种内及种间差异
2、所研究物种的单系分离
3、通过邻接法建立于与分类学一致的系统发育树
-确定这些片断在种群水平上鉴定同种的鉴别效力。
方法
1、形态学鉴定来自18个物种的50个样本。
2、DNA提取。
3、使用两个片断的引物进行PCR扩增,之后产物直接用于测序。
4、使用Kimura双参数距离模型(K2P)对核苷酸序列的分歧进行计算,通过邻接法(NJ)建立系统发育树。
结果
1、在种内和种间差异中两个片断显示了类似的结果。
2、长片断能够以高的自举值完整地区分单系物种,明显优于短片段。
3、使用长片断对这些物种建立的NJ系统发育树与分类学结果较为一致,远远优于短片段。
结论
1、COI长片段的鉴定结果更为可靠和安全,因为它能够优化分歧,特别是对那些具有低水平分歧的物种。
2、长片断另外一个优点是除了能够得到与形态学分类相一致的结果之外,还特别适用于鉴定已建立本地数据库中的新物种。
3、在种群水平上COI基因不能区分同种昆虫。
3细胞色素氧化酶基因Ⅱ：利用短片段鉴定嗜尸性双翅目昆虫
背景
由于相对较高的变异度,细胞色素氧化酶基因Ⅱ可以成功用于进化学研究、群体遗传学和分类学。然而,仅有少数文献报道使用该基因进行嗜尸性蝇类鉴定。在将任何遗传学标记用于实际案例之前,需要对这些标记进行评估。为了作为可用的法医学工具,这些标记必须含有有效的变异区能在种及种群水平上产生唯一的标识。由于短片段具有简单、低成本和很好的应用前景,最近有文献报道使用一个189-bp COⅡ片段鉴定来自中国的3种麻蝇。目前,在埃及没有使用该片断发表的数据。因此,本研究利用该片断鉴定来自两个不同地区的三科昆虫(家蝇科、麻蝇科和蝇科)14种蝇类,进而评估该片断的实用性。
目的
通过以下方法评估长度小于200-bp小片段的使用性：
1、测量种内及种间差异。
2、在科水平上证实所研究物种的单系分离及正确归类。
方法
1、形态学鉴定来自14个物种的53个样本。
2、DNA提取。
3、使用该片断的引物进行PCR扩增,之后产物直接用于测序。
4、使用Kimura双参数距离模型(K2P)对核苷酸序列的分歧进行计
算,通过邻接法(NJ)建立系统发育树。
结果
1、同种蝇类无种内差异,种间差异为2%～25%。
2、除了H. melanura和秋家蝇M. autumnalis,所有样品都正确的归类。
结论
尽管分子生物学对于鉴定嗜尸性昆虫是一种有效的方法,然而我们建议小于小于200-bp小片段用于鉴定仅仅是对传统方法的补充。
4章基于COI和COⅡ基因利用PCR-RFLP技术鉴定嗜尸性麻蝇
背景
利用麻蝇进行死亡时间推断是十分有限的,因为麻蝇的形态学鉴定十分困难,无论是在幼虫、蛹还是成虫阶段,它们都具有相似的特征。几乎所有可用的PCR-RFLP数据都来自丽蝇科昆虫。并且,对使用PCR-RFLP技术进行物种鉴定仍存在争议。目前,尚无来自中国或埃及有关被检验的物种和分子标记的数据。
目的
1、评估部分COI-COII基因的使用性；基于直接测序鉴定四种常见的嗜尸性麻蝇。
2、评估该PCR-RFLP技术的实用性；基于COI-COII片断,PCR-RFLP作为一种简单、快速和低成本的技术鉴定四种常见的嗜尸性麻蝇。
方法
1、形态学鉴定4个麻蝇物种。
2、DNA提取。
3、使用该片断的引物进行PCR扩增,之后产物直接用于测序。
4、使用限制性内切酶Hinfl和MfeI对PCR产物进行酶切,使用电泳确定条带图谱。
5、使用Kimura双参数距离模型(K2P)对核苷酸序列的分歧进行计算,通过邻接法(NJ)建立系统发育树。
结果
1、所有被检测样品都正确的归类。
2、联合使用限制性内切酶Hinfl和MfeI产生了不同的限制性片断长度多态性(RFLP)图谱,并且对3种同地区的麻蝇同样有效。
结论
本研究证实了基于部分COI-COII基因的PCR-RFLP技术可以正确的鉴定嗜尸性麻蝇。
1Seasonal succession studies for identification of forensically important arthropods
Background
Forensic entomology, the study of insects pertaining to legal investigations, is primarily concerned with estimations of post mortem interval (PMI). Insect colonization of carrion has been demonstrated to occur in a predictable sequence. There are few available Egyptian studies on the faunal succession related to stages of decomposition and their application in legal medicine. However, there are no published data on the forensically important insects in northeastern Egypt.
