Cross-specific Amplification Research Articles

Cross-specific amplification of microsatellite DNA markers in Shorea platyclados

In the megadiverse forests of Southeast Asia, hundreds of timber species are economically important but the population genetics of only a few taxa are known. Cross-specific amplification of microsatellite loci among closely related taxa could enhance our ability to study and manage previously unstudied species. We successfully utilized STMS markers in Shorea platyclados, originally developed for Shorea curtisii. The six primer pairs we tried successfully produced PCR products of expected sizes. The number of alleles observed ranged from 10 to 14 and an average of 12 alleles were detected per locus. A high expected and observed heterozygosity was observed and it ranges from 0.718 to 0.827 among all populations across all six loci tested. Microsatellite DNA markers are highly polymorphic, co-dominant, reproducible, and amenable to high throughput genetic analyses. Overall, the cross-specific amplification of microsatellite loci appears to be complicated by numerous factors. While the approach may be effective for local management and conservation of poorly known species, the results must be carefully interpreted.

Development of 26 novel microsatellite makers for the round whitefish (Prosopium cylindraceum) and successful polymorphic cross-specific amplification of seven previously developed salmonid markers

Round whitefish is a freshwater salmonid found within northern North America and north-eastern parts of Asia. Here we describe 26 novel, and seven previously described microsatellite loci that amplified in this species. Of these 33 markers, 12 were polymorphic, exhibiting between 2 and 5 alleles each. The remaining 21 loci are monomorphic. Observed and expected heterozygosities for the polymorphic markers ranged from 0.094 to 0.625 and 0.203 to 0.768 respectively. There was no evidence of linkage disequilibrium between any locus pairs, nor was there any evidence of null alleles. There were however, seven markers deviating from Hardy–Weinberg equilibrium, a result we attribute to relatively small sample and/or population size.

Microsatellite DNA markers in Shorea platyclados (Dipterocarpaceae): genetic diversity, size homoplasy and mother trees

Cross-specific amplification of microsatellite loci greatly enhances the effectiveness of this marker system. This shortcut would greatly enhance our examination of the gene flow and population structure of trees in diverse tropical rainforests. To explore the effectiveness and limitations of this approach, we examined allelic diversity at six microsatellite loci, originally developed in a congeneric species, in three populations of Shorea platyclados from Peninsular Malaysia. Fragment sizing was performed by an efficient and sensitive (1 bp resolution) technique using capillary electrophoresis, ethidium bromide detection, and minimal clean-up. Fragment size ranges were conserved between species and null allele frequencies were low. Higher overall levels of genetic diversity were detected in our study. Variation among populations was directly related to geographic distance. Fragment size class distributions suggest that each locus should be studied using different evolutionary models. Direct sequencing of SSR fragments revealed that size differences were due to changes in both the flanking regions and repeat motifs. Several clear examples of size homoplasy were observed, along with the disruption of perfect repeats, suggesting that cross-specific amplification of microsatellite loci requires an additional level of confirmation at the DNA sequence level before the influence of size homoplasy and changes in repeat structure can be assessed. Simulation studies demonstrate that the increasing intensity of timber harvest leads to higher variability in levels of potential heterozygosity and decreasing total number of alleles in the remnant "mother trees" The careful selection of "mother" trees can greatly enhance the future genetic diversity of populations.   

Development of 17 novel microsatellite markers for the longnose sucker (Catostomus catostomus) and successful cross-specific amplification of 14 previously developed markers from congeneric species

The longnose sucker (Catostomus catostomus) is a widely distributed catostomid, found throughout Canada and much of the northern United States. Although it is a species with significant ecological importance, it has been the focus of only a few studies. Here we describe 17 novel microsatellite loci, and the use of 14 previously described markers from congeneric species formerly untested on longnose sucker. Of the 31 described markers, 19 were polymorphic exhibiting between 2 and 20 alleles. Observed and expected heterozygosities ranged from 0.13 to 0.91, and 0.23 to 0.95 respectively. There was no evidence of null alleles or of deviation from Hardy–Weinberg equilibrium for any locus, and no locus pairs exhibited evidence of linkage disequilibrium.

