Investigação de variantes exônicas nos genes VPS35, EIF4G1 e LRRK2 como causa da doença de Parkinson em casuística brasileira

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente no mundo, afetando 1-2% da população acima de 65 anos, caracterizada clinicamente por tremor em repouso, bradicinesia, instabilidade postural e rigidez muscular. Essas manifestações surgem devido à degeneração neuronal progressiva e à presença de inclusões proteicas ricas em α-sinucleína. A DP é decorrente da interação entre fatores ambientais e genéticos, e entre os fatores genéticos, variantes exônicas de transmissão dominante nos genes LRRK2 (leucine-rich repeat kinase 2), VPS35 (vacuolar protein sorting 35) e EIF4G1 (eukaryotic translation initiation factor 4-gamma 1) têm sido associadas à etiologia da doença. Entretanto, estudos sobre o efeito dessas variantes na população brasileira são raros ou inexistentes. Por essa razão, neste trabalho rastreamos mutações nos genes VPS35 (p.D620N; p.R524W), EIF4G1 (p.R1205H; p.A502V) e LRRK2 (p.G2019S) em uma amostra de 582 pacientes brasileiros com DP não aparentados e 329 indivíduos controles saudáveis. Além disso, conduzimos o primeiro estudo caso-controle para análise de variantes exônicas raras (p.Q1111H, p.T1410M, p.M1646T, p.S1761R, p.Y2189C) e comuns (p.N551K, p.R1398H, p.K1423K) no gene LRRK2 em um subgrupo de 329 pacientes brasileiros com DP, não aparentados, naturais da região sudeste. Esse subgrupo foi analisado e comparado com 222 indivíduos controles saudáveis a fim de verificar associações dessas variantes e a DP. Em relação às mutações dos genes VPS35 e EIF4G1, não foram encontradas alterações nos pacientes com DP. A mutação p.G2019S no gene LRRK2 foi encontrada em 15 probandos (2,6%), dos quais 9 são do sexo feminino (64,3%). O tremor em repouso foi observado em 47,36% dos pacientes com a mutação p.G2019S como primeiro sintoma motor. As análises das variantes raras no gene LRRK2 não revelaram qualquer associação estatisticamente significante. Entre as variantes comuns, a p.K1423K mostrou evidência de associação de risco com a DP (p<0,05) na estratificação contendo o grupo de indivíduos com história familiar da doença e para as variantes p.N551K e p.R1398H não foram observadas associações. A análise do haplótipo p.N551K-p.R1398H-p.K1423K revelou associação de proteção na amostra sudeste e na estratificação Rio de Janeiro (p<0,05). Esse haplótipo não está em desequilíbrio de ligação na amostra de 222 indivíduos controles brasileiros analisados (r2≤45). Os resultados obtidos neste estudo representam contribuições valiosas ao entendimento da relação entre as variantes genéticas estudadas e o risco de desenvolvimento da doença de Parkinson, principalmente no que se refere aos endofenótipos associados.

Caracterização da distribuição de alelos de loci STR do cromossomo Y com elevada taxa de mutação em uma amostra populacional do Rio de Janeiro

