非损伤性取样法在珍稀濒危动物遗传学研究中的应用

非损伤性取样法是在不伤害珍稀濒危动物的条件下获取分析所需的DNA样本，该方法简便易行，在动物保护遗传学研究领域中得到了极其广泛的应用。就非损伤性取样的研究进展、存在问题及其应用前景进行了简要介绍。     

非损伤性取样研究进展

非损伤性取样即在不捕获、触及,甚至是未亲眼见到动物的情况下,收集不同形式的样品获取样品中的DNA。通过介绍各种类型非损伤性取样的研究进展,就该取样存在的问题及现有解决方法进行讨论,并在此基础上对非损伤性取样的研究和应用前景进行了分析,以期对进一步研究提供有价值的参考。     

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。     

一种鱼类DNA的非损伤检测方法

为了减少和改变对濒危野生鱼类的大量伤害性取样,本文探索了从非损伤性取样的鱼类体表粘液样品中提取鱼类基因组DNA的方法,并用常用的两种分子标记(Cyt b和D-Loop)检测了分离到的粘液DNA。结果表明,从非损伤性取样的鱼类粘液样品中能够分离到高质量的鱼类基因组DNA,可用于后续的研究工作。鱼类体表粘液的非损伤性取样及其DNA提取,为濒危野生鱼类遗传学研究提供了一种新的非损伤性DNA检测技术。     

体表擦拭取样在两栖动物保护遗传学研究中的应用

相比于其他取样方法,非损伤性取样对动物的伤害可以降到最低,因而在动物保护研究中有着重要的价值。在两栖类中通过体表擦拭进行非损伤性取样的方法已有报道,但是相关研究仅局限于零星物种,且均是在实验室环境下完成取样,并不适用于野外就地取样。本研究对四川申果庄省级大熊猫自然保护区内有尾两栖类及无尾两栖类6科10属11种进行了就地擦拭取样。线粒体基因序列分析表明,体表擦拭取样法获得的DNA能满足常规的分子生物学实验要求,可用于水生、陆栖等各种两栖动物类群,可在两栖类保护遗传学研究中广泛应用。     

雪豹保护遗传学和基因组学研究进展

雪豹 (Panthera uncia) 隶属于食肉目猫科豹属,是生活在青藏高原及其周边地区的旗舰物种。随着分子生物学和高通量测序技术的发展,雪豹保护遗传学和保护基因组学研究得到快速的发展,其中非损伤性遗传取样法显著推动了雪豹保护遗传学研究。本文综述了非损伤性遗传取样法在雪豹物种鉴定、个体识别和性别鉴定等研究中的应用,雪豹的系统发生地位、系统地理格局和种群遗传结构及其亚种争议、演化历史、适应性演化和基因组特征等保护遗传学和基因组学方面的研究现状和进展,并对雪豹保护遗传学和基因组学未来发展趋势进行了展望,以期促进雪豹保护生物学研究和保护对策的科学制定。     

非损伤微测技术在细胞生物学研究中的应用系列讲座(一)——技术简介

非损伤微测技术是一种选择性微电极技术,可以不损伤样品而获得进出样品的离子和分子信息,具有非损伤性,长时间、多电极、多角度测量等优势.本文介绍非损伤微测技术原理和技术特点及其在细胞生物学不同领域中的应用.     

粪便取样对亚洲黑熊脑源性神经营养因子基因全长序列分析

沿用本室改进的粪便提取方法,参照马来熊BDNF基因序列设计引物,首次从亚洲黑熊粪便DNA中扩增和克隆到包含完整核BDNF基因的753 bp片段,以毛发作阳性对照并进行重复实验,获得稳定一致结果。序列分析表明,亚洲黑熊的BDNF基因非常保守,与人相比,一致性达94.5%,与大熊猫比达98.9%。在推导的多肽序列中,其成熟区氨基酸序列与所有已报道哺乳动物的完全一致;对亚洲黑熊及其相关物种BDNF基因序列的比较分析,发现大熊猫与包括黑熊在内的熊科动物亲缘关系更近,而与小熊猫较远。文章首次采用非损伤性取样法在分子生物学水平对亚洲黑熊基因组核BDNF基因进行分析,不仅为亚洲黑熊的保护和繁育提供重要参考资料,为非损伤性取样在珍稀濒危野生动物研究中的应用拓宽了思路,也为亚洲黑熊及其近缘种的系统分类研究提供分子证据。     

