Repetitive flanking sequences (ReFS): novel molecular markers from microsatellite families

Although microsatellite markers have become exceedingly popular in molecular studies of wild organisms, their development in some taxonomic groups is challenging. This is partly because of repetitive flanking sequences, which lead to the simultaneous amplification of alleles from multiple loci. Until now, these microsatellite DNA families have been considered unsuitable for population genetics studies, but here we describe our development of these repetitive flanking sequences (ReFS) as novel molecular markers. We illustrate the utility of these markers by using them to address an outstanding taxonomic question in the moth genus Schrankia.     

DNA barcoding will frequently fail in complicated groups: An example in wild potatoes

DNA barcoding ("barcoding") has been proposed as a rapid and practical molecular method to identify species via diagnostic variation in short orthologous DNA sequences from one or a few universal genomic regions. It seeks to address in a rapid and simple way the "taxonomic impediment" of a greater need for taxonomic identifications than can be supplied by taxonomists. Using a complicated plant group, Solanum sect. Petota (wild potatoes), I tested barcoding with the most variable and frequently suggested plant barcoding regions: the internal nontranscribed spacer of nuclear ribosomal DNA (ITS) and the plastid markers trnH-psbA intergenic spacer and matK. These DNA regions fail to provide species-specific markers in sect. Petota because the ITS has too much intraspecific variation and the plastid markers lack sufficient polymorphism. The complications seen in wild potatoes are common in many plant groups, but they have not been assessed with barcoding. Barcoding is a retroactive procedure that relies on well-defined species to function, is based solely on a limited number of DNA sequences that are often inappropriate at the species level, has been poorly tested with geographically well-dispersed replicate samples from difficult taxonomic groups, and discounts substantial practical and theoretical problems in defining species.     

Cross-species amplification of microsatellite loci within the dioecious, polyploid genus Actinidia (Actinidiaceae)

Microsatellite marker transfer across species in the dioecious genus Actinidia (kiwifruit) could offer an efficient and time-effective technique for use during trait transfer for vine and fruit improvement in breeding programmes. We evaluated the cross-species amplification of 20 EST-derived microsatellite markers that were fully informative in an Actinidia chinensis mapping family. We tested all 20 markers on 120 genotypes belonging to 21 species, 5 with varieties and/or chromosome races. These 26 taxa included 16 diploids, 7 tetraploids, 2 hexaploids and 1 octaploid, and represented all four taxonomic sections in the genus. All 20 markers showed some level of cross-species amplification. The most successful marker amplified in all genotypes from all species from all sections of the genus, the least successful amplified fragments only in A. chinensis and A. deliciosa. One species, A. glaucophylla, failed to amplify with all but 2 markers. PIC (Polymorphism information content) values were high, with 14 of 17 markers recording values of 0.90 and above. Sequence data demonstrated the presence of the microsatellite in all the amplified products. Sequence homology was less 5′ of the microsatellite and increased toward the start codon of the translated region of the EST from which the marker was derived. The data confirm that EST-derived microsatellite markers from Actinidia species show cross-species amplification with high levels of polymorphism which could make them useful markers in breeding programmes.     

Microsatellite marker identification using genome screening and restriction-ligation

We have identified a fast and easy method for finding microsatellite markers that utilizes genome screening with inter-simple sequence repeat (ISSR) primers to detect microsatellite regions and to obtain sequence information flanking one side of the microsatellites and a restriction-ligation technique with a specific adaptor to allow sequence walking to obtain sequence information flanking the other side of the microsatellites. Two main alternatives of the method (with or without cloning) are presented. We successfully utilized the method when identifying microsatellite markers for 21 bryophyte species, three algal species, and for the raccoon dog. The proportion of polymorphic markers equaled 95%. We observed that microsatellites are commonly found within the sequenced ISSR amplification products (54% in the present study), in which case specific primers can be identified for the microsatellite without a further restriction-ligation step. It is evident that the DNA regions amplified by ISSR markers commonly represent microsatellite hotspots. We propose that the identified method and the knowledge of the common presence of additional microsatellite repeats within ISSR amplification products are especially attractive to researchers who conduct small-scale microsatellite identification, such as researchers in population genetics and conservation biology.     