Objectives
1. To identify forensically important species in northeastern Egypt (32°15'E and30°36'N) for the first time.
2. To document the arthropod succession pattern (record their presentation at different stages of decomposition).
3. To assess rate of decomposition in the2studied seasons.
4. To document the early arriving of Diptera (time of arriving and first colonizer).
5. To identify the dominant arthropods in each season.
Methods
1. The2seasonal experiments had been conducted in2consecutive years (2011/2012) by using exposed animal carcasses.
2. At each visit, examination of carcasses had been performed to assess the stage of decomposition. Representative samples (dead and still alive) of different life stages have been collected. Environmental variables such as ambient temperature and relative humidity were recorded during each sampling.
3. Morphological identification has been conducted by taxonomy experts with using local published keys.
Results
1. Carcasses yielded3-4orders representing7-9families with11-12collected species in summer and winter experiments respectively. Interestingly, although more species have been collected in winter study but the abundance of each species was much more in summer study.
2. To analyze succession patterns of collected species, occurrence matrices were constructed in the summer and winter studies.
3. As expected, the rate of decomposition in winter was significantly slower than in summer study.
4. There was significance delay in early arriving of Diptera in winter than in summer. The initial colonizers in summer were Musca domestica and Sarcophaga argyrostoma while the initial colonizer in winter was Chrysomya albiceps.
5. In general, Muscidae and Fanniidae were the dominant in summer study and Sarcophagidae was the dominant in winter.
Conclusion
The results of present study affirms that local surveys are critical in identifying what species are present in various areas, and therefore, what species can be expected to be most important in a medico-legal case. It also constitutes a mandatory and primary step to assess the utility of any molecular markers in identification.
2Cytochrome oxidase I gene:utility of short versus long fragments in identification of forensically significant Diptera
Background
Correct identification is an important issue from a forensic perspective because flawed forensic evidence may lead to miscarriages of justice. Molecular based method is considered as an important tool in identification toolkit guided with accurate evaluation of the chosen genetic marker. This is the first time to assess and provide molecular data of forensically important species from Egypt either by using short "272-bp"or long "1173-bp" fragment of the mitochondrial cytochrome oxidase I (COI) gene. Moreover, it is considered also the first time to assess and provide data about long fragment of COI from China because up to our knowledge up to date all available studies from china extracted from short fragment. Thus, assessment will be valuable to determine the suitability of these fragments. The collection of some species from2geographical regions is important to evaluate the dual role of these markers in identification of species and population.
Objectives
-To determine the power of short and long COI fragment in discrimination of Diptera of forensic relevance then compare between
2investigated fragments by assessing:
1. Intra and interspecific variations.
2. Monophyletic separation of investigated species.
3. NJ tree has been constructed consistent with taxonomic classification.
-To determine the ability of these fragment to discriminate within same species on population level.
Methods
1. Morphological identification (50specimens belonging to18species).
2. DNA extraction was performed for all examined specimens.
3. The2investigated markers were amplified by polymerase chain reaction (PCR) followed by direct sequencing.
4. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.
