微卫星跨物种交叉PCR扩增的一个新问题:扩增非同源产物

微卫星已被广泛应用于群体遗传学、生态学和进化生物学研究。然而,一些物种微卫星尚未克隆。为了节省时间和经费,研究人员往往使用一个物种已发表的微卫星引物扩增其近缘物种的微卫星。该研究对属于3个不同科(Clariidae、Heteropneustidae 和Pimelodidae)的7个鲶鱼物种的微卫星跨物种PCR扩增产物进行了序列分析,研究发现扩增非同源（non-orthologous）产物是微卫星跨物种PCR扩增的一个新问题。该研究共采用4对胡子鲶微卫星座位引物对7个鲶鱼物种进行了跨物种PCR扩增。对获得的204个PCR产物的序列分析结果表明,两对微卫星座位引物扩增了所有7个物种的同源特异产物。而其他两个座位的引物扩增了特异但非同源的多态产物,对近缘物种的扩增也获得类似结果。另外,除胡子鲶等位基因大小异源同型(size homoplasy)的特征不明显外,其他物种在3个微卫星座位都具有这一非常明显的特征。这些数据表明,微卫星跨物种间交叉扩增能产生非同源产物;等位基因大小异源同型与微卫星座位本身有关,而与物种间的亲缘关系无明显的相关性。微卫星跨物种扩增产生的非同源产物和等位基因大小异源同型将使系统发育、群体遗传学和进化研究明显复杂化。因此,在应用微卫星跨物种交叉扩增数据以前,最好对跨物种交叉扩增产物进行测序验证。     

Isolation of polymorphic microsatellite loci in the genus Clusia (Clusiaceae)

Microsatellite flanking region sequences may provide phylogenetically useful information. We isolated 13 polymorphic microsatellite loci from two species, Clusia minor (five loci) and Clusia nemorosa (eight loci), to aid in the determination of phylogenetic relationships within the genus Clusia. Eleven loci amplified across all 17 Clusia species tested, while two loci amplified in 10 out of 17 species. The extensive cross‐species amplification suggests that these loci may be useful for an examination of phylogenetic relationships in this genus.     

Characterization of highly conserved microsatellite loci in Araucaria cunninghamii and related species

 Ten microsatellite loci are described in Araucaria cunninghamii, the first reported in the Araucariaceae. Eight were tested in sections Eutacta and Bunya, which diverged more than 200 MYA, and to the sister genus Agathis. Specific amplification products within the expected size range were obtained for six to eight loci in section Eutacta (depending on species), five loci in section Bunya and three loci in Agathis. Two of the loci (CRCAc1 and CRCAc2, both GA repeats) produced specific amplification products in all taxa, with orthology confirmed by sequence analysis. The repeats were perfect in all taxa. The flanking sequences were extremely conserved, with sequence divergence of 0% to 2.0% within Araucaria species and 2.9% to 7.5% between Araucaria and Agathis. These microsatellites represent some of the most conserved microsatellite loci reported in plants. This may be due to a low evolutionary rate in Araucariaceae genome or the loci may be closely associated with highly conserved, unreported genes. Received January 14, 2002; accepted June 14, 2002 Published online: February 4, 2003 Current address: The Centre for Identification and Diagnostics, School of Life Sciences, The University of Queensland, Brisbane 4072, Australia.     

Evolutionary factors affecting the cross‐species utility of newly developed microsatellite markers in seabirds

Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio‐temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased nonamplification, or disruptions that may result in decreased polymorphism in nontarget species. Furthermore, high mutation rates and constraints on allele size may also with evolutionary time, promote an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next‐generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub‐Antarctic seabird, the thin‐billed prion (Pachyptila belcheri), that we tested for cross‐species amplification in other Pachyptila and related sub‐Antarctic species. We found that heterozygosity decreased and the proportion of nonamplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific F_ST provided better approximations of mtDNA relationships among the studied species than those estimated using D_C, even though F_ST was more affected by null alleles. We observed a significantly nonlinear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross‐species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa.     

Characterization of microsatellite loci in neotropical Heliconius butterflies

The Heliconius butterflies offer exceptional opportunities for the study of the ecology and evolution of mimicry. Despite previous reports of difficulties in the development of microsatellite loci in Lepidoptera, we characterize 15 polymorphic loci in H. erato that show promise for genetic mapping and population studies in this and other species. Levels of variation were high, in both numbers and size ranges of alleles. The loci showed broad amplification success across the genus and in two other genera. All loci that amplified in a population of H. melpomene were polymorphic.     

