Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae) (original) (raw)
American Journal of …, 2011
Chamaecyparis nootkatensis is an ecologically and economically important conifer of the north Pacific coastal forests. To aid in studies of clonal structure and genetic differentiation of this and related species, we isolated and characterized microsatellites from C. nootkatensis. A microsatellite-enriched library yielded 75 repeat-containing sequences for which primer pairs were designed. Only five showed reliable amplification and polymorphism, with an average of 13.7 alleles/locus and a mean expected heterozygosity of 0.592. In progeny tests with four families, few null alleles were directly detected and loci segregated according to Mendelian expectations. However, in one primer pair, high heterozygote deficiency was observed, suggesting the presence of a null allele. The ability of primer pairs to cross amplify was tested on 18 species of the Cupressaceae sensu lato; three primer pairs yielded polymorphic loci in Cupressus and Juniperus species, but not in other Chamaecyparis species. This also supports recent findings of a closer affinity of C. nootkatensis with Cupressus over other Chamaecyparis species.
Microsatellite transferability was used as a method to examine the genetic diversity and structure of populations in Pilosocereus gounellei seedling samples that have potential to implement effective restoration strategies for degraded and disturbed areas of the Caatinga biome. Genomic DNA was extracted from 85 seedlings obtained from fruit collected from plants growing in native areas in the Brazilian states of Piaui (PI), Rio Grande do Norte (RN), and Bahia (BA). Six microsatellite primers were polymorphic. AMOVA showed higher genetic variation within (72%) than among (28%) the samples from the three states. The high level of similarity between the seedlings from PI, BA, and RN indicated that samples collected at any of the three sites can be used to represent the genetic diversity of the species. Seeds of plants from the three States are recommended as samples for germplasm banks and/or the production of plantlets to i) plant in areas of strategic reserves for forage, ii) deploy new cultivation areas, iii) restore degraded areas in the semi-arid Northeast, and iv) maintain ecological reserve banks and fodder with genetically divergent plants.
Molecular Ecology Resources, 2009
The genus Conospermum Sm. (Proteaceae) represents an important component of the heathlands and woodlands of Western Australian sandplains. The genus has 53 species endemic to Australia, with its center of distribution in southwestern Western Australia (Bennett, 1995). Within the South West Australian Floristic Region, a global biodiversity hotspot (Myers et al., 2000; Hopper and Gioia, 2004), many Conospermum species are of increasing conservation concern, with four taxa already declared rare by the Western Australia government (Government Gazette, 2018). Moreover, as for many proteaceous species, various Conospermum species are widely utilized in floriculture (Bennett, 1995; Stone et al., 2006). Conospermum undulatum Lindl. is a diploid shrub with its range restricted to ca. 55 km 2 in a rapidly expanding urban zone in the metropolitan area of Perth (Close et al., 2006; Wardell-Johnson et al., 2016). This species is listed as Vulnerable under the Environment Protection and Biodiversity Conservation Act 1999. Habitat fragmentation and hybridization with sympatric Conospermum species are likely to pose a risk to the future persistence of C. undulatum. In Conospermum, studies of population genetics and reproductive biology have been undertaken using amplified fragment length polymorphism (AFLP) and random-amplified polymorphic DNA (RAPD) markers for only a few species (Stone et al., 2006; Sinclair et al., 2008). To our knowledge, no microsatellite resources have been developed for this genus to date. Considering the growing concern about this endemic genus and the number of species within it, we expect that microsatellite markers will have broad applicability for conservation and population genetic analyses. Here, we report the development and characterization of 20 microsatellite markers for C. undulatum that will be useful for the study of its genetic structure, spatial patterns of genetic diversity, and dispersal dynamics. Additionally, we tested for cross-amplification of these loci in three related Conospermum species to evaluate the utility of the marker set more broadly and specifically to allow assessment of hybridization between C. undulatum and neighboring species. METHODS AND RESULTS Genomic DNA was extracted from freeze-dried leaf material (ca. 50 mg) using a modified 2% cetyltrimethylammonium bromide (CTAB) method, with 1% polyvinylpyrrolidone and 0.1% sodium
PLOS ONE, 2015
Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as diand trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in nonmodel organisms.
