粪便DNA分析技术在动物生态学中的应用

粪便DNA分析是一项新发展起来的从粪便中获取动物DNA并用于相关研究的技术,该技术有助于分子生态学研究中所遇到的取样难题。通过对粪便DNA分析的研究方法、研究内容以及研究进展情况的介绍,提供了该技术不仅能用于分子生态学的许多研究领域,而且还能够提供诸如种群数量估计、领域边界划定等生态生态学信息,这是对分子生态学的重要补充。     

大熊猫粪便野外暴露时间对微卫星实验的影响

非损伤性取样,如从粪便中获取DNA用于分子生物学研究已经在珍稀野生动物中得到了广泛应用。DNA质量与后续研究很大程度上依赖于粪便暴露时间的长短。本研究通过分析不同新鲜程度粪便样品的DNA质量和微卫星基因分型效果探讨大熊猫粪便野外暴露时间对分子实验的影响。研究表明,暴露时间越短,粪便DNA降解程度越低,所提取的DNA质量也较高。同时粪便DNA的降解程度与季节和暴露环境也有一定的关系:春、秋季14 d以内、夏季7 d以内、冬季21 d以内的粪便样品提取的DNA质量较好,微卫星能成功分型;另外,野外卧穴口的粪便样品DNA保存时间相对较长。本研究对野外大熊猫粪便采集工作具有指导意义。     

分子粪便学及其应用—可靠性、局限性和展望

由于珍稀濒危动物的各种生存状况相当脆弱,已不允许进行破坏性的取样活动,因而长期以来,对这些动物的遗传结构及遗传多样性现状的研究一直都很薄弱。而近年来以分子粪便学为基础的粪便取样分析方法,在很大程度上解决了这一问题。传统的粪便分析方法与分子生物学技术相结合,在不干扰野生动物正常活动的情况下,可对这物种进行物种识别、性别确定、数量调查、遗传多样性、行为生态学、系统地理学、食性及疾病、巢域及分布区范围等多方面的深入研究。虽然分子粪便学由于粪便样品质量问题而具有一定的局限,从而导致其研究结果曾受到怀疑,但是该方法在近10年的发展应用中,其局限性已逐渐被克服而目趋完善,研究结果也得到了国际上的认同。因此,今后该研究方法在珍稀濒危动物行为生态学和保护生物学等研究领导中将具有很大的发展潜力,越来越多的有关珍稀濒危动物保护遗传学的研究将采用该方法进行。     

一种高效的哺乳动物粪便DNA提取通用方法

以粪便为材料提取动物DNA进行动物保护遗传学和分子生态学研究的关键是能否提取到高质量的粪便DNA.然而提取方法通用性不好和产物质量不高等问题阻碍了粪便DNA分析技术的推广.本文介绍的改进型十六烷基三甲基溴化铵提取法可广泛适用于各食性哺乳动物粪便DNA提取,在11种不同食性动物的粪便DNA提取实验中验证了它的可靠性和通用性.本方法成本低廉(3元/样),用实验室常规试剂即可完成粪便DNA提取,其产物纯度高于专用试剂盒QIAamp DNA Stool Kit,在拥有超过专业试剂盒提取效果的同时尽可能的降低了实验成本,有利于粪便DNA技术的推广.     

环境DNA metabarcoding及其在生态学研究中的应用

环境DNA metabarcoding(eDNA metabarcoding)是指利用环境样本(如土壤、水、粪便等)中分离的DNA进行高通量的多个物种(或高级分类单元)鉴定的方法。近年来,该方法引起了学者的广泛关注,逐渐应用于生物多样性研究、水生生物监测、珍稀濒危物种和外来入侵物种检测等生态学领域。介绍环境DNA metabarcoding的含义和研究方法;重点介绍环境DNA metabarcoding在物种监测、生物多样性研究和食性分析等生态学领域中的应用;总结环境DNA metabarcoding应用于生态学研究领域面临的挑战并对该方法的发展进行展望。     

环境DNA技术在鱼类生态学中的应用研究进展

全球渔业衰退是21世纪人类面临的重要挑战之一。为了有效地遏制鱼类资源的衰退,精确的鱼类生态调查是其首要任务。传统的鱼类监测以渔获物采集与形态学鉴定为主,往往耗时耗力且效果不佳,已无法满足现阶段大尺度上的精确调查。环境DNA （eDNA）技术作为一种近年来新兴的鱼类生态调查方法,其与传统方法相比具有灵敏度高、经济高效、采样受限小且对生态系统无干扰的优势,目前其已被广泛地应用于鱼类物种监测、多样性调查、生物量评估以及繁殖活动监测等方面的研究。然而,eDNA技术在鱼类生态学研究的具体应用中暴露出的一些问题将会影响其监测结果的精确性,诸如操作流程的不规范、基因数据库的不完善以及eDNA在环境中生态学过程的不明确等。鉴于上述原因,首先对eDNA技术的发展历程、分析流程以及eDNA技术在鱼类生态学研究领域中的研究进展进行了综述,而后着重分析了eDNA技术的发展当前所面临的困难与挑战,并提出了相应的解决方案,最后对eDNA技术未来在鱼类生态学研究领域中的发展趋势做出了展望。通过本研究,以期能够为eDNA技术在鱼类生态学领域中的准确应用提供理论基础。     

