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.     

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.     

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 .     

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。     

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.     

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。     

Tracing genetic differentiation of Chinese Mongolian sheep using microsatellites

The genetic consequences of population differentiation and isolation have been the subject of conservation biology. In this study, we analysed the genetic diversity and structure of Mongolian sheep in China. These animals belong to a traditional local breed with high production, extensive adaption, early maturity and roughage resistance. For this purpose, 26 microsatellites were genotyped for five Mongolian sheep populations. The Bayesian clustering indicated five clusters as the most probable genetic structure of the populations investigated. In addition, a clear genetic structure was revealed in three populations distributed at large geographical scales, while the other cluster encompassed UQ and HLBR sheep that displayed no clear differentiation, probably due to their close and small geographical distributions. Overall, our results are helpful in understanding the interplay of population dynamics in these close genetic lineages of Mongolian sheep.     

Isolation and characterization of microsatellites in Rattus rattus

We isolated and characterized 10 microsatellite loci in the black rat Rattus rattus (Muridae, Rodentia), a widespread invasive species largely known to cause serious problems in agriculture and human health. Polymorphism was studied in two populations, one from Madagascar and one from Senegal. It ranged from three to 12 alleles in Madagascar, and from two to five alleles in Senegal. Together with the loci previously adapted from Rattus norvegicus, this set of markers should allow the conduct of thorough studies on the genetic structure of natural populations of R. rattus.     

Genetic diversity and differentiation in Portuguese cattle breeds using microsatellites

Genotype data from 30 microsatellites were used to assess genetic diversity and relationships among 10 native Portuguese cattle breeds, American Charolais and the Brazilian Caracú. Hardy–Weinberg equilibrium was observed for all loci/population combinations except for five loci in Brava de Lide and one locus in Alentejana that exhibited heterozygote deficiency. Estimates of average observed and expected heterozygosities, total number of alleles (TNA) per breed and mean number of alleles (MNA) per locus/population were obtained. A total of 390 alleles were detected. TNA among Iberian cattle ranged from 170 to 237 and MNA ranged from 5.67 to 8.07. The highest observed heterozygosities were found in the Caracú, Maronesa, Garvonesa and Arouquesa and the lowest in Brava de Lide and Mirandesa. Estimation of population subdivision using Wright's F_ST index showed that the average proportion of genetic variation explained by breed differences was 9%. Neighbour‐joining phylogenetic trees based on D_A distances showed that the genetic relationships of present‐day Portuguese native breeds are consistent with historical origins in the Brown Concave (Arouquesa, Mirandesa, Marinhoa) and Red Convex (Mertolenga, Alentejana, Garvonesa, Minhota) evolutionary groups. The Iberian Black Orthoide group, represented by Brava de Lide and Maronesa, and the Barrosã breed appeared to be more closely related to the Brown Concave group but may represent a separate lineage. The Caracú breed was not found to be closely associated with any of the native Portuguese breeds.     

PERMANENT GENETIC RESOURCES: Cross-family amplification: microsatellites isolated from Macropodidae are polymorphic in Potoroidae

The superfamily Macropodoidea consists of two families - the Macropodidae and Potoroidae. Cross-species amplification and polymorphism of microsatellite loci is widely recognized within the macropodid family; however, the success of macropodid loci in potoroid species has not been as widely published. In this study, we tested the amplification and polymorphism of 17 cross-species microsatellite loci isolated from macropodids and potoroids in Bettongia lesueur (a potoroid). Success varied between loci and was not predicted by genetic distance from the species of isolation.     

Molecular, morphological and behavioural data reveal the presence of a cryptic species in the widely studied Drosophila serrata species complex

The Drosophila serrata species complex from Australia and New Guinea has been widely used in evolutionary studies of speciation and climatic adaptation. It is believed to consist of D. serrata, D. birchii and D. dominicana, although knowledge of the latter is limited. Here we present evidence for a previously undescribed cryptic member of the D. serrata species complex. This new cryptic species is widespread in far north Queensland, Australia and is likely to have been previously mistaken for D. serrata. It shows complete reproductive isolation when crossed with both D. serrata and D. birchii. The cryptic species can be easily distinguished from D. serrata and D. birchii using either microsatellite loci or visual techniques. Although it occurs sympatrically with both D. serrata and D. birchii, it differs from these species in development time, viability, wing size and wing morphology. Its discovery explains patterns of recently described mitochondrial DNA divergence within D. serrata, and may also help to clarify some ambiguities evident in early evolutionary literature on reproductive incompatibility within the D. serrata species complex.     

Cross-species testing of 27 pre-existing microsatellites in Podarcis gaigeae and Podarcis hispanica (Squamata: Lacertidae)

We tested 27 microsatellite loci for cross-species amplification in the lacertids Podarcis gaigeae and Podarcis hispanica. We detected 11 and 15 polymorphic loci in the former and the latter species, respectively. In a larger sample of individuals from a single population of each species, the number of alleles ranged from five to 23 in 10 of the polymorphic loci in P. gaigeae, and between four and 13 in nine of polymorphic loci in P. hispanica. Two locus deviated from Hardy-Weinberg equilibrium in P. hispanica. Between 11 and 16 of the 27 loci also amplified successfully in three other Podarcis species.     

Morphometrical evolution in a Drosophila clade: the Drosophila obscura group

Five morphometrical traits (wing and thorax length, ovariole number, and thoracic and female abdomen pigmentation) were investigated in laboratory stocks of 20 species belonging to the Drosophila obscura group (subgenus Sophophora). These species originated from four biogeographical regions and represent all five of the presently recognized, taxonomic subgroups. Size‐related traits (wing and thorax length) were highly variable across species, and interspecific variation explained more than 90% of total variability. In both traditional and phylogenetic analyses, wing size was positively correlated with latitude of origin. These interspecific correlations were however notably weaker than those for intraspecific correlations. Wing/thorax ratio, which may be related to flight capacity, showed little variation. Ovariole number was highly variable (range 27–53) both within and between species, and was positively correlated with the wing/thorax ratio, suggesting that species with relatively large ovaries have relatively low wing loading. Although many species are completely dark, 11 had some regions of light coloration. A light thorax with a median darkening was observed in six species. A variable pigmentation of abdominal tergites, in females only, was found in nine species, belonging to three subgroups only. With respect to both molecular phylogeny and morphometrical evolution, the D. obscura subgroup is probably now the best investigated clade in Drosophila.