北方冬季有蹄类动物4种数量调查方法的比较

科学的种群数量调查方法的探索一直是困扰北方有蹄类动物种群资源有效管理工作的重要问题。目前,北方野生有蹄类调查所采用的方法主要有样线法、样带法、大样方法和非损伤性CMR法4种。然而,不同的调查方法基于的统计学假设和生态学原理不同,调查结果往往会出现很大差异,迫切需要对北方冬季有蹄类动物的这4 种调查方法的有效性进行评估。以驼鹿种群数量调查为例,采用样线法、样带法、大样方法和非损伤性CMR调查法,于2012年3月和2012年12月对内蒙古汗马国家级自然保护区约120 km²的区域驼鹿种群数量进行了调查和评估。 结果显示,以上4 种方法得到的驼鹿种群数量分别为:样线法168(109-227) 只,样带法237(165-309) 只,大样方法37(23-50) 只,非损伤性CMR法55(43-68) 只,表明样线法和样带法的调查结果远大于大样方法和非损伤性CMR法,并探讨了不同调查方法应用的科学性、限制性和适用性,为北方冬季有蹄类动物种群资源调查方法的选择和应用提供了科学参考。     

一种从鸟类剥制标本提取DNA的改进方法

应用非损伤性取样的方法,收集鸟类剥制标本的皮肤组织和羽毛,用无水乙醇、浸泡液预处理的方法抽提DNA,结果两者都可提取DNA供PCR扩增。将PCR产物经序列测定和比对分析,证明提取的DNA为目的DNA,表明本试验方法可行。鸟类剥制标本的皮肤组织和羽毛可以作为研究种群遗传学的资源。     

快速、无损大豆种子连续取样技术及其DNA制备

建立简便、快速和无损种子连续取样技术流程及基因型鉴定技术体系, 可节约种植成本及缩短鉴定周期, 提高基因功能研究和育种效率。该研究利用微型电钻和空气泵等简单装置设计了一种连续且无损钻取大豆(Glycine max)种子组织的方法, 并优化了利用384深孔微孔板高通量提取DNA及基因型鉴定技术体系。该方法也可用于水稻(Oryza sativa)和玉米(Zea mays)等多种主要作物的种子取样及基因型鉴定。     

Jetski-based plankton towing as a new method of sampling larval fishes in shallow marine habitats

I tested jetski-based plankton towing as a new method of sampling larval fishes in a shallow marine environment in warm temperate South Africa. Larval fishes were collected by towing a stainless-steel ring net behind a three-man jetski. The sampling technique was designed and tested to overcome problematic sampling of shallow sandy beach surf zones and the adjacent, shallow nearshore immediately seaward of the surf zone (behind the breaker line) with the same type of equipment. The composition, density, stage of development, estuary association, and length frequency of larval fish catches obtained by the new sampling method in the surf zone and in the adjacent shallow nearshore were compared. The success of the technique was evaluated by determining the extent of replication of known trends observed in other surf studies in the same climatic region of South Africa using either a seine or pushnet. Preflexion and postflexion larval fishes were collected. Larvae of the family Sparidae dominated the catches. Trends in catch composition, estuary association, and length frequency of the larvae were similar to those found in other studies of warm temperate South African surf zones. Density of larvae varied depending on the type of equipment used, although these results are tentative given the subjectivity of calculation of the water volume sampled and the extent of nursery use by larvae in the habitats sampled in each study. Increased towing times are recommended. Jetski-based plankton towing is a useful alternative technique for sampling larval fishes in shallow habitats and can easily be applied in surf zones, shallow nearshore areas, and estuaries. The technique greatly increases sampling agility in previously problematic sampling areas.     

A portable pump sampler for lotic periphyton

A sampling technique for collecting lotic periphyton on sedimentary substrates using a peristaltic pump is described. Quantitative samples of periphyton standing crop and colonization rate are collected by the same procedure. The technique eliminates human disturbance problems associated with floating artificial samplers by establishing permanent sampling sites directly on submerged substances.     

Accurate determination of vegetational change in meadows by successive point quadrat analysis

The point quadrat method can be used for determination of vegetational change in meadows of great species diversity. An appropriate sampling technique is described comprehending an apparatus of high rigidity and a shelter to keep off wind and rain. Sample size related to sampling time expense affects the number of species recorded and methodical error which is empirically determined by repetitive sampling. A setup of fixed points leads to higher accuracy than random sampling. When methodical error is quantitatively known, significant vegetational change can be detected by sampling at successive times.For fluctuation studies of plant populations in meadows the point quadrat technique should be preferred to visual estimates of plant cover because of its higher accuracy.Abbreviations f= frequency - s= standard deviation - Sr= variation coefficient - DWLS= distance weighted least squares     

Fetal blood sampling in baboons (Papio spp.): Important procedural aspects and literature review

Background  The baboons ( Papio cynocephalus ) have similarities with human placentation and fetal development. Fetal blood sampling allows investigators to assess fetal condition at a specific point in gestation as well as transplacental transfer of medications. Unfortunately, assessing fetal status during gestation has been difficult and fetal instrumentation associated with high rate of pregnancy loss. Our objectives are to describe the technique of ultrasound guided cordocentesis (UGC) in baboons, report post-procedural outcomes, and review existing publications.
Methods  This is a procedural paper describing the technique of UGC in baboons. After confirming pregnancy and gestational age via ultrasound, animals participating in approved research protocols that required fetal assessment underwent UGC.
Results  We successfully performed UGC in four animals (five samples) using this technique. Animals were sampled in the second and third trimesters with fetal blood sampling achieved by sampling a free cord loop, placental cord insertion site or the intrahepatic umbilical vein. All procedures were without complication and these animals delivered at term.
Conclusions  Ultrasound guided fetal umbilical cord venipuncture is a useful and safe technique to sample the fetal circulation with minimal risk to the fetus or mother. We believe this technique could be used for repeated fetal venous blood sampling in the baboons.     