Marcadores genéticos presentes no cromossomo Y, como os microssatélites (Y-STRs) e polimorfismos de único nucleotídeo (Y-SNPs) são utilizados na caracterização de linhagens masculinas, visto que são transmitidos às gerações seguintes sem alterações, a menos que ocorram mutações (Singh et al., 2011; Mitchell & Hammer, 1996; Butler, 2009). Por isso, esses marcadores são amplamente empregados em diversas situações, destacando-se o uso constante dos Y-STRs na genética forense por apresentarem alta capacidade de discriminar linhagens. Recentemente, foram descritos 13 marcadores com taxas de mutação substancialmente superiores àquelas verificadas para loci STR do cromossomo Y, denominados Rapidly Mutating (RM) Y-STRs (Ballantyne et al., 2010; Kayser et al., 2012). Devido às taxas de mutação elevadas, os RM-YSTRs apresentam maior eficiência na discriminação entre indivíduos proximamente relacionados, pertencentes à mesma linhagem patrilínea. O presente trabalho buscou aprofundar o conhecimento acerca das características populacionais e mutacionais dos loci RM-YSTRs em amostra do Rio de Janeiro, contribuindo com estudos desta natureza na população brasileira. Realizou-se a análise de 13 loci do cromossomo Y em 258 indivíduos do sexo masculino, compondo 129 pares de pais e filhos, nascidos no estado do Rio de Janeiro. O DNA das amostras foi extraído, conforme os protocolos vigentes na rotina do LDD-UERJ. As sequências genéticas de interesse foram amplificadas pela técnica de reação em cadeira da polimerase (PCR) através da realização de três PCR multiplex, cujos produtos de amplificação foram separados por eletroforese em sequenciador automático ABI-3500 (Applied Biosystems). Para os pares pai/filho que apresentaram haplótipos mutados, empregou-se a técnica de sequenciamento para confirmação das mutações. Os loci RM-YSTR geraram um poder de discriminação de 1,0 na amostra analisada, o que significa que todos os 129 indivíduos da amostra populacional apresentaram haplótipos diferentes para tais marcadores, com frequências de 0,0077 e diversidade haplotípica igual a 1. Além disso, foram obtidos valores elevados de diversidade gênica para os 13 marcadores. A análise de distância genética e os resultados de AMOVA baseados nos valores de Fst demonstraram que os RM-YSTR não indicam subdivisão populacional e traços ancestrais comuns. Tais valores estão associados às elevadas taxas de mutação encontradas, cuja média foi de 2,11 x 10-2. Foi possível observar que os loci RM-YSTR são muito discriminativos na amostra miscigenada analisada, além de terem maior capacidade de diferenciar indivíduos do que outros conjuntos de marcadores normalmente usados em estudos populacionais e análises forenses. Sendo assim, é possível concluir que os marcadores RM-YSTR são promissores para discriminar indivíduos da mesma linhagem patrilínea, visto que devido às suas elevadas taxas mutacionais e poder de discriminação, são capazes de diferenciar indivíduos de maneira mais eficiente do que os outros conjuntos de STR. Porém, é necessário maior número de estudos para melhor caracterização destes loci em diferentes populações.

长花马先蒿的居群遗传变异与生物地理学

青藏高原地区是我国植物物种多样性和特有性最高的地区，且作为东亚植物区系的一个现代分化中心受世人瞩目。长花马先蒿是青藏高原高山草甸的广布种，其地理分布格局的形成与高原的隆升和气候变迁有着密切关系。因此，对长花马先蒿进行谱系生物地理学研究，有助于探讨青藏高原地区物种快速分化的机制和群体建立过程。 本研究对长花马先蒿11个居群、188个个体的叶绿体DNA trnT-trnF区进行了序列分析，发现该片段的长度变异范围为1441-1472bp。对位排列后的矩阵（含外类群）长1534bp，内类群中含33个碱基替换和 17 个插入/缺失,可分为20种单倍型。11个地理居群的总核苷酸多态性（π）为0.00468，单倍型多态性（Hd）为0.853，居群间的遗传变异（FST）高达88.2％，说明长花马先蒿具有很高的遗传多样性，且居群间发生了强烈遗传分化。 系统发育和遗传多样性分析发现长花马先蒿的20种叶绿体单倍型可归于四个地理单元——川西高海拔地区、川北地区、云南德钦地区及川藏地区。进一步分析发现：4个地理单元间存在着显著的遗传分化, 说明长花马先蒿具有明显的谱系地理分布格局。其中川西高海拔地区的四种主导单倍型构成了系统发育树最基部的一支，而以川西地区为中心、向南扩展至云南的两个居群所包含的几种单倍型均属于比较进化的类型。单倍型的网络关系(Network) 显示出西藏、青海、云南及四川北部的一些单倍型间遗传差异很小，亲缘关系很近。上述结果表明：长花马先蒿群体在冰期后的重新扩张过程主要表现为由南往北的递进式扩散，第四纪冰期气候的反复波动导致了该物种居群随生境变化而不断扩张或收缩,形成了现今的分布格局。初步推测川西地区很可能曾是长花马先蒿在第四纪冰期时的重要避难所，瓶颈效应和奠基者效应对其遗传多样性分布格局有重要影响。

