Isolation and characterization of microsatellites in Drosophila virilis and their cross species amplification in members of the D. virilis group

We report the isolation and cross species amplification of 42 Drosophila virilis microsatellite loci. Nine loci were isolated from mapped P1 bacteriophage clones and 33 were obtained from genomic DNA or GenBank searches. Cross species amplification was tested for all members of the D. virilis group. The amplification success was high (varying from 45% to 100%) and most of the loci were polymorphic. This set of loci can be applied for several genetic studies such as mapping behavioural quantitative trait loci (QTL) and for studying population structure in a phylogeographical framework in D. virilis group species.     

Isolation and characterization of microsatellites in Drosophila montana and their cross‐species amplification in D. virilis

We report the isolation of 20 microsatellite loci from Drosophila montana and their cross amplification in the relative D. virilis. All microsatellite loci were polymorphic in the focal species D. montana, with gene diversities ranging from 0.23 to 0.93. In D. virilis only eight loci (40%) amplified and two loci were polymorphic (10%). These markers represent the first report of microsatellites isolated in D. montana. They could be applied for studying population structure and phylogeography. The largest benefit, however, will be their use in studies of quantitative trait loci, such as the mapping of behavioural quantitative trait loci.     

黑腹果蝇种组的核型与进化研究

观察了国内黑腹果蝇种组34种果蝇的有丝分裂中期核型,其中首次描述了一些新核型。系统地分析了黑腹果蝇种组8个种亚组之间的核型进化关系及种间亲缘关系。结果是:elegans种亚组的核型为A型;eugracilis、melanogaster和ficusphila种亚组的核型为C型;takahashii和suzukii种亚组的核型为C型和D型;montium种亚组的核型为B、C、C’、D、D’、和E型;ananassae种亚组的核型为F、G和H型。从核型分化的角度可以将黑腹果蝇种组分为5个谱系:elegans,eugracilis-melanogaster-ficusphila,takkahashii-suzukii,montium,ananassae。这与2004年Yang等的观点基本一致,正好从核型进化的角度验证了Yang通过DNA序列分析所得到的结果。差别只在于elegans种亚组,作者把它单独列为一支,认为是祖先种亚组。通过选取同一种果蝇的几个不同地域单雌系的核型分析,结果表明:同一种果蝇的核型存在地域差异。这种差异可能是由于不同生境造成,也可能是本身进化程度的差异,或是两种因素相互作用的结果。     

基于微卫星标记的桃蚜种群寄主遗传分化

桃蚜Myzus persicae(Sulzer)是寄主范围最广、危害最大的蚜虫种类之一。为了探明桃蚜在不同寄主上的遗传分化特点,采用微卫星分子标记技术,对西兰花、桃树、辣椒上的桃蚜种群进行遗传多样性和遗传结构研究。结果表明,在所选用的5个微卫星位点上共检测到38个等位基因,平均每个位点的等位基因数达到7.6个,桃树种群遗传多样性最高,这可能是因为各种夏寄主上的桃蚜迁回桃树上越冬,从而使多种等位基因和基因型得以聚集的原因。等位基因频率差异分析显示西兰花种群、桃树种群和辣椒种群两两之间(除了桃树06种群和辣椒06种群之间没有遗传分化外)都出现了明显遗传分化,相比之下桃树种群和辣椒种群的分化程度要比桃树种群和西兰花种群的分化程度低,这可能预示着西兰花寄主上的桃蚜正在向远离桃树和辣椒种群的方向进化。     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

Isolation and characterization of microsatellites in giant freshwater prawn Macrobrachium rosenbergii

Microsatellite loci were characterized in a freshwater prawn from enriched genomic library using six biotinylated probes: (AG)₁₀, (TG)₁₀, (CAA)₁₀, (CAG)₁₀, (GAT)₁₀ and (TAC)₁₀. Primers for DNA amplification were designed and synthesized for 20 loci. Ten loci were polymorphic with the number of alleles ranging from five to 17 alleles per locus and the observed heterozygosity ranging from 0.27 to 0.83 per locus. Developed microsatellite primers should prove useful for selective breeding programs and population genetic studies of freshwater prawn.     