Conserved Microsatellites in Ants Enable Population Genetic and Colony Pedigree Studies across a Wide Range of Species

Broadly applicable polymorphic genetic markers are essential tools for population genetics, and different types of markers have been developed for this purpose. Microsatellites have been employed as particularly polymorphic markers for over 20 years. However, PCR primers for microsatellite loci are often not useful outside the species for which they were designed. This implies that a new set of loci has to be identified and primers developed for every new study species. To overcome this constraint, we identified 45 conserved microsatellite loci based on the eight currently available ant genomes and designed primers for PCR amplification. Among these loci, we chose 24 for in-depth study in six species covering six different ant subfamilies. On average, 11.16 of these 24 loci were polymorphic and in Hardy-Weinberg equilibrium in any given species. The average number of alleles for these polymorphic loci within single populations of the different species was 4.59. This set of genetic markers will thus be useful for population genetic and colony pedigree studies across a wide range of ant species, supplementing the markers available for previously studied species and greatly facilitating the study of the many ant species lacking genetic markers. Our study shows that it is possible to develop microsatellite loci that are both conserved over a broad range of taxa, yet polymorphic within species. This should encourage researchers to develop similar tools for other large taxonomic groups.     

Development and transferability of apricot and grape EST microsatellite markers across taxa

EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.     

Development of microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

Studies of the transferability of microsatellites derived from Triticum tauschii to hexaploid wheat and to diploid related species using amplification,hybridization and sequence comparisons

Hexaploid wheat (Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes, respectively. We recently isolated microsatellites from a T. tauschii library enriched for various motifs and evaluated the transferability of these markers to several diploid species carrying the A, B or D genomes. All of the primer pairs amplifying more than one locus on bread wheat and half of those giving D-genome-specific loci gave an amplification product on A-and/or B-diploid species. All of the markers giving a single amplification product for T. tauschii and no amplification on the other diploid species were D-genome-specific at the hexaploid level. The non-specific microsatellite markers (which gave an amplification product on diploid species carrying the A, B or D genome) gave either a complex amplification pattern on bread wheat (with several bands) or generated a single band which mapped to the D genome. Southern blot hybridizations with probes corresponding to the microsatellite flanking regions gave a signal on all diploid and hexaploid species, whatever the specificity of the microsatellite. The patterns observed on bread wheat were generally in accordance with those observed for diploid species, with slight rearrangements. This suggests that the specificity of microsatellite markers is probably due to mutations in microsatellite flanking regions rather than sequence elimination during polyploidization events and that genome stringency is higher at the polyploid than at the diploid level.     

Lessons learned from microsatellite development for nonmodel organisms using 454 pyrosequencing

Microsatellites, also known as simple sequence repeats (SSRs), are among the most commonly used marker types in evolutionary and ecological studies. Next Generation Sequencing techniques such as 454 pyrosequencing allow the rapid development of microsatellite markers in nonmodel organisms. 454 pyrosequencing is a straightforward approach to develop a high number of microsatellite markers. Therefore, developing microsatellites using 454 pyrosequencing has become the method of choice for marker development. Here, we describe a user friendly way of microsatellite development from 454 pyrosequencing data and analyse data sets of 17 nonmodel species (plants, fungi, invertebrates, birds and a mammal) for microsatellite repeats and flanking regions suitable for primer development. We then compare the numbers of successfully lab‐tested microsatellite markers for the various species and furthermore describe diverse challenges that might arise in different study species, for example, large genome size or nonpure extraction of genomic DNA. Successful primer identification was feasible for all species. We found that in species for which large repeat numbers are uncommon, such as fungi, polymorphic markers can nevertheless be developed from 454 pyrosequencing reads containing small repeat numbers (five to six repeats). Furthermore, the development of microsatellite markers for species with large genomes was also with Next Generation Sequencing techniques more cost and time‐consuming than for species with smaller genomes. In this study, we showed that depending on the species, a different amount of 454 pyrosequencing data might be required for successful identification of a sufficient number of microsatellite markers for ecological genetic studies.     

Nine novel microsatellite markers for the hog deer (Axis porcinus)

Hog deer (Axis porcinus) is listed as an endangered species in many countries and its taxonomic status remains ambiguous. In this study, we developed nine novel microsatellite markers using enrichment methods. These polymorphic microsatellite loci showed allele numbers of 2–3 with an average of 2.22 in a group of 26 individuals and observed and expected heterozygosity ranging from 0.044 to 0.619 (average 0.397) and 0.194 to 0.632 (average 0.433) respectively. Linkage disequilibrium was not detected. These markers could be used in the genetic study and taxonomic identification of this species.     