蕨属的系统发育及谱系地理学研究

蕨属（Pteridium Gled. ex Scop）分布范围极广，几乎遍布于全世界。关于此属的属下分类一直争论不休，主要是因为此属的分布广，形态变异比较大，变异类型之间形态差异不显著，缺乏明确的鉴别性状，或者变异类型之间存在过渡。 本研究利用叶绿体rps4-trnS DNA序列和trnS-G DNA序列构建了世界不同地区蕨属植物的系统关系及中国区域蕨属植物的系统关系，同时构建了世界不同地区蕨属植物的单倍型关系。我们发现，（1）南美洲和澳洲的蕨属植物分化最早;（2）北美洲的蕨属植物遗传变异最丰富，有多条相互独立的进化线（evolutionary lineages）;（3）欧亚大陆和非洲的蕨属植物都与北美洲具有密切关系;（4）大洋洲的蕨属植物同时受南美洲和亚洲蕨属植物的影响;（5）东亚的蕨属植物直接受北美洲蕨属植物的影响，与欧洲和非洲没有直接的关系;（6）非洲和欧洲的蕨属植物关系密切，有着相同的起源。 根据不同地点的蕨属植物的系统关系和单倍型Network关系，我们认为，蕨属植物起源于南美洲，而后向北美洲和澳洲扩散。在北美洲东部和东南部蕨属植物得到繁荣并分别向三个方向扩散与分化。第一个方向是朝北美西部扩散，第二个方向是两次独立扩散到亚洲，第三个方向是扩散到非洲，进而由非洲向欧洲扩散。 根据叶绿体基因所揭示的世界不同地区蕨属植物的发生和发展规律结合前人的研究，我们认为蕨属包含四个物种。Pteridium aquilinum (L.) Kuhn，分布于北美洲、亚洲、非洲和欧洲。该种分布广泛，存在一定的地理分化，可进一步划分8个亚种。Pteridium esculentum (G. Forst.) Cokayne，分布于南美洲，并扩散到澳大利亚，甚至东南亚国家。Pteridium caudatum (L.) Maxon，分布于中美洲和南美洲，是个异源四倍体。Pteridium semihastatum (Wall. ex Ag.) S.B.Andrews，分布于澳洲和东南亚，是个异源四倍体。 通过分子数据和形态分析相结合，作者认为中国蕨属包括两个亚种，即蕨[Pteridium aquilinum (L.) Kuhn subsp.japonicum (Nakai) Á.Löve & D.Löve]和毛轴蕨[Pteridium aquilinum (L.) Kuhn subsp. revolutum (Bl.) X. Q. Chen, stat. nov.]。作者认为，《中国植物志》中记载的食蕨，特有种云南蕨、糙轴蕨、镰羽蕨和长羽蕨并不存在或只是蕨或毛轴蕨的变形，或者毛轴蕨与蕨的杂交个体。 为了今后更进一步研究蕨属植物的系统与进化关系、居群的遗传结构等问题，作者还分离了8个具有多态性的微卫星位点，探讨了微卫星引物开发设计过程中如何提高效率等问题。

云南僰人源流的父系和母系遗传研究

对云南僰人32份男性DNA样本进行Y染色体单倍型以及mitochondrial DNA (mtDNA)单倍型分析,结果发现云南僰人的父系和母系遗传组分都表现出典型的南方人群的遗传特征.由僰人的数据结合已经发表的东亚人群的Y染色体和mtDNA单倍型(haplotype)数据进行Multidimensional Scaling(MDS)分析,结果表明,在MDS分布图中僰人群体的Y染色体单倍型和mtDNA单倍型都与南方人群聚在一起.这一结果支持僰人的遗传族源为东亚南方人群后裔,与考古学的推论相一致.结合历史和考古学证据来探讨僰人的起源和史前迁移,为揭开"僰人悬棺"这种独特的考古文化的起源和史前传播提供遗传学的研究证据.

Y chromosome haplotypes reveal prehistorical migrations to the Himalayas

By using 19 Y chromosome biallelic markers and 3 Y chromosome microsatellite markers, we analyzed the genetic structure of 31 indigenous Sino-Tibetan speaking populations (607 individuals) currently residing in East, Southeast, and South Asia. Our results showed that a T to C mutation at locus M122 is highly prevalent in almost all of the Sino-Tibetan populations, implying a strong genetic affinity among populations in the same language family. Furthermore, the extremely high frequency of H8, a haplotype derived from M122C, in the Sino-Tibetan speaking populations in the Himalayas including Tibet and northeast India indicated a strong bottleneck effect that occurred during a westward and then southward migration of the founding population of Tibeto-Burmans. We, therefore, postulate that the ancient people, who lived in the upper-middle Yellow River basin about 10,000 years ago and developed one of the earliest Neolithic cultures in East Asia, were the ancestors of modem Sino-Tibetan populations.