2009
Chamaecyparis nootkatensis is an ecologically and economically important conifer of the north Pacific coastal forests. To aid in studies of clonal structure and genetic differentiation of this and related species, we isolated and characterized microsatellites from C. nootkatensis. A microsatellite-enriched library yielded 75 repeat-containing sequences for which primer pairs were designed. Only five showed reliable amplification and polymorphism, with an average of 13.7 alleles/locus and a mean expected heterozygosity of 0.592. In progeny tests with four families, few null alleles were directly detected and loci segregated according to Mendelian expectations. However, in one primer pair, high heterozygote deficiency was observed, suggesting the presence of a null allele. The ability of primer pairs to cross amplify was tested on 18 species of the Cupressaceae sensu lato; three primer pairs yielded polymorphic loci in Cupressus and Juniperus species, but not in other Chamaecyparis species. This also supports recent findings of a closer affinity of C. nootkatensis with Cupressus over other Chamaecyparis species.
Genetic relationships in Opuntia Mill. genus (Cactaceae) detected by molecular marker
Plant Science, 2003
The Opuntia genus includes over 181 species comprising, on the basis of morphological traits, a total of 29 series [The Cactaceae (1919)]. Starting from this classification, several authors have investigated the Opuntia genus taxonomy but the large morphological variation within different species, suggests that phenotypical characteristics will not serve to produce a stable classification. In this work chloroplastic simple sequence repeat (cpSSR) and amplified fragment length polymorphism (AFLP) were used to evaluate the usefulness of molecular markers in Opuntia species characterization and to study the relationships among different species. Results show that the combination of cpSSR and AFLP markers provide a quantitative estimation of genetic relationships among several Opuntia species. Both molecular analyses reveal a genetic similarity among species of series 20 and 21 [The Cactaceae (1919)] as suggested also by morphological traits. Particular attention was focused on the genetic relationship between Opuntia ficus-indica and Opuntia megacantha : individuals from different populations of the two species were analyzed with both molecular markers. A common genetic constitution of O. ficus-indica and O. megacantha was detected. On the basis of molecular data, morphological traits and biogeographical distribution, we suggest that O. ficus-indica should be considered as a domesticated form of O. megacantha. Our results suggest the importance of a revision of Opuntia genus classification using several tools: molecular, morphological and biogeographical analysis.
Molecular Ecology Resources, 2008
We have developed a set of eight polymorphic nuclear microsatellite markers for the Mediterranean shrub Pistacia lentiscus by means of an enriched library method. Characterization for the eight loci was carried out on 42 individuals from two populations sampled in southern Spain. The overall number of alleles detected was 59, ranging from three to 13 per locus. Expected heterozygosity per locus and population ranged from 0.139 to 0.895. Two loci albeit only in one population (Seville) departed significantly from Hardy-Weinberg equilibrium expectations and no linkage disequilibrium between pairs of loci was detected. These markers will be used in studies of gene flow across a fragmented landscape.
Conservation Genetics
Fourteen polymorphic microsatellite loci were isolated from the genomic DNA of Pistacia weinmannifolia, using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) method, and screened on 12 individuals from each of two wild populations. The 14 polymorphic loci had an average of 4.1 alleles per locus varying from 1 to 9. The observed (H o) and expected (H e) heterozygosities across the two populations ranged from 0.000 to 0.933 and from 0.000 to 0.906, respectively. Tests for departure from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) were conducted for each of the two populations separately. It was found that no locus significantly deviated from HWE proportions and no significant LD was detected between loci (p < 0.001). In the test of cross-species utility, we successfully amplified nine (64.2%) of 14 loci in P. chinensis and four (28.6%) in P. mexicana. The relatively high level of polymorphism for these markers will facilitate further studies of gene flow, population structure and evolutionary history of P. weinmannifolia and its congeners.
Plant Omics J, 2011
The genetic diversity for twenty species belonging to four genera of Cacataceae (Rebutia, Aylostera, Mediolobivia and Sulcorebutia) was analyzed employing taxonomic methods, chromosomes number and RAPD markers. The botanical classification that describes the phenotypic aspects of different characters, such as plant diameter, number of spines/areole, mean spines length, flower diameter, and flower color, was employed. Chromosome analysis revealed both diploids and polyploids in the studied species. ...
