四种鲟鱼线粒体PCR-RFLP鉴定方法的研究

利用线粒体序列开发高效准确的分子鉴定方法广泛应用于水产品易混物种的鉴定中。应用PCR-RFLP技术,对鲟鱼易混种开展了分子生物学鉴定方法研究。结果表明,利用一组引物对8种鲟鱼线粒体基因进行PCR扩增,分别应用限制性内切酶TaqαI、Ava II和Eag I-HF、Nae I对扩增产物进行酶切,并用3.0%的琼脂糖凝胶检测PCR产物的酶切结果,可从8种鲟鱼易混种中分别鉴别出中华鲟、小体鲟、达氏鳇以及欧鳇。所建立的方法操作简单,在保证鱼种存活基础上只需剪取少量鳍条,便可快速准确地进行常见鲟鱼和不同鲟鱼产品的鉴别,大大增加了鉴定结果的准确性和可信度,极大地提高了工作效率。     

5种鲟鱼免疫球蛋白重链恒定区序列研究

为了探讨几种鲟鱼免疫球蛋白(IgM)所包含的信息与其亲缘和进化之间的关系, 分别对俄罗斯鲟(Acipenser. gueldenstaedtii)、小体鲟(A. ruthenus)、施氏鲟(A. schrenckii)、中华鲟(A. sinensis)和欧鳇(Huso huso)的IgM重链(IgH)恒定区进行了研究。采用RT-PCR的方法对IgH核酸序列进行了克隆, 通过软件获得了相应的IgH氨基酸序列。在分别对这5种鲟鱼免疫球蛋白重链恒定区4个区(CH1～CH4)进行研究后发现, 其CH4区氨基酸序列相似性最高。通过对CH4区序列氨基酸变异期望值(Kaa), 物种分化时间(T)及物种间系统进化树(Phylogenetic Tree)等参数的分析, 将克隆的5种鲟鱼IgH恒定区序列与已发表的西伯利亚鲟同源序列比对(源于NCBI序列)后发现: 西伯利亚鲟与俄罗斯鲟、施氏鲟与欧鳇、小体鲟各构成一个分支, 并与中华鲟相对。实验结果从体液免疫系统的演化这个角度, 反映了被研究的鲟鱼物种间的分类地位、地理分布及进化关系之间的联系。     

蚯蚓对瓦氏黄颡鱼摄食达氏鳇卵的干扰实验

自然水域中中华鲟(Acipenser sinensis)卵常受到敌害鱼类捕食的威胁,瓦氏黄颡鱼Pelteobagrus vachelli(Richerdson)是中华鲟卵的重要敌害鱼类。以中华鲟卵的敌害鱼类瓦氏黄颡鱼为研究对象,选择环毛蚓(Pheretima tschiliensis)为瓦氏黄颡鱼的饵料,以达氏鳇卵(Kalugasturgeon)替代中华鲟卵进行室内实验,通过研究适合瓦氏黄颡鱼摄食的环毛蚓规格,以及环毛蚓干扰瓦氏黄颡鱼摄食达氏鳇卵投放比例,探讨中华鲟卵的保护方法。在水温为(20.0±1.5)℃的条件下,研究了3种不同体长规格[体长(15.3±1.4)cm、(12.0±2.1)cm、(7.7±0.5)cm]实验鱼个体分别摄食3种不同体长规格环毛蚓(体高为0.3cm,体长分别为8、4、2cm)和达氏鳇卵的摄食率;研究了体长为4cm的环毛蚓对3种不同体长实验鱼摄食达氏鳇卵的干扰效果。实验结果表明:3种体长规格瓦氏黄颡鱼均摄食达氏鳇卵;3种体长规格瓦氏黄颡鱼共同摄食的替代饵料规格为体长4cm及以下的环毛蚓;适宜干扰实验所选体长实验鱼摄食达氏鳇卵的饵料为体长4 cm的环毛蚓;当环毛蚓与达氏鳇卵数量比例为1∶1时,可实现达氏鳇卵的保护。研究证明在实验条件下可实现环毛蚓对中华鲟卵敌害鱼类——瓦氏黄颡鱼实施摄食干扰,提示下一步可以尝试选择环毛蚓作为替代中华鲟卵的饵料,进行干扰食卵鱼类摄食中华鲟卵的野外验证试验。     