Drosophila subpulchrella, a new species of the Drosophila suzukii species subgroup from Japan and China (Diptera: Drosophilidae)

Drosophila (Sophophora) subpulchrella Takamori and Watabe, sp. nov., of the D. suzukii subgroup in the D. melanogaster species group, is described from Japan and southern China, and compared with its sibling species, D. pulchrella Tan et al. distributed in the Yun‐Gui Highland, south‐western China. The results of cross‐experiments show a complete pre‐mating isolation between D. subpulchrella and D. pulchrella.     

Rapid isolation of CA microsatellites from the tilapia genome

We have developed (CA)n microsatellite markers for the cichlid fish, Oreochromis niloticus using a variation of the hybrid capture method. The resulting genomic library was highly enriched in repetitive DNA with 96% of clones containing CA repeats. The number of repeats ranged from four to 45 with an average of 19. Two-thirds of the sequenced clones had 12 or more repeats and sufficient flanking sequence to design primers. The resulting markers were tested in an F2 cross of O. niloticus x O. aureus. Nearly 90% of the markers amplified in this cross and 74% of these were informative. This work demonstrates the importance of minimizing the number of polymerase chain reaction (PCR) amplification cycles before and after the enrichment steps to reduce PCR recombination and the generation of chimaeric clones.     

中国云南果蝇属暗果蝇种组的核型分化

观察了新近发现于我国云南的果蝇属暗果蝇种组(Drosophila obscura species group)种类D．luguensis、D．dianensis和D．limingi的有丝分裂中期核型,并将3个种的核型与各自的近缘种类进行了比较。D．luguensis具2n=12条染色体,包括3对中央着丝粒(V形)染色体、2对近端着丝粒(棒状)染色体以及1对微小(点状)染色体。其中X和Y染色体均为中央着丝粒染色体。D．dianensis和D．limingi具2n=10条染色体,包括1对大的V形常染色体,1对小的V形常染色体,2对J形(亚中着丝粒型)常染色体和1对点状染色体。其中X染色体为J形,Y染色体为短棒状。基于核型比较的结果以及D．sinobscura亚组地理分布的资料,结合种间系统发育关系研究结果,认为D．luguensis可能保留了该亚组祖先种类的核型。D．sinobscum的核型(2n=12：2V,1J,2R,1D)可能由一个pre-“sinobscura-hubeiensis”谱系的一个分支通过臂间倒位演化而来,而D．hubeiensis的核型(2n=10：4V,1D)可能由该谱系的另一分支通过着丝粒融合(2对近端着丝粒常染色体的融合)而形成。推测在D．dianensis和近缘欧洲种D．subsilvestris(2n=12：3V,2R,1D)间、D．limingi和东亚近缘种D．tsukubaensis(2n=12：3V,2R,1D)间的物种分化过程中,可能有相似的染色体变异类型发生。     

Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat

Microsatellites, or simple sequence repeats (SSRs), have become the markers of choice for genetic studies with many crop species including wheat. Currently an international effort is underway to enrich the repertoire of available sequence tagged microsatellite site (STMS) markers in wheat. As a part of this effort, we have sequenced 43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones contained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide repeats) were classified as 19 simple perfect, 18 simple imperfect and 4 compound imperfect types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16 microsatellites could be designed, since the flanking sequences of the others were either too short or were otherwise not suitable for designing the microsatellite specific primers. Microsatellite loci of the expected size and polymorphism were successfully amplified from 15 of these 16 primer pairs using three wheat varieties. 14 loci detected by 12 out of the 15 functional primer pairs were assigned to 11 specific chromosomes. An erratum to this article is available at .     