Applications in Plant Sciences, 2016
Next-generation sequencing (NGS)-based methods have allowed the quick development of microsatellite primers specific to nonmodel organisms (e.g., Duwe et al., 2015; González et al., 2015). Here, microsatellite markers are presented for the grass genus Anthoxanthum L., comprising around 20 species often affected by reticulation (Pimentel et al., 2010, 2013). The phylogeny of Anthoxanthum defines a Euro-Siberian (as well as Macaronesian and Afroalpine) polyploid complex of species (Pimentel et al., 2013). It includes four diploid taxa: (i) the Mediterranean A. aristatum Boiss.-A. ovatum Lag. complex (Pimentel et al., 2010), (ii) the Macaronesian A. maderense Teppner, and (iii) the Arctic-alpine A. alpinum Á. Löve & D. Löve (Pimentel et al., 2013). The clade also includes at least three polyploid lineages (Chumová et al., 2015): the Iberian endemic A. amarum Brot. (16x-18x); the East African A. nivale K. Schum. (4x, 6x), and the Eurasian A. odoratum L. (4x). Fifteen microsatellite markers that can be applied to the Euro-Siberian complex of Anthoxanthum are presented here. These markers will be used to determine the geographic patterns of gene flow within and among the different diploid lineages in the complex, as well as to unravel the origin of its polyploid groups. METHODS AND RESULTS Microsatellite development-A microsatellite-enriched genomic library (motifs AC, AG, ACC, AGG, and ACG) was constructed at AllGenetics & Biology SL (A Coruña, Spain) from an equimolar mix of DNA extracts from the diploid A. aristatum-A. ovatum (two individuals) and the tetraploid A. odoratum (one individual; Appendix 1) using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, California, USA). Given the difficulty in morphologically distinguishing Anthoxanthum cytotypes (Chumová et al., 2015), between one and five individuals per population were assessed using flow cytometry following Galbraith et al. (1983). DNA was extracted from silica-dried leaves using the DNAeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The enriched genomic library was sequenced in a fraction of an Illumina MiSeq PE300 run (Illumina), and the reads were processed using the software Geneious 7.1.5 (Biomatters, Auckland, New Zealand). Five hundred loci were detected containing a microsatellite and flanked by regions adequate to design PCR primers using Primer3 (Untergasser et al., 2012). Primer pairs were multiplexed with Multiplex Manager 1.0 (Holleley and Geerts, 2009). Forty microsatellite loci were combined so that differences in annealing temperatures were minimized and spacing between markers was maximized. Primers were tested for polymorphism on six diploid and two tetraploid samples (Appendix 1) that belonged to the different Anthoxanthum lineages and came from geographically distant populations. Each PCR reaction was performed following Schuelke (2000) with three primers (one of them fluorescently labeled using FAM or HEX; Table 1). PCR reactions were conducted in a final volume of 12.5 μL, containing 1 μL of DNA (10 ng/μL), 6.25 μL Type-it Microsatellite PCR Kit (QIAGEN), 4 μL PCR-grade water, and 1.25 μL of the primer mix (Schuelke, 2000). The optimal PCR protocol consisted of an initial denaturation step at 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 56°C for 90 s, and 72°C for 30 s; eight cycles of 95°C for 30 s, 52°C for 90 s, and 72°C for 30 s; and a final extension step at 68°C for 30 min. Labeled PCR products were then subjected to fragment analysis by Macrogen (Seoul, Republic of Korea). The resulting .fsa files were manually analyzed using Geneious 7.1.5 (Biomatters). Fifteen primers were selected based on amplification success and the number of alleles generated (Table 1).
American Journal of Botany, 2012
• Premise of the study: Twelve novel polymorphic microsatellite loci were developed and characterized from a repeat-enriched genomic library of Crocus sativus to study population and conservation genetics of this economically and medically important species. • Methods and Results: The microsatellite loci were isolated using a modifi ed Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) method. The average number of alleles per locus was 2.6. The observed and expected heterozygosities varied from 0.07 to 0.92 and 0.1 to 0.58, respectively. Polymorphic information content value ranged from 0.09 to 0.55 with an average of 0.34. Four out of twelve loci showed signifi cant departures from Hardy-Weinberg equilibrium. • Conclusions: The microsatellite markers reported here will be useful for evaluating genetic diversity and will likely serve as an ideal resource for use in marker-assisted breeding programs, germplasm analysis, and varietal identifi cation.