12种鲟形目鱼类mtDNA ND4L-ND4基因的序列变异及其分子系统学

鲟形目鱼类是一类在鱼类乃至脊椎动物进化史上占有很重要地位的古老濒危鱼类 ,长期悬而未决的系统演化关系为世人瞩目 .采用DNA测序技术首次测定了包括中国特有鲟形目鱼类在内的 1 2种鲟形目鱼类的mtDNA ND4L和ND4基因 ( 70 3bp)的序列 ,并进行了分子系统学分析 .从分子系统学的角度得出了如下结论 :( 1 )鳇属可以归并到鲟属 ;( 2 )达氏鲟与中华鲟的亲缘关系最近 ,很有可能为中华鲟的一陆封类型 ;( 3)环太平洋地区的鲟科鱼类可能有共同的起源 ;( 4)ND4L和ND4基因是进行鲟形目鱼类系统演化研究很好的遗传标记 .     

利用微卫星遗传标记探讨达氏鳇的多倍体倍性

鲟形目(Acipenseriformes)鱼类是一类多倍体起源的鱼类,种问易于杂交,且由于存在大量微型染色体,其染色体数目和倍性也难于确定.到目前为止包括达氏鳇(Huso dauricus)在内的一些鲟鱼物种基因组大小和染色体倍性仍旧存在争议.本实验采用微卫星遗传标记技术,通过观察9个微卫星位点的等位基因数目,发现达氏鳇在大部分位点中显示的倍性大于二倍性,同时利用多倍体分析软件POLYSAT推断了26尾达氏鳇的个体倍性.结果表明,26尾达氏鳇中有20尾显示为六倍体,占总数的76.92％;4尾显示为八倍体,占总数的15.38％;2尾显示为四倍体,占总数的7.79％.因此,我们认为达氏鳇应该为八倍体物种,结果中出现的四倍体和六倍体个体,是由于这些个体在我们选取的微卫星位点中存在部分纯合等位基因,导致了多数个体显示为六倍体.这一结论与近年来支持达氏鳇为进化性八倍体物种的研究结果一致.     

鲟鳇鱼网箱养殖环境微生物菌群结构及潜在病原菌分析

为探究鲟鳇鱼网箱养殖环境中的细菌群落结构组成和潜在致病菌的种类与数量, 文章用高通量测序对中华鲟Acipenser sinensis、施氏鲟Acipenser schrenckii、达氏鳇Huso dauricus、大杂交鲟A. schrenckii♂×H. dauricus♀等网箱养殖水体的细菌群落结构特征和潜在病原菌属进行分析, 同时应用实时荧光定量PCR方法对鲟鳇鱼养殖水体中的典型病原菌种进行定量检测。研究结果表明, 在同一水域同一管理条件下, 不同品种鲟鳇鱼网箱养殖水体中细菌群落结构组成相似度高, 优势菌门由Proteobacteria、Actinobacteria、Bacteroidetes、Verrucomicrobia和Planctomycetes组成, 占到所有样品序列总数的88.81%—93.94%; 在属水平上, 各样品未分类的细菌(unclassified)占比较大, 共发现鲟鳇鱼潜在致病细菌菌属7个: Aeromonas、Acinetobacter、Chryseobacterium、Edwardsiella、Flavobacterium、Pseudomonas和Vibrio。实时荧光定量PCR结果显示, 养殖水体中鲟鳇鱼病害常见的病原菌种Aeromonas hydrophila的丰度最高, 丰度值为(7406±1892) copies/mL; 其次为Flavobacterium columnar[(4889±1649) copies/mL]和Pseudomonas fluorescens[(3259±1117) copies/mL]。研究结果为该区域的鲟鳇鱼集约化养殖及疾病防控提供科学依据。     