中国黑腹果蝇种组40种果蝇的核型多样性研究

通过传统的敲片、Giemsa染色的方法制片对中国黑腹果蝇种组(Drosophilamelanogasterspeciesgroup)8个种亚组40种果蝇的染色体进行了分析,共发现18种核型,即A、A′′、C、C′、C′′、C′′′、C′′′′、D、D′、D′′、E、E′、E′′、F、F′、G、H和I,其中A、A′′、C′′′、C′′′′、D′′和F′为新发现的核型。8个种亚组的基本核型分别是:嗜凤梨果蝇种亚组(D.ananassaesubgroup)的核型为F、F′、G和H型;牵牛花果蝇种亚组(D.eleganssubgroup)的核型为A和A′′型;细针果蝇种亚组(D.eugracilissubgroup)的核型为C型;嗜榕果蝇种亚组(D.ficusphilasubgroup)的核型为C′型;黑腹果蝇种亚组(D.melanogastersubgroup)的核型为C和C′型;山果蝇种亚组(D.montiumsubgroup)的核型为C、C′、C′′、D、D′、D′′、E、E′、E′′和I型;铃木氏果蝇种亚组(D.suzukiisubgroup)的核型为C′′′和C′′′′型;高桥氏果蝇种亚组(D.takahashiisubgroup)的核型为C、C′′′和C′′′′型。透明翅果蝇(D.lucipennis)雌性核型2n=8,雄性核型2n=7,雄性Ⅳ号染色体为染色体单体。此外还发现,吉川氏果蝇(D.kikkawai)、林氏果蝇(D.lini)、奥尼氏果蝇(D.ogumai)、拟嗜凤梨果蝇(D.pseudoananassae)和叔白颜果蝇(D.triauraria)5种果蝇有B染色体。本文确定了D.sp.likeelegans、D.sp.likenyinyii、D.sp.liketrapezifrons1、D.sp.liketakahashii、D.sp.liketrapezifrons2和D.sp.likeauraria等6个未描述种的核型和1个新记录种吉里果蝇(D.giriensis)的核型。本研究证明了在黑腹果蝇种组内、亚组内、种内和单雌系内的核型多样性,为果蝇遗传和进化提供了进一步的细胞学证据。     

Allelic variation,fragment length analyses and population genetic models: a case study on Drosophila microsatellites*

The allelic variation of 16 microsatellite loci from selected species of the Drosophila melanogaster and D. obscura group was determined. Intra‐ and interspecific sequence comparisons allowed discrimination of mutations affecting the repetitive microsatellite from those affecting the flanking regions. The hypotheses that slippage needs a minimum number of repeats in order to become efficient with respect to microsatellite variability, and of an increased mutation rate with increased length of the microsatellite are supported by the results of our analyses. There is in particular at the interrupted complex microsatellite locus BICOID in the species of the D. obscura group, extensive variation in the flanking regions in addition to length and sequence variation of the repetitive microsatellite. The allelic variation at this locus can hardly be explained by slippage alone. Estimates of microsatellite variability by fragment length analyses will pick up only a minor fraction of allelic variation at such loci, and conclusions that are based on the stepwise mutation model will not hold.     

兔(AG)n微卫星DNA富集文库的构建与鉴定

目的:微卫星遗传标记具有数量大、分布广且多态信息含量高等优点,因而被广泛用于动植物遗传图谱的构建、QTL定位、标记辅助选择及亲缘关系鉴定等领域。方法:采用亲和捕捉法。结果:构建了兔(AG)n微卫星DNA序列的富集文库,文库含重组克隆4 850个,其中含有(AG)n微卫星DNA序列的阳性克隆占66.7%。     