Study of closely related species within the Physalaemus cuvieri group (Anura): contribution of microsatellite markers

Various species of the Physalaemus cuvieri group of frogs are difficult to distinguish morphologically, making molecular analysis an attractive alternative for indentifying members of this group, which is considered to be at risk because of loss of habitat. The genetic structure of natural populations of P. ephippifer and P. albonotatus species was investigated and analyzed, together with that of five previously studied populations of P. cuvieri. Nine microsatellite loci were used in the analyses. The overall G(ST) value (0.46) revealed high genetic variation among the populations, as expected for different species. Bayesian analysis implemented by the STRUCTURE software clustered the seven populations into seven groups (K = 7). All the P. albonotatus and P. ephippifer specimens were grouped into a single cluster, both species showing clear differentiation from P. cuvieri. The different grouping based on these microsatellites of some P. cuvieri individuals from Porto Nacional and from Passo Fundo suggests that they could be a new species, indicating a necessity for taxonomic reevaluation. Despite the intrinsic difficulties in analyzing closely related species, the nine microsatellite loci were found to be adequate for distinguishing these three species of the P. cuvieri group and their populations.     

Development, characterization, and transferability to other Solanaceae of microsatellite markers in pepper (Capsicum annuum L.).

A novel set of informative microsatellite markers for pepper (Capsicum annuum L.) is provided. Screening of approximately 168 000 genomic clones and 23 174 public database entries resulted in a total of 411 microsatellite-containing sequences that could be used for primer design and functional testing. A set of 154 microsatellite markers originated from short-insert genomic libraries and 257 markers originated from database sequences. Of those markers, 147 (61 from genomic libraries and 86 from database sequences) showed specific and scoreable amplification products and detected polymorphisms between at least 2 of the 33 lines of a test panel consisting of cultivated and wild Capsicum genotypes. These informative markers were subsequently surveyed for allelic variation and information content. The usefulness of the new markers for diversity and taxonomic studies was demonstrated by the construction of consistent phylogenetic trees based on the microsatellite polymorphisms. Conservation of a subset of microsatellite loci in pepper, tomato, and potato was proven by cross-species amplification and sequence comparisons. For several informative pepper microsatellite markers, homologous expressed sequence tag (EST) counterparts could be identified in these related species that also carry microsatellite motifs. Such orthologs can potentially be used as reference markers and common anchoring points on the genetic maps of different solanaceous species.     

The Wagner Parsimony using morphological characters: a new method for palaeosynecological studies

The limits and difficulties related to the tools currently in use for palaeosynecological comparisons of faunas or floras of different geological periods are discussed. The new method of the Wagner parsimony Applied to Palaeosynecology Using Morphology (WAPUM method), is defined and tested on morphological characters gathered from two insect groups Odonatoptera and Thripida. The difficulties related to the monophyly of the taxonomic groups used in the more traditional approaches are no longer a problem when using the WAPUM method. In the WAPUM a character is ‘presence versus absence of species bearing a morphological structure’. The results obtained from use of the WAPUM minimize the number of changes among character states. Application of the WAPUM could reveal signals to confirm or object the currently available scenarios for the global changes in the evolution of past diversity and disparity of organisms (major changes or global crises of diversity).     

A method to incorporate the effect of taxonomic uncertainty on multivariate analyses of ecological data

Researchers in ecology commonly use multivariate analyses (e.g. redundancy analysis, canonical correspondence analysis, Mantel correlation, multivariate analysis of variance) to interpret patterns in biological data and relate these patterns to environmental predictors. There has been, however, little recognition of the errors associated with biological data and the influence that these may have on predictions derived from ecological hypotheses. We present a permutational method that assesses the effects of taxonomic uncertainty on the multivariate analyses typically used in the analysis of ecological data. The procedure is based on iterative randomizations that randomly re‐assign non identified species in each site to any of the other species found in the remaining sites. After each re‐assignment of species identities, the multivariate method at stake is run and a parameter of interest is calculated. Consequently, one can estimate a range of plausible values for the parameter of interest under different scenarios of re‐assigned species identities. We demonstrate the use of our approach in the calculation of two parameters with an example involving tropical tree species from western Amazonia: 1) the Mantel correlation between compositional similarity and environmental distances between pairs of sites, and; 2) the variance explained by environmental predictors in redundancy analysis (RDA). We also investigated the effects of increasing taxonomic uncertainty (i.e. number of unidentified species), and the taxonomic resolution at which morphospecies are determined (genus‐resolution, family‐resolution, or fully undetermined species) on the uncertainty range of these parameters. To achieve this, we performed simulations on a tree dataset from southern Mexico by randomly selecting a portion of the species contained in the dataset and classifying them as unidentified at each level of decreasing taxonomic resolution. An analysis of covariance showed that both taxonomic uncertainty and resolution significantly influence the uncertainty range of the resulting parameters. Increasing taxonomic uncertainty expands our uncertainty of the parameters estimated both in the Mantel test and RDA. The effects of increasing taxonomic resolution, however, are not as evident. The method presented in this study improves the traditional approaches to study compositional change in ecological communities by accounting for some of the uncertainty inherent to biological data. We hope that this approach can be routinely used to estimate any parameter of interest obtained from compositional data tables when faced with taxonomic uncertainty.     