短尾猴陈旧粪便中DNA的提取

分子粪便学（Molecular scatology）是一门将传统粪便分析方法与分子生物学技术相结合,以动物粪便为实验材料进行多领域研究的学科（魏辅等,2001）。虽然该方法已在野生濒危动物保护遗传学和分子生态学研究中发挥了很大作用（Kohnand Wayne,1997）,但目前大多数分子粪便学研究中使用的材料是新鲜粪便,从保存时间很长的陈旧粪便中很难提取到高质量的DNA用于PCR扩增以及序列分析,严重制约了分子粪便学的广泛应用（Wasser et al．,1997;Constable et al．,2001;Murphy et al．．2002）。     

利用rbcL-a序列模拟检测小鼠食性

动物食性是研究动物生活习性中非常重要的一环,利用DNA条形码进行检测变得非常快捷和方便。利用这项技术,模拟生态环境研究动物食性,来检测小白鼠的食物组成。首先,给小白鼠喂食了6种植物(白菜、玉米、胡萝卜、小麦、生菜、花椰菜)并收集其粪便,然后对这些样本进行DNA提取、PCR扩增与克隆,最后测序并获得来自质体的rbcL基因(ribulose-1,5-bisphosphate carboxylase large subunit gene)的部分序列(rbcL-a)。粪便样本中得到的序列分别与GenBank数据库和实验中得到的作为食物的5种植物的rbcL-a序列(玉米中未发现)进行比对,确定其分类单元。利用GenBank数据库,共鉴定出了3科和1属。与实验中的5种植物rbcL-a序列比对能鉴定出3个植物物种。表明实验用的rbcL-a序列在检测粪便中物种方面具有很大的潜力,对野生动物的食性分析及分子生态学的研究具有重要的意义。     

岩羊(Pseudois nayaur)研究概述

野生动物管理需要生境质量、行为学、种群动态以及种间关系等数据支持。岩羊(Pseudois nayaur)作为我国Ⅱ级保护动物,是雪豹(Panthera uncia)、狼(Canis lupus)、棕熊(Ursus arctos)等珍稀食肉目动物的重要食物来源。20世纪,岩羊作为重要的狩猎物种被大量捕杀贩卖,影响了该物种的种群规模和分布,也影响了雪豹等动物的食物资源。当前,由于缺乏野生动物管理所需数据,岩羊保护与人类利益发生冲突十分常见。岩羊主要分布于青藏高原及周边地区,种群分布及行为学相关研究主要集中在国内的宁夏、甘肃、青海和新疆等省份;在境外地区,印度、尼泊尔等国也有岩羊的反捕食栖息方面研究的报道,而高原中部地区岩羊种群的研究却非常少。当前研究存在主要问题:分类学上对亚种间分化争议较大,栖息地质量评估无法满足岩羊有效管理,网围栏架设等人类活动干扰其生存并间接影响濒危物种雪豹的保护等。此外,有关岩羊动物疫病流行病学、肠道微生物及动物营养学等报道,尚缺乏对岩羊与家畜交叉感染相关案例的深度分析。已有的研究有助于认识物种分化和对环境的适应机制,但对岩羊宏观生态学研究不够系统,仍存在大部分地区种群动态数据不足、亚种分类和地理划分不清等问题。近年来,随着新技术的发展,应用分子识别、计算机识别、无人机技术和红外相机技术等对岩羊进行宏观生态学研究成为热点,建议加大对矮岩羊种群和贺兰山岩羊种群的保护;对保护区内应用新技术构建全面的野生动物及环境监测系统,从而对野生动物的种群分布动态和生存状态能够进行有效监测;增加对区外野生动物的调查频率,及时建立保护区、扩大保护区范围并建立相应的监测站。     