中国马兜铃属和关木通属(马兜铃科)概览

马兜铃属(广义) (Aristolochia sensu lato)具有花单被、花萼管状、合蕊柱、子房下位、中轴胎座、胚珠多数、蒴果等主要特征, 广布于全世界的热带、亚热带和温带地区, 约有600种, 是马兜铃科中种类最多的属。依据Flora of China, 我国有本属植物45种, 其中33种为中国特有。近些年, 国内大量的新类群被相继报道, 特别是云南、广西两地, 而另一些类群则得到了确认、恢复、重新发表或修订。最近, 基于形态和分子证据, 关木通属(Isotrema)因其花萼管急剧弯曲; 合蕊柱3裂; 雄蕊6, 成对与合蕊柱裂片对生; 蒴果由上而下开裂等区别特征而从广义马兜铃属中被分出独立成属。本文基于大量的野外调查、标本鉴定、数码照片考证和相关文献的仔细研究, 重新梳理了中国马兜铃属和关木通属的种类及分布情况, 确认现阶段中国马兜铃属17种和关木通属58种1亚种, 并一一记述同时编制了最新的检索表。其中, 柔叶关木通(I. mollis)、线叶关木通(I. neolongifolia)的种级地位得到重新确认, 并提供了图版。探讨了优贵马兜铃(A. gentilis)、川滇马兜铃(A. chuandianensis)和纤细马兜铃(A. gracillima)的关系, 昆明关木通(I. kunmingense)与波氏关木通(A. bonatii)的关系, 以及卵叶关木通(I. ovatifolium)、葫芦叶关木通(I. cucurbitoides)、西藏关木通(I. griffithii)、过石珠(I. versicolor)、大别山关木通(I. dabieshanensis)等复合群和袋形关木通(I. saccata)等物种存在的问题。     

鲟鱼心外膜脓肿的病理学初步研究

作为新的养殖对象,我国各地先后开展了中华鲟(Acipenser sinensis Gray)、施氏鲟(A.schrenckii Brandt),以及引进的俄罗斯鲟(A.gueldenstaedti Brandt)、杂交鲟等鲟鱼人工养殖,由于养殖刚刚起步且养殖集约化程度较高等诸多因素,出现了多种新的非寄生性疾病,并造成大量死亡,经济损失极其严重,成为当前鲟鱼试养阶段的制约因素。1999年北京市一家鲟鱼养殖试验场鲟鱼患病,经检验和诊断,确诊为“鲟鱼心外膜脓肿”.本文报道了该病的病理解剖和组织病理学研究结果,为疾病的临床检验与诊断及有效控制提供科学依据。       

几种鲟鱼基因组大小、倍体的特性及鲟形目细胞进化的探讨

采用Ｆｅｕｌｇｅｎ－显微分光光度计方法,以鸡红细胞为标准ＤＮＡ（３．２２ｐｇ／２Ｃ）测定了长江白鲟、达氏鲟、中华鲟、史氏鲟和北美匙吻鲟的体细胞基因组大小（ＤＮＡ含量）。结果表明,上述五种鲟鱼ＤＮＡ含量分别为４．１１、８．２６、９．０７、６．０７和３．９６ｐｇ。长江白鲟和北美匙吻鲟均属于四倍体鱼类,分布在长江水系的中华鲟和达氏鲟两种鲟属鱼类为八倍体类型。史氏鲟初步判断为八倍体,据分析可能存在四倍体的     

中华鲟(Acipenser sinensis)及相关种类的mtDNA控制区串联重复序列及其进化意义

采用 PCR技术和 DNA测序技术 ,发现了我国一级珍稀保护动物中华鲟 ( Acipensersinensis)线粒体 DNA( mt DNA)的控制区 ( D- loop)存在数目不等的串联重复序列 ,该重复序列造成了中华鲟广泛的异质性现象 .从分子水平进行了不同类型重复序列变化规律的研究 ,同时还初探了重复序列在我国其它几种鲟鱼类的存在情况 ,发现在白鲟 ( Psephurus gladius)、达氏鲟 ( A.dabryanus)和史氏鲟 ( A.schrenckii)均存在类似的重复序列结构 .序列比较分析表明 ,不同鲟鱼类重复序列在鲟鱼类进化过程中扮演着一定的角色 ,很有可能碱基差异大小与它们的亲缘关系的远近呈正相关 .     