Mitochondrial DNA variation in cattle of South China: origin and introgression

Ten restriction endonucleases were used to investigate the mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) of 11 native cattle breeds and one cultivated cattle breed in South China. Twenty-three restriction morphs were detected, which can be sorted into five haplotypes, A phylogenetic tree of the haplotypes was constructed by using the 'upgma' method. Our study showed that haplotype I and II are identical to the zebu (Bos indicus) and taurine (Bos taurus) haplotypes, respectively. Zebu and taurine were the two major origins of cattle populations in South China, and the zebu probably had more influence on the native cattle population than taurine did. Haplotype III is identical to haplotype I of yak (Bos grunniens), which was only detected in the Diqing cattle breed. Haplotype IV was detected for the first time. This haplotype, found only in Dehong cattle, might be from an independent domestication event, probably from another Bos indicus population. Divergence of haplotypes I and IV occurred about 268,000-535 000 years ago, much earlier than the 10,000-year history of cattle husbandry. Our results also suggest a secondary introgression of mtDNA from yak to Diqing cattle.

Evolutionary history of the mtDNA 9-bp deletion in Chinese populations and its relevance to the peopling of east and southeast Asia

In total, 1218 Chinese from twelve ethnic groups and nine Han geographic groups were screened for the mtDNA 9-bp deletion motif. The frequency of the 9-bp deletion in all samples was 14.7% but ranged from 0% to 32% in the various ethnic groups. Three individuals had a triplication of the 9-bp segment. Phylogenetic and demographic analyses of the mtDNA hypervariable segment 1 (HVS1) sequences suggest that the 9-bp deletion occurred more than once in China. The majority of the Chinese deletion:haplotypes (about 90%) have a common origin as a mutational event following an initial expansion of modem humans in eastern Asia. Other deletion haplotypes and the three haplotypes with a 9-bp triplication may have arisen independently in the Chinese, presumably by replication error. HVS1 haplotype analysis suggests two possible migration routes of the 9-bp deletion in east and southeast Asia. Both migrations originated in China with one route leading to the Pacific Islands via Taiwan, the other to southeast Asia and possibly the Nicobar Islands. Along both routes of peopling, a decrease in HVS1 diversity of the mtDNA haplotypes is observed. The "Polynesian motif (16217T/C, 16247A/G, and 16261C/T)" and the 16140T/C, 16266C/A, or C/G polymorphisms appear specific to each migration route.

Origin of rabbit (Oryctolagus cuniculus) in China: evidence from mitochondrial DNA control region sequence analysis

A fragment of mitochondrial DNA (mtDNA) control region (similar to700 bp) was sequenced in 104 individuals from 20 breeds (three Chinese domestic breeds, five recently derived breeds and 12 introduced breeds) of domestic rabbits, Oryctolagus cuniculus . Nineteen sites were polymorphic, with 18 transitions and one insertion/deletion, and eight haplotypes (A1, A2, A3, A4, A5, A6, A7 and A8) were identified. Haplotype A1 was the most common and occurred in 89 individuals. In the 25 Chinese rabbits, only haplotype A1 was observed, while four haplotypes (A1, A3, A5 and A6) were found in 26 recently derived individuals. Haplotype A2 was shared by seven individuals among three introduced strains. The other six haplotypes accounted for 0. 96-1. 92% of the animals. Combined with the published sequences of European rabbits, a reduced median-joining network was constructed. The Chinese rabbit mtDNAs were scattered into two clusters of European rabbits. These results suggest that the (so-called) Chinese rabbits were introduced from Europe. Genetic diversity in Chinese rabbits was very low.