中国特有果蝇 Drosophila curviceps 种亚组在Drosophila immigrans种组中的种系发生地位

果蝇immigrans种组中的curviceps种亚组是1992年新建立的中国特有果蝇类群。该种亚组中的物种主要分布在中国大陆和台湾。目前除了形态学水平的研究外,还没有其他证据支持建立该种亚组的合理性及其起源和种系发生地位。为了在DNA分子水平上探讨果蝇curviceps种亚组在果蝇immigrans种组中的种系发生地位,从而为今后更深入地研究中国特有果蝇,甚至为果蝇亚属的进化遗传学提供理论依据,测定了immigrans种组5个种亚组（nasuta、immigrans、hypocausta、quadrilineata、curviceps)中12个代表物种的rDNA的ITS1和部分Adh基因的序列。其中ITS1序列的长度为513－587bp,共有191个信息位点;Adh基因片段的长度在714－747bp之间,共99个信息位点。考虑到单个分子提供的信息较少,将两个分子的序列综合起来,组成一个较长的复合序列。分别根据ITS1,Adh和两个分子的复合序列排比（Alignment)结果,和最大简约法和邻接法构建分子系统树,其中根据复合序列构建的系统树与形态学研究结果最为一致。分子树显示curviceps种亚组的特种确定单独形成一个分枝,为种亚组级的分类阶元,支持了形态学将其建立为一个新种亚组。根据Kimura距离,估算了复合分子的替换速率约为每百万年1．48％,进而计算出5个种亚组的分 歧年代。结合各物种的地理分布,推测了immigrans种组的进化历史：curviceps种亚组与quadrilineata种亚组的亲缘关系最近,主要分布在中国南部的温带地区。它们之间的分歧时间大约为3．4百万年,是最年轻的两个种亚组。主要分布在苏门答腊及附近的热带地区的hypocausta种亚组的物种是最早分化出来的,与其他种亚组的分歧时间约为9．2百万年。该结果与形态学和生物地理学研究相吻合。值得一提是的,目前归属仍存在争议的物种D.neohypocausta,在分子系统树中与hypocausta种亚组的物种相距较远,而与immiagrasn种亚组的关系较近,但分枝置信度较低（＜50％）。由于还缺乏其他方面的证据,因此D.neohypocausta的归属有待今后的研究来作定论。     

Microsatellite loci in the cactophilic species Drosophila antonietae (Diptera; Drosophilidae)

Morphometric and isoenzymatic data have showed little variation among Drosophila antonietae populations. This suggests historic gene flow, due to the distribution of these populations, which are associated with cacti along rivers, and natural selection on these markers. Microsatellite loci have high variability and are ideal for detecting gene flow and analysing population structure. Here we describe the isolation of seven polymorphic microsatellite loci in D. antonietae.     

Characterization of a species-specific satellite DNA of Drosophila buzzatii

The pBuM189 satellite DNA family was found to be species specific for Drosophila buzzatii . It consists of slightly AT-rich tandemly arranged repeats with a high copy number in the genome and shows a very high level of intraspecific sequence similarity. pBuM189 repeats cannot be detected in the genomes of closely related species such as Drosophila serido , Drosophila borborema and Drosophila koepferae . The data support the marginal systematic position of D. buzzatii within the buzzatii cluster of the Drosophila repleta group.     

Isolation and characterization of microsatellites from a marine foodfish species ribbonfish Trichiurus haumela

The ribbonfish, Trichiurus haumela, is an important marine foodfish species. Supply of this fish relies on capture from the wild. No information about genetic diversity and population structure of wild stocks is available. Therefore, we developed the first set of 10 polymorphic microsatellites. The average allele number was 13.2 per locus ranging from six to 20. The expected heterozygosity was 0.84, whereas the observed heterozygosity was 0.71. This set of microsatellites provides a tool for assessing genetic diversity and population structure of this species.     

黑腹果蝇种组五种核型的报道

通过传统的敲片、Giemsa染色的方法, 本文首次对果蝇属黑腹果蝇种组的5种果蝇 （D. constricta、D. ohnishii、D. ogumai、D. pseudobaimaii、D. tani）染色体的数目和形态进行了分析报道。分析发现：这5个种具有相同的染色体数目（2n=8）和不同的形态。D. pseudobaimaii和D. tani 为2V,1R,1D型;D. constricta染色为2V,1R,1D型且其点状染色体难以辨认;D. ohnishii和D. ogumai 具有相同形态为2V,2R。另外,还发现核型与亲缘关系之间有一定的对应性。