中华鲟养殖池背角无齿蚌和鲢鳙鱼生态修复效果比较

2009年5月31日-6月20日在广东省大沙河水库利用微型生态系统比较不同放养密度的褶纹冠蚌(Cristaria plicata)和背角无齿蚌(Anodonta woodiana)对水体氮、磷及浮游植物的影响,探讨两种蚌在控制南亚热带水库富营养化水体藻类水华上的可行性。实验结果表明,在褶纹冠蚌和背角无齿蚌处理组中,总氮、总磷和叶绿素a浓度显著增加,而铵氮的浓度显著下降;褶纹冠蚌和背角无齿蚌导致了浮游植物群落组成结构的改变和数量的增加,实验过程中绿藻所占的比例迅速上升。两种蚌之间没有显著的差异,只是在不同的作用强度下,时间上的响应不同。综合实验结果,褶纹冠蚌和背角无齿蚌难以有效地运用于我国华南地区水库的水质改善与富营养化控制。     

Polyphyly of mtDNA lineages in the Russian sturgeon, Acipenser gueldenstaedtii: forensic and evolutionary implications

Molecular species identification methods are an important component of CITES monitoring programs for trade in sturgeon and caviar. To date, obtaining molecular evidence for distinguishing caviar from four closely related Eurasian sturgeon species Acipenser baerii (Siberian sturgeon), A. gueldenstaedtii (osetra), A. persicus (Persian sturgeon), A. naccarii (Italian sturgeon) remains problematic. Using approximately 2.3 kb of mtDNA sequence data (cytochrome b, NADH5, control region), we find this to be attributable to the polyphyletic nature of these mitochondrial DNA markers in the Russian sturgeon, A. gueldenstaedtii. Two mitochondrial lineages are present within this species: one is phylogenetically affiliated with A. persicus and A. naccarii, while the other clusters with A. baerii. These findings have a direct impact on molecular testing of commercial caviar and demonstrate the necessity of using large sample sizes when constructing forensic databases. Furthermore, the results affect current taxonomic designations for these species as well as hypotheses concerning their evolutionary origins.     

Responses in allometric growth of two dominant species of subalpine meadow—Arundinella anomala and Miscanthus sinensis—to climate warming in Wugongshan Mountains,China

AimsArundinella anomala and Miscanthus sinensis are dominant species in the subalpine meadow of Wugongshan Mountains. Here we studied the effects of climate warming on allometric relationships among different growth components in the two species through simulated warming in natural habitat.
Methods The warming experiments were conducted with open-top chambers (OTCs) in natural habitat in a subalpine meadow community of Wugongshan Mountains. The two main Gramineae species, Arundinella anomala and Miscanthus sinensis, were selected as the study materials. Two sizes of OTCs were set up to create contrasting levels of warming (i.e. TD for low warming and TG for high warming). The morphological variables, such as the plant height and the basal diameter of shoots, were measured. Allometric analysis was conducted with the Smart Package in R software.
Important findings Significant or highly significant correlations and significant allometric relationships were found between and among growth components in both species. The allometry of the growth in most morphological features was strengthened and modified by simulated warming. Stem diameter and plant height, and spike length in A. anomala and M. sinensis changed from isometric to allometric following warming. The relationship The synchronized growth between stem diameter and spikelet in A. anomala was weakened, but their allometry was enhanced with increased warming. The allometric relationship between plant height and leaf length in A. anomala transformed into isometric growth, but it was in reversed pattern in M. sinensis. Warming promoted the plant height and leaf sheath length in A. anomala, whilst higher warming changed the growth relationship between plant height and leaf sheath length in M. sinensis. Similar allometric relationships among the leaf traits were observed, but warming did not significantly impact their allometric exponents. The results suggested that climate warming could have varied effects on different plants, and the differences are often related to the adaptability of plants.     