Origin and dispersal of atypical aldehyde dehydrogenase ALDH2*487Lys

The East Asian respond with a marked facial flushing and mild to moderate symptoms of intoxication after drinking the amounts of alcohol that has no detectable effect on European. The alcohol sensitivity in Orientals is due to a delayed oxidation of aceta

Evidence for Positive Selection on the Osteogenin (BMP3) Gene in Human Populations

Background: Human skeletal system has evolved rapidly since the dispersal of modern humans from Africa, potentially driven by selection and adaptation. Osteogenin (BMP3) plays an important role in skeletal development and bone osteogenesis as an antagonist of the osteogenic bone morphogenetic proteins, and negatively regulates bone mineral density. Methodology/Principal Findings: Here, we resequenced the BMP3 gene from individuals in four geographically separated modern human populations. Features supportive of positive selection in the BMP3 gene were found including the presence of an excess of nonsynonymous mutations in modern humans, and a significantly lower genetic diversity that deviates from neutrality. The prevalent haplotypes of the first exon region in Europeans demonstrated features of long-range haplotype homogeneity. In contrast with findings in European, the derived allele SNP Arg192Gln shows higher extended haplotype homozygosity in East Asian. The worldwide allele frequency distribution of SNP shows not only a high-derived allele frequency in Asians, but also in Americans, which is suggestive of functional adaptation. Conclusions/Significance: In conclusion, we provide evidence for recent positive selection operating upon a crucial gene in skeletal development, which may provide new insight into the evolution of the skeletal system and bone development.

Prion protein gene (PRNP) polymorphisms in native Chinese cattle

Le polymorphisme au sein de quatre regions du gene codant pour la proteine prion bovine (PRNP) confere la susceptibilite a l'encephalopathie bovine spongiforme (BSE). Ceux-ci comprennent un polymorphisme d'insertion/deletion (indel) de 23 pb dans le promoteur, un indel de 12 pb dans l'intron 1, un octapeptide repete ou un indel de 24 pb au sein du cadre de lecture, et un polymorphisme mononucleotidique (SNP) dans la region codante. Dans ce travail, les auteurs ont examine la frequence des genotypes, des alleles et des haplotypes pour ces indel au sein de 349 bovins d'origine chinoise, de meme que la sequence nucleotidique de ce gene chez 50 de ces animaux. Leurs resultats montrent que l'allele ayant la deletion de 12 pb et l'haplotype combinant la deletion de 23 pb et la deletion de 12 pb, lesquels ont ete suggeres comme etant importants pour la susceptibilite a la BSE, sont rares au sein des bovins du sud de la Chine. Une difference significative a ete observee entre les bovins affectes par la BSE et les bovins chinois sains pour ce qui est de l'indel de 12 pb. Au total, 14 SNP ont ete observes dans la region codante du gene PRNP chez les bovins chinois. Trois de ces SNP etaient associes a des changements d'acides amines (K3T, P54S et S154N). La substitution E211K qui a ete rapportee recemment chez un cas atypique de la BSE chez un bovin americain n'a pas ete detectee dans ce travail.

More evidence for non-maternal inheritance of mitochondrial DNA?

Background: A single case of paternal co-transmission ofmitochondrial DNA (mtDNA) in humans has been reported so far. Objective: To find potential instances of non-maternal inheritance of mtDNA. Methods: Published medical case studies (of single patients) were searched for irregular mtDNA patterns by comparing the given haplotype information for different clones or tissues with the worldwide mtDNA database as known to date-a method that has proved robust and reliable for the detection of flawed mtDNA sequence data. Results: More than 20 studies were found reporting clear cut instances with mtDNAs of different ancestries in single individuals. As examples, cases are reviewed from recent published reports which, at face value, may be taken as evidence for paternal inheritance of mtDNA or recombination. Conclusions: Multiple types (or recombinant types) of quite dissimilar mitochondrial DNA from different parts of the known mtDNA phylogeny are often reported in single individuals. From re-analyses and corrigenda of forensic mtDNA data, it is apparent that the phenomenon of mixed or mosaic mtDNA can be ascribed solely to contamination and sample mix up.

