不同保藏处理的昆虫标本DNA提取及其随机扩增多态DNA反应

实验利用CTAB法对柳二十斑叶甲Chrysomelavigintipunctata (Scopoli)、异色瓢虫HarmoniaaxyridisPollas、七星瓢虫Coc cinellaseptempunctataLinnaeus、小地老虎Agrotisypsilon (Rottemberg)、红蜻CrocothemisserviliaDrury、无齿稻蝗OxyaabentataWil lemse和中华稻蝗Oxyachinensis (Thunberg)等 7种昆虫进行了基因组DNA提取。从自然干燥标本、烘干标本及酒精浸泡标本获得的DNA均可用于RAPD PCR反应 ,且烘干标本、酒精浸泡标本提取效果优于自然干燥标本。这种提取方法简便易行 ,容易掌握 ,且耗资小于其它分子生物学方法。     

九种蝗虫核型似近系数的聚类分析研究

应用核型似近系数聚类分析方法,研究了小稻蝗Oxya hyla intricata、山稻蝗O.Agav-isa、上海稻蝗O.Shanghaiensis无齿稻蝗O.Adentata、中华稻蝗O.Chisensis、日本稻蝗O.Japonica、短额负蝗Atractomorpha sinensis、奇异负蝗A.Pergrina和日本蚱Tetrix japonica等9种蝗虫的亲缘关系。结果显示,9种蝗虫分为3类：稻蝗,负蝗和蚱。6种稻蝗之间的核型似近系数(λ)在0.961～0.5695之间,2种负蝗的λ＝0.5867,日本蚱与这8种蝗虫的λ在0.5318～.0322。聚类图直观地反映出它们的亲缘关系与形态分类学的分类结果相一致。从9种蝗虫 的核型演化上看,日本蚱是较原始的类型,负蝗分化也较早,而稻蝗则是较进化的类型。     

六斑月瓢虫模板DNA的制备及16SrDNA序列扩增

本文对酒精浸泡的六斑月瓢虫Menochilus sexmaculata(Fabricius)标本进行基因组DNA的提取,用线粒体16 SrDNA引物扩增出长度约为500bp的PCR产物,为进一步开展瓢虫的分子系统学研究打下基础.     

湖南蝗虫二新种(直翅目：斑腿蝗科)

记述斑腿蝗科Catantopidae 卵翅蝗属Caryanda Stal ,1878一新种雪峰山卵翅蝗C. xuefengshanensis sp.nov. 和蹦蝗属Sinopodisma Chang,1940 一新种大围山蹦蝗S. daweishana sp. nov.。前者标本采自湖南省黄桑自然保护区（绥宁县）;后者标本采自湖南省大围山国家森林公园(浏阳市)。     

陕西长安及汉中两地中华稻蝗的比较。

通过对采自陕西长安及汉中地区的中华稻蝗Oxya chinensis(Thunberg)之11个数量性状进行了多元分析。在主成分分析的第1、2主轴所构成的平面上,生活在两个地区的稻蝗个体被明显分成两个集团。此外,两地稻蝗在卵囊平均含卵量、卵粒大小及有效积温等生物学特性上均有显著的差异,这意味着长期的地理阻隔(秦岭山脉)致使两地的稻蝗演化成两个地理宗。由于使用传统的形态方法无法准确区分这两个地区的稻蝗,我们为这两个居群的雌性和雄性成虫分别建立了线性判别函数,以便为今后进一步的小进化研究奠定一个基础。V(雌性)＝-11.67+195.44H+1.91E/F+8.663MX+22.80F/C+0.34P一3.50MZ-102.76H/C-42.51F;y(雄性)＝-17.06+30.183MZ+4.313F,这里,当v(雌性或雄性)<0时,个体属于长安县的居群,否则,属于汉中的居群。     

氟虫脲对东亚飞蝗和中华稻蝗几丁质合成酶基因表达的影响

为了探讨氟虫脲可能的作用靶标及毒性机制, 本研究以重要农业害虫东亚飞蝗Locusta migratoria manilensis (Meyen)和中华稻蝗Oxya chinensis (Thunberg)为材料, 采用简并引物扩增中华稻蝗几丁质合成酶1基因（OcCHS1）的部分cDNA序列; 以氟虫脲浸渍法处理2龄中期中华稻蝗及1, 2和3龄东亚飞蝗若虫为处理组, 丙酮处理为对照组, 使用RT-PCR和实时荧光定量PCR方法分析氟虫脲对蝗虫几丁质合成酶基因mRNA表达的影响。结果获得的OcCHS1部分cDNA序列, 其长度为312 bp, 编码104个氨基酸, GenBank登录号为HM214491, 与东亚飞蝗几丁质合成酶1基因（LmCHS1）在氨基酸水平上相似度达95％。RT-PCR结果显示, 处理组几丁质合成酶1扩增带均强于对照组。实时荧光定量PCR结果表明: 与对照组相比, 处理组中华稻蝗2龄中期若虫OcCHS1 mRNA表达提高了1.02倍, 东亚飞蝗1, 2, 3龄若虫LmCHS1 mRNA表达分别提高了34%, 82%和89%, 差异显著（P<0.05）。分析基因表达提高的原因是几丁质合成受阻后基因表达水平的一种代偿性增加, 由此推测几丁质合成酶可能是氟虫脲作用的靶标之一。     