Mislabeled and counterfeit sturgeon caviar from Bulgaria and Romania

A market survey was performed on caviar from Romania and Bulgaria, two of the last range states of critically endangered sturgeons threatened by poaching and illegal trade. Twenty‐seven samples were collected between April 2011 and February 2012, including two samples of Bulgarian farm caviar bought in an Austrian fish shop. Mitochondrial sequences of the cytochrome b gene and microsatellites were used for species identification. Ten samples were in agreement with the species code on their CITES labels, but four samples were mislabeled, six samples were counterfeit and four samples were offered as ‘wild origin,’ ignoring the fishing bans in place. At least one sample was upgraded from a lower priced species to a more expensive species. These results demonstrate the weakness of sturgeon protection in these Member States of the European Union and key range states. Therefore, we recommend intensifying conservation and enforcement efforts, including also DNA testing of caviar in containers with apparently correct CITES labels.     

Gonadal Maturity in Wild Sturgeons, Huso dauricus, Acipenser mikadoi and A. schrenckii Caught near Hokkaido, Japan

We investigated the occurrence and gonadal maturity of wild sturgeons, which are rarely caught off Hokkaido island in Japan. Based on their morphological features, combined with the data on their cell DNA content, 48 sturgeons were classified into three species and one hybrid. These were the kaluga, Huso dauricus; the Japanese (Sakhalin) sturgeon, Acipenser mikadoi (historically, this species has been considered conspecific with the American green sturgeon, A. medirostris); the Amur sturgeon, A. schrenckii; and the kaluga–Amur sturgeon hybrid; with a ratio of 36:10:1:1. All of them were caught in saline waters, rather than in the fresh-water of the river, although some of them were in saline waters in the mouth of the river. Most of them were smaller than adult size; however, early juvenile fish were not detected. Histological analysis of gonadal development revealed early stages of gametogenesis in the majority of the fish of both genders. These results indicate that several sturgeon species are still occurring in the coastal waters of northern Japan, at different stages of gonadal maturity. In contrast, the lack of reports of both mature and early juvenile sturgeon capture in the Hokkaido rivers may suggest that native spawning populations of Japanese sturgeon have become extinct in Japan.     

High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

鼠类对栓皮栎和锥栗种子的贮藏:验证高单宁假说

种子中的单宁影响鼠类的取食和贮藏策略。高单宁假说认为鼠类倾向于优先贮藏单宁含量高的种子、取食单宁含量低的种子。同域分布的鼠类对种子的取食和贮藏选择是否符合高单宁假说的预测尚缺乏足够的实验证据。本研究通过围栏实验研究了四川都江堰地区亚热带常绿阔叶林中的小泡巨鼠（Leopoldamys edwardsi）、针毛鼠（Niveventer fulvescens）、北社鼠（N.confucianus）、高山姬鼠（Apodemus chevrieri）、中华姬鼠（A.draco）对单宁含量高的栓皮栎（Quercus variabilis）种子和单宁含量低的锥栗（Castanea henryi）种子的取食和贮藏偏好,旨在进一步验证高单宁假说。结果表明：（1）5种鼠均偏好取食锥栗种子,很少取食或不取食栓皮栎种子;（2）仅具集中贮藏习性的针毛鼠和北社鼠偏好集中贮藏锥栗种子;兼具集中和分散贮藏习性的小泡巨鼠和高山姬鼠也倾向于贮藏锥栗种子;（3）兼具集中和分散贮藏习性的中华姬鼠偏好分散贮藏栓皮栎种子,集中贮藏锥栗种子。研究结果并不完全支持高单宁假说,表明同域分布的鼠类对种子取食和贮藏偏好表现出种间分化,高单宁假说在同域分布鼠类中可能不具有普遍性。     

Genetic variation and relationships of seven sturgeon species and ten interspecific hybrids

Background

Sturgeon cultivation is important for both industry and aquaculture in China. To date, more than 17 species or strains have been farmed for fillets and caviar production. Crossbreeding among different sturgeon species is frequent and the F2 hybrids are fertile. However, large-scale farming can have negative impacts on wild populations i.e. escape of exotic sturgeons and must be taken into consideration. Escape of exotic sturgeons can cause severe ecological problems, including threatening native sturgeon species once the exotic varieties become established or hybridize with native individuals. However, little is known about their genetic resources and variation.