Genetic diversity study of Caspian brown trout (Salmo trutta caspius Kessler, 1877) population in five rivers in the southern part of Caspian Sea, Iran

For study the genetic diversity of Caspian brown trout population in five rivers in the southern part of Caspian Sea in Iran 182 number generators in the fall and winter of 1390 were collected in Chalus, Sardab Rud, Cheshmeh Kileh, Kargan Rud and Astara rivers. Then about 3-5 g of soft and fresh tissue from the bottom fin fish removed and were fixed in ethanol 96°. Genomic DNA was extracted by using ammonium acetate, then quantity and quality of the extracted DNA were determined by using spectrophotometry and horizontal electrophoresis in 1% agarose gel. The polymerase chain reaction was performed by using 16 SSR primers and sequencing primers (D-Loop) and the quality of PCR products amplified by SSR method were performed by using horizontal electrophoresis in 2% agarose gel. Alleles and their sizes were determined by using vertical electrophoresis in 6% polyacrylamide gel and silver nitrate staining method. Gel images were recorded by gel documentarian, the bands were scored by using Photo- Capt software and statistical analysis was performed by using Gene Alex and Pop Gene software. Also the PCR sequencing products after quality assessment by usinghorizontal electrophoresis in 1.5% agarose gel were purified and sent to South Korea Bioneer Corporation for sequencing. Sequencing was performed by chain termination method and the statistical analysis was performed by using Bio- Edit, Mega, Arlequin and DNA SP software. The SSR method, 5 pairs of primers produced polymorphic bands and the average real and effective number of alleles were calculated 5.60±1.83 and 3.87±1.46 in the Cheshmeh Kileh river and 7.60±1.75 and 5.48±1.32 in the Karganrud river and the mean observed and expected heterozygosity were calculated 0.44 ±0.15 and 0.52 ±0.16 in the Cheshmeh Kileh river and 0.50 ±0.11 and 0.70±0.13 in the Karganrud river. Analysis of Molecular Variance results showed that significant differences in genetic diversity between and within populations and between and within individuals in the studied rivers (P<0.01). The sequencing method identified 35 different haplotype, the highest number of polymorphic position (251) and haplotype (14) were observed in the Chalus river. The highest mean observed number of alleles (2.24±0.48) was calculated in the Sardabrud river, the highest mean observed heterozygosity (1.00±0.03) was calculated in the Chalus river and the highest mean nucleotide diversity (0.13±0.07) was observed in the Sardabrud river and mean haplotype diversity was obtained (1) in three studied rivers. The overall results show that there are no same population of this fish in the studied rivers and Karganrud and Chalus rivers in the SSR and sequencing methods had the highest levels of genetic diversity.

Investigation of polymorphism by PCR-RFLP method in Cobia fish Rachycentron canadum in the Persian Gulf and Oman Sea

Cobia is a native fish species in Iranian waters in the Persian Gulf and Sea of Oman and has a good internal and foreign market. This fish is a fast growing species and for this reason Iranian Fisheries is considering to go for it culture practices. To go for any utilization such as fishing from wild stocks or culture activities, needs a better understanding of its peculiarities and genetic characteristics of its natural resources. Therefore, this project was discribed and conducted. In this investigation, cuts 2 or 3 cm of fin tissue of  specimen of Cobia obtained from Sistan and Bluchestan, Hormozgan, Bushehr and Khuzestan water provinces, were collected. DNA was extracted by Phenol-chlorophorm method and produced PCR product in length of 1060 and 1450 base pair of two mitochondrial genes COI and NADH2. Using 13 cutting enzymes (4 enzymes were subscriber for both of genes), 205 base pair (from 2510 base pair, equal with %3.8 from gene regains) were directly investigated. But binding patterns of enzymatic digestion of PCR products of both COI and ND genes from electrophoresis were monomorph in all samples and no polymorphism was observed. This may be attributed to the unsuitable choice of COI and ND2 genes for showing of intra specific divergence. But in general non-existence of genetic diversity or noticeable decrease of that among individuals has been reported in regions were fish migration exist and they can freely move between two regions. Therefore, non-observation of polymorphism in the study area might be the case and indicates represents the area. On the other hand, some scientists believe that the distributions of populations in different regions are greatly affected by environmental and physical and ecological factors. Althoug Cobia is a migratory fish, but with regard to the fact that the environmental conditions are different (specially temperature and salinity) between east and west of Persian Gulf and Oman sea, there is a possibility that different genetic groups of this species exist in the regions. Of course It is clear that using more samples and enzymes from other genetically regions could produce better results. Since none of the two investigated genes didn’t show genetic divergence or polymorphism amongst the individuals of one region or between different regions, therefore, statistic analysis for estimating of haplotype diversity or nucleotide diversity and drawing of relationship tree among individuals using available softwares was not possible.