分子倒置探针技术的研究进展及应用

分子倒置探针技术是一项新发展起来的用于目标序列捕获的分子生物学技术,该技术通过设计特异的探针对已知的特定目的基因组序列进行捕获,将目标序列DNA富集后再利用芯片杂交或测序进行检测。此技术有助于研究人员对大样本中的基因组重要区域进行研究,避免了全基因组研究费用高、分析困难等问题。并且,分子倒置探针技术弥补了杂交捕获技术、PCR捕获技术等分子捕获手段的不足,为动植物及病原菌重要DNA片段的研究提供强有力的技术支持。目前,分子倒置探针技术广泛应用于单核苷酸多态性(SNP)分型、外显子测序、拷贝数变异、杂合性丢失、体细胞突变、DNA甲基化和可变剪接等方面的研究。由于其特异性强、重复性好、操作简单、费用低廉,并对DNA完整度要求不高,适用于福尔马林石蜡包埋样本分析等特点,分子倒置探针技术的应用越来越广泛。然而,分子倒置探针技术在探针设计及数据分析软件研发等方面仍存在一些不足,还需要进一步的优化完善。为促进相关领域学者全面了解该技术,综述了分子倒置探针技术的基本原理、发展历程、技术特点及在疾病研究领域的应用,讨论了分子倒置探针技术的价值及存在的问题。     

Testing the reliability of noninvasive genetic sampling by comparing analyses of blood and fecal samples in Barbary macaques (Macaca sylvanus).

Genetic studies of wild animal populations are often hindered by difficulties in obtaining blood samples. Recent advances in molecular biology have allowed the use of noninvasive samples as sources of DNA (e.g., hair or feces), but such samples may provide low-quality DNA and prevent the determination of true genotypes in subsequent DNA analysis. We present a preliminary study aimed at assessing the reliability of using fecal samples for genotyping in Barbary macaques (Macaca sylvanus). The test was performed on samples of blood and feces from 11 captive animals, using three dinucleotide microsatellites. The CTAB DNA extraction method was found to be the most relevant for Barbary macaque feces, yielding successful amplification at all loci for 70% of PCRs. All the fecal samples tested gave correct genotypes at least once for each locus when referenced against blood-derived genotypes. An average of 18.3% of PCRs displayed spurious genotypes (false homozygous or false allele). The minimum theoretical probability required to obtain a 100% accurate genotype is 0.74, based on the criterion that a correct genotype is assessed only if it was observed at least twice. The observed probability of obtaining a correct genotype from three PCRs, based on our genotyping results, was greater (0.81 on average) than the minimum threshold. In conclusion, our comparison of blood and fecal samples showed that fecal sampling is a reliable tool for the further study of wild Barbary macaque populations.     

非损伤性取样样品中富集宿主DNA的研究进展

非损伤性取样被广泛应用在动物保护遗传学、分子生态学和分子进化等研究领域。随着基因组测序技术的发展和基因组学时代的到来,如何从非损伤性取样样品中获取能够用于进行基因组测序的高质量DNA是研究者面临的难题。本文总结和比较了非损伤性取样中最常用的粪便样品和考古材料或博物馆标本两类样品中富集宿主DNA的方法及应用,以期为非损伤性取样在动物基因组学的研究和应用提供重要参考。     

粪样在野生动物研究中的作用

野生动物数量少,取样难度大;在野外工作时,动物肌肉和血液样品还难以保存。这些给野生动物研究带来很大不便。由于动物粪样容易收集,易于保存,对动物的影响小,在野生动物研究中得到了广泛的应用。粪样分析已经应用在动物的领域、食性、消化动态、疾病与寄生虫、种群数量和遗传结构、有效种群大小、食物链与食物网、能量流与物质流的研究等方面。研究证明,在野生动物的研究和保护中,通过粪样可以得到许多关键性问题的答案。     

Non-invasive genotyping of transgenic animals using fecal DNA

For genotyping of transgenic animals, many IACUC guidelines recommend the use of fecal DNA when possible because this approach is non-invasive. Existing methods for extracting fecal DNA may be costly or involve the use of toxic organic solvents. Furthermore, feces contain an abundance of PCR inhibitors that may hinder DNA amplification when they are co-purified with fecal DNA. Here the authors describe a cost-effective, non-toxic method for genotyping transgenic animals by using the reagent AquaStool to extract fecal DNA and remove PCR inhibitors. Genotyping results obtained from fecal DNA samples extracted using AquaStool were reliably accurate when compared with results obtained from tail DNA samples. Because it is non-invasive, the authors believe that use of this method for genotyping transgenic animals using fecal DNA samples may improve animal welfare.     