Factors affecting avian cross-species microsatellite amplification

Compilation and analysis of information from the literature regarding cross-species microsatellite amplification and polymorphism success, and relating this to source-target species genetic distance as estimated by pairwise cytochrome b ( cytb ) divergence, enabled an in-depth investigation of factors affecting avian cross-species microsatellite amplification. Source-target species cytb distances provided accurate estimates of cross-species microsatellite amplification/polymorphism success rates not only in birds, but also in taxa where microsatellites cross-amplify across contrasting levels of taxonomic classification (frogs and cetaceans). As cytb is one of the most commonly sequenced DNA regions, pairwise cytb genetic distances should therefore be useful for predicting cross-species microsatellite success across a range of taxonomic groups. While the most important factor affecting cross-species microsatellite amplification/polymorphism success was a negative association with source-target species genetic distance, associations with additional features affecting cross-species amplification/polymorphism success included: decreasing PCR annealing temperature significantly increasing the chance of successful cross-species amplification, and a significant positive association between source species polymorphism and the proportion of target species in which a locus revealed polymorphism. No association between cross-species amplification and repeat motif (di-, tri-, or tetranucelotide) or repeat structure (perfect, imperfect, or compound) was observed. A set of nine loci which cross-amplified across an unusually broad range of passerine bird species were also identified, and could serve as a good starting point for cross-species amplification testing in passerine species for which insufficient loci are available.     

Isolation and characterization of microsatellite loci in the ant Myrmica scabrinodis

Five polymorphic microsatellite loci were developed for the ant Myrmica scabrinodis using a magnetic bead hybridization selection protocol. The number of alleles per locus varied between three and six. Cross‐species amplification of four of the loci yielded positive amplification products in four Myrmica species, suggesting their general suitability for microsatellite analysis within this taxonomic group.     

A set of microsatellite markers for Heliconius melpomene and closely related species

The butterflies in the genus Heliconius offer an exceptional opportunity for the study of the ecology and genetics of an adaptive radiation due to their extensive intra‐ and interspecific variation in wing colour patterns and mimetic associations. Here, we characterize 22 polymorphic microsatellite loci in Heliconius melpomene that have been shown to be useful for linkage mapping and population studies in this and other species. Levels of variation were high, although heterozygosity deficiencies were found in most loci, probably due to null alleles. The loci showed broad amplification success on six other species across the genus.     

Isolation of highly polymorphic microsatellite loci for a species with a large genome size: sharp-ribbed salamander (Pleurodeles waltl)

Fourteen highly polymorphic microsatellite markers were developed and characterized for the sharp-ribbed salamander, Pleurodeles waltl. Isolating microsatellites with more than 12 single repeat type units was only successful for a tetranucleotide repeat (ATAG). Compared to microsatellite libraries constructed simultaneously for two anuran amphibian species, a greater number of primer pairs designed for P. waltl had to be discarded, due to consistent amplification problems. Low amplification success rate for P. waltl may be due to its larger genome size. Consequently, to avoid nonspecific binding and to increase amplification success, polymerase chain reaction programmes with touchdown cycles were used. For 14 microsatellite markers, amplification was successful and consistent with number of alleles and expected heterozygosity ranging from seven to 22 and from 0.79 to 0.94, respectively. All 14 microsatellite markers will be extremely useful for metapopulation studies of this unique amphibian species.     

Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.     

Microsatellite markers in the primitive termite Mastotermes darwiniensis

We developed 10 microsatellite loci in the primitive termite Mastotermes darwiniensis. The number of alleles per locus ranged from four to 15, and the expected heterozygosites spanned from 0.21 to 0.90, in a sample of 40 workers collected from the Northern Territory, Australia. We also determined that only two loci amplified in five other termite species. The low frequency of cross‐amplification probably resulted from the high level of phylogenetic divergence between M. darwiniensis and the other taxa. Thus, although the loci are not widely applicable, they should prove effective in elucidating the genetic structure of M. darwiniensis populations.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Cross-amplification and characterization of microsatellite loci for the Neotropical orchid genus Epidendrum

In this study we tested the cross-amplification of 33 microsatellite loci previously developed for two closely related Neotropical orchid genera (Epidendrum and Laelia). A set of ten loci were polymorphic across five examined species (20 individuals each) with 2 to 15 alleles per locus. The mean expected and observed heterozygosity (average across species) ranged from 0.34 to 0.82 and from 0.27 to 0.85, respectively. In addition we tested all loci in 35 species representative of the genus Epidendrum. Of these, 26 loci showed successful amplification. Cross-application of these loci represent a potential source of co-dominant markers for evolutionary, ecological and conservation studies in this important orchid genus.     

New microsatellite markers for the bush rat,Rattus fuscipes greyii: characterization and cross‐species amplification

We describe here 16 new microsatellite markers for the bush rat, Rattus fuscipes greyii, and characterize their cross‐species amplification within the Australian Rattus and at a greater level of divergence in Rattus rattus and Rattus norvegicus. Within R. f. greyii, all of the loci are highly polymorphic, with six to 24 alleles per locus across the species range and expected heterozygosity ranging from 0.48 to 0.90 per locus within a sample of 24 rats from a large population on Kangaroo Island. Cross‐species amplification rates were approximately 87% within the Australian Rattus and approximately 50% within R. rattus and R. norvegicus. These loci are highly polymorphic with a high success rate of cross‐species amplification, making them potentially useful for a wide range of genetic studies.     