Methods

Genetic diversity and introgression of seven sturgeon species were analyzed using mitochondrial DNA cytochrome oxidase subunit I (COI) and nine microsatellite markers. This study included 189 individuals from seven sturgeon species and 277 individuals from ten lineages of F2 hybrid strains.

Results

MtDNA COI sequences (632 bp long) were generated from 91 individuals across the 17 sturgeon strains and produced 23 different haplotypes. Haplotype diversity was high (h = 0.915 ± 0.015) and nucleotide diversity was low (π = 0.03680 ± 0.00153) in the seven sturgeon species and ten interspecific hybrids. Phylogenetic analyses resulted in almost identical tree topologies, and different haplotype structures were mainly related with sturgeons of different female parents. Analysis of molecular variance revealed that 81.73% of the genetic variance was due to matrilineal differences, while 9.40% resulted from strain variation. Pairwise Fst values obtained with POLYSAT software, were high among strains and ranged from 0.031 to 0.164. Admixture analysis assigned seven distinct groups and ten genotypes of admixed clusters composed of hybrid strains using STRUCTURE when assuming K = 7.

Conclusions

The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions. These relationships were also supported by the results from the microsatellite analysis and contributed to unambiguously identify seven sturgeon species and ten F2 hybrid strains from sturgeon farms in China. Moreover, we found that introgressive hybridization is pervasive, exists in both purebred and hybrid sturgeons, and may reflect widespread mismanagement in sturgeon breeding in China.     

猕猴桃种间杂种三倍体形态学和减数分裂观察

本文报道中华猕猴桃(2n=116)×毛花猕猴桃(2n=58)F₁代形态学和减数分裂观察结果。F₁代植株部分性状同父本(如花色)或母本(如雄蕊数),部分性状居双亲变异范围之间,综合形态特征大致为双亲的中间型。根尖细胞染色体数为87。花粉母细胞减数分裂中期Ⅰ染色体构型为29Ⅱ+29Ⅰ,应为异源三倍体。雌雄株均高度不育。     

种植模式对当归根际细菌群落多样性及代谢通路的影响

连作障碍严重限制了当归产业的可持续发展。为了探索当归(Angelica sinensis)高效栽培技术,本研究在当归主产区甘肃省渭源县设置5种种植模式(A: 豌豆-当归-当归,对照;B: 豌豆-小麦-当归;C: 豌豆-蒙古黄芪-当归;D: 豌豆-马铃薯-当归;E: 豌豆-休耕-当归),于采挖期分别测定不同种植模式下,当归根际土壤理化特性和细菌基因组DNA相对丰度,分析不同种植模式对当归根际土壤理化特性、细菌群落多样性和代谢通路的影响。结果表明: 1) 5种种植模式下当归根际土壤理化特性差异较大。与对照相比,C模式根际土壤的电导率显著增加,B、D和E模式的电导率略有下降,B、C、D和E模式的土壤CO₂呼吸速率显著提高。2) 5种种植模式的当归根际土壤细菌隶属于26门368属,其中,芽单胞菌门的芽单胞菌属、变形菌门的鞘脂单胞菌属和酸杆菌门的Subgroup_6属为优势菌属。与对照相比,B、C模式变形菌门和放线菌门的相对丰度显著增加,D模式酸杆菌门的相对丰度显著降低;E模式变形菌门、酸杆菌门和放线菌门的相对丰度显著增加。3) 5种种植模式下,当归根际土壤的pH值、电导率、有机质、碱解氮、有效磷和速效钾含量与变形菌门细菌的相对丰度呈显著负相关。4) 5种种植模式下,当归根际土壤中6种代谢通路细菌的相对丰度差异显著。C模式对当归根际土壤理化特性和细菌群落有较好的调节作用,是克服当归连作障碍的主要种植模式。