Feasibility and Recommendations for Swift Fox Fecal DNA Profiling

Abstract: Genetic profiling using fecal samples collected noninvasively in the wild can provide managers with an alternative to live-trapping. However, before embarking on a large-scale survey, feasibility of this methodology should be assessed for the focal species. Costs associated with fecal genotyping can be high because of the need for multiple amplifications to prevent and detect errors. Assessing the relative amount of target DNA before genotyping means samples can be eliminated where error rates will be high or amplification success will be low, thereby reducing costs. We collected fecal samples from an endangered population of swift fox (Vulpes velox) and employed target-DNA quantification and a screening protocol to assess sample quality before genetic profiling. Quantification was critical in identifying samples of low quality (68%, <0.2 ng/μL). Comparison of the amplification, from a subset of loci in 25 samples that did not meet the screening criteria, confirmed the effectiveness of this method. The protocol, however, used a considerable amount of DNA, and an assessment of the locus and sample variability allowed us to refine it for future population surveys. Although we did not use <50% of the samples we collected, the remaining samples provided 36 unique genotypes, which corresponded to approximately 70% of animals estimated to be present in the study area. Although obtaining fecal DNA from small carnivores is challenging, our protocol, including the quantification and qualification of DNA, the selection of markers, and the use of postgenotyping analyses, such as DROPOUT, CAPWIRE, and geographic information, provides a more cost-effective way to produce reliable results. The method we have developed is an informative approach that wildlife managers can use to conduct population surveys where the collection of feces is possible without the need for live-trapping.     

gemini: software for testing the effects of genotyping errors and multitubes approach for individual identification

Improvements in the determination of individual genotypes from samples with low DNA quantity and quality are of prime importance in molecular ecology and conservation for reliable genetic individual identification (molecular tagging using microsatllites loci). Thus, errors (e.g. allelic dropout and false allele) appearing during samples genotyping must be monitored and eliminated as far as possible. The multitubes approach is a very effective but a costly and time‐consuming solution. In this paper, we present a simulation software that allows evaluation of the effect of genotyping errors on genetic identification of individuals and the effectiveness of a multitubes approach to correct these errors.     

False alleles derived from microbial DNA pose a potential source of error in microsatellite genotyping of DNA from faeces

Microsatellite genotyping of wild animals using DNA extracted from noninvasive samples such as faeces is a powerful means to identify individuals within a population and examine aspects of genetic social structure, such as relatedness and paternity. However, the use of the low quantities of poor quality DNA typically obtained from noninvasive samples can result in genotyping errors. Here we report the first instance of artefactural ‘alleles’ resulting from specific co‐amplification of microorganismal DNA present in the total DNA derived from faeces.     

Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

Species identification,molecular sexing and genotyping using non-invasive approaches in two wild bovids species: Bos gaurus and Bos javanicus

Since the second Indochina war, habitat destruction and overhunting has resulted in fragmentation of the remaining populations of Bos javanicus and B. gaurus. Nowadays, both species are in serious danger, especially the gaur. In Vietnam, where these species have become almost impossible to capture in the wild, non-invasive investigations are the only feasible approach to obtain data on populations. However, non-invasive derived DNA, especially in tropical areas, is usually characterized by low concentrations, poor quality and/or contamination from alien DNA. To assist in tropical conservation management, baseline information is provided here on assessing the reliability of species identification, molecular sexing and microsatellite genotyping using fecal DNA from B. gaurus and B. javanicus. For species identification using bovine fecal samples, cytochrome b fragment between positions 867 and 1140 was found to contain species diagnostic sites, which distinguishes the four species encountered in the region: B. gaurus, B. indicus, B. javanicus and B. taurus. For sex determination, primers were initially tested on DNA obtained from blood. Then, these primers were successfully used on DNA derived from fecal material. Finally, we also evaluate the feasibility of non-invasive microsatellite genotyping on fecal samples collected in Vietnamese nature reserves. The results presented here improve on current molecular methods based on fecal material obtained from tropical areas. Zoo Biol 28:127–136, 2009. © 2008 Wiley-Liss, Inc.     

Molecular tracking of jaguar melanism using faecal DNA

Major evolutionary questions remain elusive due to persistent difficulties in directly studying the genetics of variable phenotypes in natural populations. Many phenotypic variants may be of adaptive relevance, and thus important to consider in the context of conservation genetics. However, since the dynamics of these traits is usually poorly understood in the wild, their incorporation in conservation strategies is difficult to accomplish. For animals which exhibit intriguing phenotypic variation but are difficult to track in the wild, innovative approaches are required to investigate such issues. Here we demonstrate that non-invasive DNA sampling can be used to study the genetics and ecology of melanism in the jaguar, by directly genotyping the molecular polymorphism underlying this coloration trait. These results open new prospects for the in-depth investigation of this polymorphism, and highlight the broader potential of non-invasive DNA-based phenotype tracking for wildlife in general.