Utility of pairwise mtDNA genetic distances for predicting cross-species microsatellite amplification and polymorphism success in fishes

In many studies involving microsatellites cross-species amplification, primers designed for one (source) species are used to amplify homologous loci in related (target) species. However, it is not clear how closely related the species must be to attain significant success. Genetic divergence is a clear and easy way to assess similarity between species and provides an accurate measure of their evolutionary distance. Eight Mediterranean target species of the family Serranidae were analysed using twelve primers developed for Serranus cabrilla. Additionally, two mitochondrial genes (12S rRNA and 16S rRNA) were chosen on the basis of their extensive use in phylogenetic and evolutionary analyses to compute genetic divergence between the species. Significant negative correlations were found between genetic divergence and both cross-species amplification and maintained polymorphism of microsatellite markers, which could be generalized by gathering information from different fish studies. The success of obtaining amplifiable and polymorphic microsatellite loci can be a priori approximated knowing the mtDNA genetic divergence between a given source and target species using our inferred regression equations. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microsatellites of <Emphasis Type="BoldItalic">Laminaria digitata</Emphasis> tested in <Emphasis Type="BoldItalic">Lessonia nigrescens</Emphasis>: Evaluation and improvement of cross amplification between kelps of two different families

The use of primers designed originally to amplify DNA for one species in a different one can save time and resources, particularly for microsatellite loci. Microsatellite amplification improvements across two kelp families are reported, where loci originally described in Laminaria digitata (Laminariaceae) were tested in Lessonia nigrescens) was observed in two localities affected by massive mortality events. Nei’s distances among five populations presented similar patterns to those of 30 multilocus dominant loci (RAPD) evaluated in the same localities. Although some success might be achieved in cross-species microsatellite amplifications, the strong mutations detected between these two Laminarian families suggests that better results of cross-amplifications should be expected at much lower taxonomic levels. Thus, although more expensive, construction of new gene libraries is strongly recommended.     

Isolation and characterization of polymorphic microsatellite loci in the field vole,Microtus agrestis,and their cross‐utility in the common vole,Microtus arvalis

We developed nine polymorphic microsatellite loci for the field vole, Microtus agrestis. The number of alleles ranged from five to 15 and observed heterozygosities ranged from 0.40 to 1.00. We also tested the microsatellite loci for amplification and polymorphism in the congeneric species Microtus arvalis. Five of the nine loci were successfully analysed in this species. The microsatellite markers will be employed in studies of reproductive success and fine‐scale spatial genetic structure.     

Development of high‐resolution DNA barcodes for Dioscorea species discrimination and phylogenetic analysis

The genus Dioscorea is widely distributed in tropical and subtropical regions, and is economically important in terms of food supply and pharmaceutical applications. However, DNA barcodes are relatively unsuccessful in discriminating between Dioscorea species, with the highest discrimination rate (23.26%) derived from matK sequences. In this study, we compared genic and intergenic regions of three Dioscorea chloroplast genomes and found that the density of SNPs and indels in intergenic sites was about twice and seven times higher than that of SNPs and indels in the genic regions, respectively. A total of 52 primer pairs covering highly variable regions were designed and seven pairs of primers had 80%–100% PCR success rate. PCR amplicons of 73 Dioscorea individuals and assembled sequences of 47 Dioscorea SRAs were used for estimating intraspecific and interspecific divergence for the seven loci: The rpoB‐trnC locus had the highest interspecific divergence. Automatic barcoding gap discovery (ABGD), Poisson tree processes (PTP), and generalized mixed Yule coalescence (GMYC) analysis were applied for species delimitation based on the seven loci and successfully identified the majority of species, except for species in the Enantiophyllum section. Phylogenetic analysis of 51 Dioscorea individuals (28 species) showed that most individuals belonging to the same species tended to cluster in the same group. Our results suggest that the variable loci derived from comparative analysis of plastid genome sequences could be good DNA barcode candidates for taxonomic analysis and species delimitation.     

Potentials and limitations of the cross-species transfer of nuclear microsatellite marker in six species belonging to three sections of the genus Populus L.

The genus Populus is classified into six different sections, and depending on the declaration of hybrids, the number of species varies between 22 and 85. Species within one section, and sometimes between sections, are crossable to each other, resulting in many naturally but also artificially produced hybrids. Morphological attributes for a clone characterisation are often difficult to evaluate when different poplar species or even hybrids are crossed; thus, molecular markers are needed to characterise the different species. Taking advantage of the large microsatellite resource developed for Populus trichocarpa, however, amplification of these microsatellite markers in other Populus species either often fails, or in the case of amplification, unrelated genomic regions are amplified. To meet this obvious problem of the species transferability of microsatellite markers, in total, 305 microsatellite loci, mainly from P. trichocarpa but also few from Populus tremuloides and Populus nigra, were tested for their transferability to diverse genotypes of six species belonging to three sections of the genus Populus. Ultimately, 209 microsatellite loci could be amplified with varying sizes in the different species. The PCR products of selected loci were separated in a polyacrylamide gel and sequenced to assure that the expected loci were derived from the database genome of P. trichocarpa. The present results constitute a large study for microsatellite transferability for Populus species. The documented microsatellite loci can be applied to species-, hybrid- and clone-specific diagnostic approaches or as universal markers for comprehensive ecological studies.