Screening of New Microsatellite DNA Markers from the Genome of Platyeriocheir formosa

The catadromous Platyeriocheir formosa is a crab endemic in Taiwan. To conserve P. formosa population diversity and ensure the sustainable use of this natural resource, we have developed new genetic markers, 17 polymorphic microsatellite loci, to promote the study of its population genetics in the future. In this study, more than 70 microsatellite sequences were found. Among these, 18 loci were selected to analyze the genetic diversity of P. formosa. With the exception of the Pfo15 locus, all of the remaining loci were polymorphic with allelic numbers ranging from 3–14. Heterozygosity within all 17 polymorphic loci ranged from 0.2–0.95 with an average of 0.55, which suggested that these loci are proper markers for studying population genetics. After we tested cross-specific amplification, eight and six primer sets could be successfully used for the amplification of microsatellite loci in morphologically similar Eriocheir sinensis and E. japonica, respectively; this suggests that they are useful markers for closely related species.

Microsatellite DNA markers for population‐genetic studies of blue mackerel (Scomber australasicus) and cross‐specific amplification in S. japonicus

Blue mackerel (Scomber australasicus) is targeted by large-scale purse-seiners in the western North Pacific, and its stock structure is still contentious. Herein, we described 10 polymorphic microsatellite loci for blue mackerel. The number of alleles among 32 individuals surveyed ranged from five to 27 (average of 16.2 alleles per locus). Departures from Hardy-Weinberg expectation were observed at two loci. Cross-specific amplification in the congener, S. japonicus, was successful, except for one locus, revealed to be diagnostic for these congeners. These microsatellite loci will be useful tools to address queries in population genetic structure, fishery management unit and taxonomic species status in the genus Scomber.

Polymorphic microsatellite markers of freshwater prawn Palaemon paucidens (De Haan, 1844) (Crustacea: Palaemonidae)

We report 11 polymorphic microsatellite loci developed from the freshwater prawn Palaemon paucidens. Their genetic characteristics were assessed in 48 individuals selected from six Korean populations. The number of alleles ranged from two to 21, and the observed and expected heterozygosities were between 0.13 and 0.83, and 0.46 and 0.95, respectively. We examined the cross-specific amplification of each locus in three species of palaemonid prawn and one species of atyid prawn.

Characterization of six microsatellite loci in Myrica faya (Myricaceae) and cross amplification in the endangered endemic M. rivas-martinezii in Canary Islands, Spain.

Six novel polymorphic microsatellite markers were isolated from enriched libraries in Myrica faya Ait., recently renamed Morella faya, (fayatree, firetree, or firebush) in order to examine the genetic diversity in natural populations. Also, test cross-specific amplification and genetic diversity in Myrica rivas-martinezii, which is endemic on the Canary islands. Microsatellite loci were screened in 225 individuals of both species from different islands of the Canarian archipelago. All markers were successfully amplified from both Myrica species, with an average number of 6.5 and 9.3 alleles per locus in M. rivas-martinezii and M. faya, respectively. There was no evidence for linkage disequilibrium between loci, and the probability of null alleles ranged from 0.01 to 0.17.

Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

AbstractMolecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of perennial ryegrass, two cultivars of Italian ryegrass, two cultivars of meadow fescue, two cultivars of tall fescue and 10 accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA‐derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes. The efficiency of cross‐specific amplification for LPSSR markers varied from 38% in meadow fescue to 93% in two cultivars of Festulolium and from 57% in meadow fescue to 87% in Italian ryegrass for LMSSR markers. Of 40 amplified markers, 14 (35%) produced species‐difference alleles in the relation to cultivars used in the present study. Thirty‐five LPSSR locus‐derived alleles were found to be specific to Lolium species, four to meadow fescue and six to tall fescue. For LMSSR alleles, eight were specific to Lolium species and five were only associated with Italian ryegrass, and null alleles were detected for meadow fescue in all instances. These species‐difference markers could clearly identify different accessions of Festulolium. Cluster analysis separated the individual taxa and showed grouping of intergeneric hybrids based on genomic composition. The data distinguished between the species and reflected the known pedigree of the cultivars and the differences between the species. The dendrogram also distinguished between the Festulolium accessions and clearly demonstrated the relations between Festulolium hybrids and their parent species.

Transpecific microsatellites for hard pines

Microsatellites are difficult to recover from large plant genomes so cross-specific utilisation is an important source of markers. Fifty microsatellites were tested for cross-specific amplification and polymorphism to two New World hard pine species, slash pine ( Pinus elliottii var. elliottii) and Caribbean pine ( P. caribaea var. hondurensis). Twenty-nine (58%) markers amplified in both hard pine species, and 23 of these 29 were polymorphic. Soft pine (subgenus Strobus) microsatellite markers did amplify, but none were polymorphic. Pinus elliottii var. elliottii and P. caribaea var. hondurensis showed mutational changes in the flanking regions and the repeat motif that were informative for Pinus spp. phylogenetic relationships. Most allele length variation could be attributed to variability in repeat unit number. There was no evidence for ascertainment bias.