Microsatellite loci for Paspalum atratum (Poaceae) and cross-amplification in other species

? Premise of the study: Paspalum atratum is a perennial, cespitose, tropical grass native to Central and South America. This species belongs to a polyploid complex (Plicatula group) little known at the genetic level. The characterized microsatellite markers provide new informative tools for further studies of the hybridization, mating systems, and structure of the population. ? Methods and Results: Using the microsatellite-enriched library method, we isolated and characterized 19 microsatellite markers from P. atratum. Eleven of them were polymorphic, showing a variable degree of variation, while eight were monomorphic in the samples analyzed. Additionally, the transferability of these microsatellite markers was tested in other species. ? Conclusions: These results suggest that the characterized markers have enough discriminatory potential to be used in genetic characterizations of Paspalum taxa, which are based on an understanding of their mating systems and genetic structure, as well as in understanding the evolutionary processes involved in the evolution of groups of Paspalum.     

Diversity in coffee assessed with SSR markers: structure of the genus Coffea and perspectives for breeding.

The present study shows transferability of microsatellite markers developed in the two cultivated coffee species (Coffea arabica L. and C. canephora Pierre ex Froehn.) to 15 species representing the previously identified main groups of the genus Coffea. Evaluation of the genetic diversity and available resources within Coffea and development of molecular markers transferable across species are important steps for breeding of the two cultivated species. We worked on 15 species with 60 microsatellite markers developed using different strategies (SSR-enriched libraries, BAC libraries, gene sequences). We focused our analysis on 4 species used for commercial or breeding purposes. Our results establish the high transferability of microsatellite markers within Coffea. We show the large amount of diversity available within wild species for breeding applications. Finally we discuss the consequences for future comparative mapping studies and breeding of the two cultivated species.     

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.     

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.     

Genetic diversity and population structure of the Korean field mouse(Apodumus peninsulae) in South Korea: from 17 previously and newly developed microsatellite markers

To provide more reliable genetic information on species and minimize experimental errors, biologists increase the number of genetic markers available and then carefully select optimal markers from a large candidate pool. We developed nine novel microsatellite markers from the Korean field mouse (Apodemus peninsulae), which is one of the most dominant forest animals in South Korea. The mean observed and expected heterozygosities across nine markers were 0.65 and 0.73, respectively, with an average polymorphic information content of 0.70. Using 17 microsatellite markers (nine polymorphic markers in this study, in combination with eight previously reported for the species), we conducted genetic analysis on the animals from six sampling locations. These locations are divided into the eastern (EAST) and the western (WEST) sides of the Taebaek mountain ranges in South Korea. Genetic diversity was high at both groups, with the mean expected heterozygosity of 0.77 in EAST and 0.78 in WEST. However, we did not observe strong evidence of genetic divergence between two groups. Future genetic research with more samples incorporating ecological study may clarify population structure in the species and the hypothesis of the mountains discontinuity of gene flow.     

An AFLP marker approach to lower-level systematics in Eucalyptus (Myrtaceae)

Genus Eucalyptus, with over 700 species, presents a number of systematic difficulties including taxa that hybridize or intergrade across environmental gradients. To date, no DNA marker has been found capable of resolving phylogeny below the sectional level in the major subgenera. Molecular markers are needed to support taxonomic revision, assess the extent of genetic divergence at lower taxonomic levels, and inform conservation efforts. We examined the utility of 930 amplified fragment length polymorphisms (AFLPs) for analyzing relationships among Tasmanian taxa of subgenus Symphyomyrtus section Maidenaria. Phenetic and cladistic analyses resolved species into clusters demonstrating significant genetic partitioning, largely concordant with series defined in the most recent taxonomic revision of Eucalyptus. Some departures from current taxonomy were noted, indicating possible cases of morphological convergence and character reversion. Although the resolution obtained using AFLP was greatly superior to that of single sequence markers, the data demonstrated high homoplasy and incomplete resolution of closely related species. The results of this study and others are consistent with recent speciation and reticulate evolution in Maidenaria. We conclude that a combination of phylogenetic and population genetic approaches using multiple molecular markers offers the best prospects for understanding taxonomic relationships below the sectional level in Eucalyptus.