Comparison of two methods for sampling invertebrates: vacuum and sweep‐net sampling

ABSTRACT With numerous invertebrate sampling techniques available, deciding which technique to use under certain circumstances may be difficult. Many researchers interested in invertebrate abundance and availability relative to the foraging ecology of birds may use a technique (e.g., vacuum sampling or sweep‐netting) without understanding the impacts their choice may have on the samples collected and the ability of the method to meet research objectives. We compared the characteristics, including overall biomass, morphospecies richness, average size, diversity, and body length categories, of invertebrates collected using a sweep‐net and a Dietrick vacuum sampler along paired transects in Woodward County, Oklahoma, from May to July 2007 and 2008. These sampling techniques differed in the taxa collected, with the orders Diptera, Homoptera, and Hymenoptera dominating vacuum samples and the orders Homoptera, Orthoptera, and Araneae dominating sweep‐net samples. Although morphospecies richness was similar for the two techniques, the mean size of invertebrates collected and overall invertebrate biomass were greater for sweep‐netting than vacuum sampling. Vacuum sampling was more effective at collecting small (e.g., <5 cm) invertebrates, whereas sweep‐netting captured large (>5 cm) Orthopteran and Lepidopteran larvae at higher rates. Thus, our results indicate that neither sampling method effectively sampled all invertebrate families and investigators should be aware of the potential biases of different sampling techniques and be certain that the technique selected will allow study objectives to be met.     

Theoretical Calculation of the Liquid—Vapor Coexistence Curve of Water,Chloroform and Methanol with the Cavity-Biased Monte Carlo Method in the Gibbs Ensemble

Abstract

The Gibbs ensemble computer simulation method of Panagiotopoulos is combined by the cavity-biased sampling technique used previously in the grand-canonical ensemble. The combined technique is applied to the determination of the liquid—vapor coexistence curve of the Lennard—Jones fluid as a test case, two water models (SPC and TIP4P) as well as methanol and chloroform, both described with the OPLS model. The application of the virial-based sampling technique, used earlier in the isobaric ensemble is also discussed.     

Conventional sampling plan for the green leafhopper Empoasca kraemeri in common beans

Abstract:  A conventional sampling plan for the leafhopper Empoasca kraemeri Ross & Moore (Auchenorrhyncha: Cicadellidae) in common beans (Phaseolus vulgaris L.) is presented based on samples from 51 commercial fields. The research encompassed three phases. The first phase had the objective of determining the most suitable leaf to be used as a precise and representative sampling unit. In the second phase of the project, the available sampling methods for E. kraemeri were compared, including the approach identified in the first phase. In the third phase, the theoretical frequency distribution of the sampling data was assessed and the number of samples necessary for the sampling plan was established. The best leaves to sample adult leafhoppers were the fourth and the fifth from the apex, while the best leaf for sampling nymphs was the fifth from the apex. The best sampling technique for nymphs and adults was the beating of the apical leaves against a plastic tray. The sampling data obtained with this technique were fit to a negative binomial distribution with common aggregation parameter for adults ( K _common = 1.04) and nymphs ( K _common = 0.47). This sampling plan required 16.8 and 31.2 min to be carried out at an expense of US$ 0.31 and US$ 0.54 for sampling adults and nymphs in a field, respectively.     

A photographic method and a ranking procedure for estimating numbers of the rose aphid,Macrosiphum rosae (L.), on rose buds

Two sampling methods for aphids are described. The first is a photographic method that allows sampling with replacement for use in field experiments and in which a linear regression technique is used to estimate the errors of sampling. The second method is a ranking procedure for sampling natural populations in which regressions of aphid numbers on ‘colony length’ are used to minimize bias.     

SOIL SAMPLING FOR POTATO ROOT EELWORM CYSTS

The National Agricultural Advisory Service (Advisory Entomologists) has adopted a uniform soil-sampling technique for potato root eelworm. In Part I the recommended technique is quoted in full. In Part II an account is given of some experimental sampling done by the Advisory Entomologists, on which the recommendations are partly based. In trying to estimate the average cyst content of the soil of a field, a statistical problem is encountered of deciding on an adequate sampling procedure. A similar problem was investigated in the wartime wireworm survey, but the rather different features of eelworm sampling call for fresh consideration of points of detail.