不同地域小稻蝗mtDNA部分序列及其相互关系

测定了9个不同地理区域的小稻蝗Oxya intricata (Stal)共33个个体的线粒体DNA (mtDNA)细胞色素b基因部分序列,比较其同源性,计算核苷酸组成,并用瘤锥蝗科的曲尾似橄蝗Pseudomorphacris hollisi和斑腿蝗科的芋蝗Gesonula punctifrons作外群构建NJ分子系统树。在获得的小稻蝗432 bp的序列中, A+T约占70.3%,其中30个核苷酸位点存在变异(约占所测核苷酸的6.9%),有5个位点引起了氨基酸的变异。就每个氨基酸密码子来看,第3位点的A+T含量较高,达90.4%。由NJ树显示,小稻蝗mtDNA 细胞色素b序列不同单倍型之间有一定的分歧,形成不同的簇类关系,其分枝与地理分布没有直接的对应关系,但总体上看,这种簇类关系基本上呈平行分布,没有明显的地域性差别。     

吉林雏蝗属一新种（直翅目：蝗总科）

记述了采自松嫩草原的雏蝗属Chorthippus Fieber一新种,即吉林雏蝗Chorthippus jilinensis sp. Nov.,并与其近似种翠饰雏蝗C.dichrous (Ev.)做了比较。模式标本保存于东北师范大学生命科学学院动物标本室。     

一种高效快速的鱼类标本基因组DNA提取方法

以95%酒精保存的黄鳝(M onopterus albus)和斑鳢(Channa maculates)标本为材料,采用先沉降DNA再去除杂质的方法从鱼类标本中提取基因组DNA。基因组DNA的琼脂糖凝胶电泳和紫外分光光度法检测以及PCR扩增结果显示,本方法提取的鱼类基因组DNA的电泳主带清晰明亮;A260/A280值在1.7830-2.0144之间;PCR扩增产物条带清晰明亮,且单一整齐没有拖带,表明本方法可从酒精保存的鱼类标本中提取比较纯净的DNA,能够满足一般分子生物学试验需要。与传统苯酚/氯仿法相比,本方法操作简单快速,避免了苯酚等物质对后续实验的影响,可作为一种常规动物组织DNA提取方法。     

福尔马林保存鱼类标本中大片段DNA的提取

从福尔马林保存的鱼类标本中获得高质量DNA是比较困难的。我们对前人的方法进行了如下改进：1）在标本的前处理过程中,通过长时间的缓冲液浸泡、短暂的加温、真空干燥来消除福尔马林对样品的影响;2）在样品消化过程中,加入相对过量的蛋白酶K和还原剂;3）提取DNA后立即进行PCR反应,并增加反应的循环次数和提高退火温度。通过这些改进,我们成功地从福尔马林保存的鱼类标本中提取出了高质量DNA;通过对比不同方法（福尔马林、酒精及冰冻）处理过的标本的DNA测序结果,表明该方法是值得信赖的;标本从死亡到用福尔马林处理之间的时间延搁可能是影响所提取的DNA质量的重要因素。     

Dry weight carbon and nitrogen content of some euphausiids from the north Atlantic Ocean and the Celtic Sea

Euphausiids were identified, measured and then dried duringa research cruise to sites in the north-eastern Atlantic Oceanand shelf waters of the Celtic Sea in late summer. The lengthsof furcilia larvae and later stages were measured in three ways,and regressions relating each pair of measures were calculatedfor different developmental stages of each species. Four hundredand ninety-three dried specimens of nine species were weighedand length–weight regressions were calculated. A subsetof 187 specimens was selected for analysis of carbon (C) andnitrogen (N) content soon after return to the laboratory. TheC and N contents of a further 122 specimens were determinedafter the specimens had been stored for 9 years. No consistentdifferences in percentage C and N content were found betweenthe two groups, indicating no systematic change during prolongedstorage in desiccators. Calyptopes of Nyctiphanes couchi containeda lower percentage of both C (mean 36.2%) and N (mean 8.6%)than later stages (means: 40.6% C, 11.0% N). Carbon contentand C:N ratios of a group of specimens of Thysanoessa longicaudata,including some juveniles, adult males and adult females, weredistinctly higher (>46% and >5.0, respectively) than thoseof the majority of specimens of the same species.     

Panicum spikelets from the Early Holocene Takarkori rockshelter (SW Libya): Archaeo-molecular and -botanical investigations

This paper deals with the extraction, amplification and sequencing of ancient DNA (aDNA) from spikelets of wild cereals dated at ca. 9000 cal yr BP, representing the most ancient plants with preserved genetic material from the Sahara desert. The sub-fossil records were collected from the archaeological excavation carried out at Takarkori, an archaeological site located in south-western Libya. Morphological and genetic analyses were made on 100 well preserved dried spikelets. Ten DNA extraction protocols were performed to evaluate nucleic acid recovery in terms of DNA yield, purity and amplification success of the chloroplast barcode region matK. The extraction protocol that returned the most suitable DNA to be amplified is the Kistler and Shapiro (2011: J Archaeol Sci 38: 3549-3554) modified protocol. In our study, the results from matK amplification suggested that four specimens are the most appropriate number of spikelets for these analyses. DNA was then used for PCR amplifications of four chloroplast barcode genes: rbcL, matK, trnH-psbA and trnL. A phylogenetic analysis shows the strict relation between the archaeological specimens and modern Panicoideae, supporting the morphological identification. The results indicate that spikelets have a close relation to Panicum laetum Kunth, a wild cereal still collected in tropical Africa.     