Results
1. Both tested fragment displayed overlapping between intra and interspecific variations.
2. Long marker outperforms short one in completeness of monophyletic separation with high bootstrap support.
3. NJ tree based on long fragment clustered species more in accordance with their taxonomic classification than in short fragment.
Conclusion
1. Identification based on long COI fragment is more reliable and safer because it has the ability to optimize divergence particularly for species characterized by low levels of divergence.
2. The other advantage of using long fragment is obtaining phylogenetic data that in accordance with that based on morphological taxonomy. That will help in correct identification even new cryptic species when local databases had been constructed.
3. COI gene displayed inability to discriminate within same species on population level.
3Cytochrome oxidase Ⅱ gene:identification of forensically significant Diptera based on short fragment
Background
Although it has been reported that cytochrome oxidase Ⅱ (COII) gene is useful in evolution studies, population genetics and systematics due to the relatively high degree of variation in the region, but there are few publications about identification of forensically relevant flies based on COII alone. Before introducing any genetic marker to practice on real cases, we need to assess that marker. In order to be useful as a forensic tool, it must contain enough variation to generate unique identifiers. Because of the value of short DNA fragment as being simple and cheap as well as promising result of recent study which assessed189-bp COII in discrimination between3Sarcophagidae species in China. There is no data up to date about that fragment from Egypt. Based on these circumstances, the present study assessed the utility of189-bp COII fragment in identification of14species; belonging to Calliphoridae, Sarcophagidae and Muscidae, originating from2different geographical areas.
Objectives
To evaluate the utility of too small fragment (<200-bp in length) by assessmg:
1. Intra and interspecific variations.
2. Monophyletic separation of investigated species and correct assignment on family level.
Methods
1. Morphological identification (53specimens belonging to14species).
2. DNA extraction was performed for all examined specimens.
3. The investigated marker was amplified by polymerase chain reaction (PCR) followed by direct sequencing.
4. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.
Results
1. No intraspecific variation within same species (0%), while interspecific variation ranged from2%to25%.
2. All specimens were properly assigned into correct species and families apart from H. melanura and M. autumnalis.
Conclusion
Although molecular methods are very useful to identify forensically important insect, we propose to use this fragment (too small fragment<200-bp) only in addition to the conventional methods.
4Applicability of PCR-RFLP in identification of forensically important Sarcophagidae (Diptera) based on COI and COII genes
Background
The use of Sarcophagidae (Diptera) for PMI estimation is limited since morphological determination is often hampered due to similar characteristics in the larval, pupal and even adult stage. Almost all available PCR-RFLP data extracted from Calliphoridae. Furthermore, there is a debate about the usefulness of PCR-RFLP technique in identification. Therefore, we aimed to assess the utility PCR-RFLP; as simple and inexpensive technique, based on partial COI-COII genes in identification of4common forensically important Sarcophagidae (Flesh flies) in Egypt and China.
Objectives
1. To assess the utility of partial COI-COII genes; based on direct sequencing, in identification of4common forensically important Sarcophagidae (Flesh flies).
2. To assess the utility of PCR-RFLP; as simple, fast and inexpensive technique based on partial COI-COII genes in identification of4common forensically important Sarcophagidae.
Methods
1. Morphological identification of4species belong to Sarcophagidae.
2. DNA extraction was performed for all examined specimens.
3. The investigated marker was amplified by polymerase chain reaction (PCR) followed by direct sequencing.
4. Both restriction enzymes Hinfl and MfeI has been used on amplified PCR products then electrophoresis to determine the banding profile.
5. Nucleotide sequence divergences were calculated using the Kimura two-parameter (K2P) distance model and neighbour-joining (NJ) phylogenetic trees generated.
Results
1. All examined specimens were assigned to the correct species.
2. Combinations of the restriction enzymes Hinfl and MfeI provide4different restriction fragment length polymorphism (RFLP) profiles even among3sympatric species belong to Sarcophaga genus.
Conclusion
This study demonstrates that PCR-RFLP based on partial mt COI-COII genes was accurate to identify forensically important flesh fly species.
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