2012
The study of the genetic structure of wild plant populations is essential for their management and conservation. Several DNA markers have been used in such studies, as well as isozyme markers. In order to provide a better comprehension of the results obtained and a comparison between markers which will help choose tools for future studies in natural populations of Oryza glumaepatula, a predominantly autogamous species, this study used both isozymes and microsatellites to assess the genetic diversity and genetic structure of 13 populations, pointing to similarities and divergences of each marker, and evaluating the relative importance of the results for studies of population genetics and conservation. A bulk sample for each population was obtained, by sampling two to three seeds of each plant, up to a set of 50 seeds. Amplified products of eight SSR loci were electrophoresed on non-denaturing polyacrylamide gels, and the fragments were visualized using silver staining procedure. Isozyme analyses were conducted in polyacrylamide gels, under a discontinuous system, using six enzymatic loci. SSR loci showed higher mean levels of genetic diversity (A=2.83, p=0.71, A P =3.17, H o =0.081, H e =0.351) than isozyme loci (A=1.20, p=0.20, A P =1.38, H o =0.006, H e =0.056). Interpopulation genetic differentiation detected by SSR loci (R ST =0.631, equivalent to F ST =0.533) was lower than that obtained with isozymes (F ST =0.772). However, both markers showed high deviation from Hardy-Weinberg expectations (F IS =0.744 and 0.899, respectively for SSR and isozymes). The mean apparent outcrossing rate for SSR ( =0.14) was higher than that obtained using isozymes ( =0.043), although both markers detected lower levels of outcrossing in Amazonia compared to the Pantanal. The migrant number estimation was also higher for SSR (Nm=0.219) than isozymes (Nm=0.074), although a small number for both markers was expected due to the mode of reproduction of this species, defined as mixed with predominance of self fertilization. No correlation was obtained between genetic and geographic distances with SSR, but a positive correlation was found between genetic and geographic distances with isozymes. We conclude that these markers are divergent in detecting genetic diversity parameters in O. glumaepatula and that microsatellites are powerful for detecting information at the intra-population level, while isozymes are more powerful for inter-population diversity, since clustering of populations agreed with the expectations based on the geographic distribution of the populations using this marker. Rev. Biol. Trop. 60 (4): 1463-1478. Epub 2012 December 01.
2007
Knowledge of the genetic structure and diversity of natural populations is important in developing strategies for in situ and ex situ conservation. We used eight microsatellite loci to estimate genetic structure and investigate within and between population genetic variation in eleven Brazilian wild rice (Oryza glumaepatula) populations. The study showed the following genetic diversity parameters: average number of 3.1 alleles per locus; 77.3% polymorphic loci; 0.091 observed heterozygosity and 0.393 gene diversity. F-statistics detected by microsatellite loci were: F ST = 0.491 (and R ST = 0.608), F IS = 0.780 and F IT = 0.888. No population was in Hardy-Weinberg equilibrium. The estimated apparent outcrossing rate (t a = 0.143) indicated a predominance of self-fertilization. The gene flow values were low (Nm = 0.259 and 0.161 for F ST and R ST , respectively). Populations were spatially structured but without a correlation between genetic and geographic distances. Five populations (PG-4, PG-2, PU-1, SO-4, NE-18) were identified as priorities for conservation strategies. Populations from the Amazon biome showed heterogeneity with respect to intrapopulation diversity (H e ). The high level of genetic differentiation between populations and the high number of private alleles suggested that sampling should be carried out on a large number of O. glumaepatula populations for ex situ conservation purposes.
Genetic Relationships Among Pistacia Species Using AFLP Markers
Plant Systematics and …, 2009
This study addresses the phylogenetic relationship between Pistacia species by amplified fragment length polymorphism (AFLP). The plant materials of this study consisted of a total of 44 accessions belonging to P. vera, P. eurycarpa, P. khinjuk, all subspecies of P. atlantica (atlantica, mutica, kurdica and cabulica), three unknown genotypes and three accessions, proposed to be hybrid from P. eurycarpa 9 P. atlantica. The accessions were from Iran, Turkey, USA and Syria. Six AFLP primer combinations produced a total of 475 fragments, with average of 79.16 fragments per primer pair, of which, 336 bands were polymorphic. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on jaccard's similarity coefficient matrix and also average similarity of each species. According to the results, two main clusters were developed and P. vera, P. eurycarpa, P. atlantica (subsp. atlantica, kurdica, mutica, cabulica) and the hybrid genotypes located in the first main cluster. P. khinjuk accessions from Iran and USA localized in second main cluster. The hybrid accessions located between eurycarpa and atlantica species and their hybrid nature between these two species were confirmed. One of the unknown accessions clustered with the hybrid ones and the two other were grouped closely with P. Khinjuk. According to this study, the closest species toP. vera was Eurycarpa group, followed by P. atlantica. UPGMA analysis separated P. atlantica subsp. mutica and cabulica from P. atlantica and P. eurycarpa. Subspecies mutica and cabulica were two closest genotypes; hence, P. atlantica subsp. mutica could be classified as a distinct species as P. mutica and the cabulica as a subspecies of P. mutica. This study revealed that P. eurycarpa is synonym for P. atlantica subsp. kurdica and should be considered distinct from P. atlantica; however, P. atlantica showed a closer genetic similarity to P. eurycarpa than the other species.