Comparison of Preservation Methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for Reliable DNA Amplification by PCR

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 °C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.     

Amplification of cox2 (～620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens,Comparing 19 Extraction Methods and 15 Polymerases

During the past years an increasing number of studies have focussed on the use of herbarium specimens for molecular phylogenetic investigations and several comparative studies have been published. However, in the studies reported so far usually rather large amounts of material (typically around 100 mg) were sampled for DNA extraction. This equals an amount roughly equivalent to 8 cm² of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amounts of tissue are usually not available or would irretrievably damage the specimens. Through systematic comparison of 19 DNA extraction protocols applied to only 2 mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; ∼620 bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.     

Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.     

Museum fish specimens and molecular taxonomy: A comparative study on DNA extraction protocols and preservation techniques

Museum fish specimens are invaluable resources for genetic studies, but extraction of high quality DNA is often problematic. In this study, hairtail fishes of the genera Trichiurus and Lepturacanthus (family: Trichiuridae) representing a wide range of preservation histories and three different methods of preservation were analyzed for mitochondrial DNA (mtDNA) extraction, amplification and sequencing of marker genes. A total of six protocols, including a commercially available kit, were compared in this study. Amplification of conserved genes such as16S rRNA and 12S rRNA were done using polymerase chain reaction with sequence analyses using automated capillary sequencing techniques. The results show that mtDNA extraction, amplification and sequencing of conserved genes could be obtained successfully from frozen (?20°C) preserved specimens (1–5 years) and also from ethanol (95%) fixed specimens (2–5 years) but not from any of the formalin (10%) fixed specimens (3–4 years). However, specimens that have been fixed for only 7 days in buffered formalin (10% formalin with phosphate buffer containing 173 mm salt) and ethanol (95%) could yield successful mtDNA extraction, amplification and sequence information of both 16S rRNA and 12S rRNA.     

How old can we go? Evaluating the age limit for effective DNA recovery from historical insect specimens

Historical museum specimens are valuable for exploring population genetics and evolutionary questions because they can provide snapshots of morphological and genetic characteristics from populations over space and time. Unfortunately, DNA found in older museum specimens is frequently degraded, so obtaining genotypes from many individual samples necessary for rigorous molecular population genetic studies is challenging. Previous studies have varied greatly in their success at obtaining genotypes from older preserved insect material. Many well-intentioned collection curators have used research results showing poor preservation of DNA preserved in museum specimens to inform curatorial best practices, in some cases choosing not to allow DNA extraction by destructive sampling because, in their estimation, the likelihood of success would be low. Recent methodological advances in DNA extraction, amplification, and genotyping have allowed some researchers to include mid-19th century samples in molecular genetic analyses. Here we present a robust, high-throughput, and low-cost DNA extraction and genotyping protocol for historical insect specimens employing restriction digests of PCR products followed by high sensitivity electrophoresis. Using this technique, we obtained mitochondrial haplotypes for 100% of 48 New World Junonia butterfly specimens (Nymphalidae) ranging in age from pre-1813 to 1909 and show that the haplotype frequencies obtained are statistically indistinguishable from 20th-century and contemporary reference populations of Junonia (1632 specimens) matched by geographic region. As most extant insect specimens were collected after 1813, based on our findings we would expect that many or even most pinned specimens preserved in museum collections contain usable DNA for mitochondrial haplotyping.     

DNA EXTRACTION METHODS FOR KELP (LAMINARIALES) TISSUE1

Newly adapted extraction methods for harvesting total cellular DNA from kelp (Laminariales; Phaeophyta) exploit the life-history stages of these heteromorphic algae. Earlier techniques, developed primarily for chloroplast DNA extraction, were time consuming and labor intensive and required large quantities of fresh sporophyte tissue. In contrast, the new methods expedite DNA extraction by employing dried sporophyte laminae or fresh gametophyte filaments and meiospores. The current methods require less tissue and procedural manipulation, reducing the time and labor involved in DNA extraction. Additionally, the current methods were successfully employed to extract DNA from herbarium specimens up to 22 years old. Total cellular DNA yields were 12.8 and 26.4 μg:g^?1 from dried laminae,: 1.9 and 11 μg from small volumes of concentrated gametophyte filaments (25 μL), and 43 to 54.5 μg from pellets of isolated meiospores (50 μL). Following purification on Sepharose columns, DNA from all three sources was sufficiently pure for molecular applications, including restriction endonuclease digestion, Southern blot-hybridizations, and amplification via the polymerase chain reaction.     

DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.