A suite of twelve single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow trout

A suite of 12 subspecies and species-specific single nucleotide polymorphism (species-specific SNP) markers was developed to distinguish rainbow trout (RT) Oncorhynchus mykiss from the four major subspecies of cutthroat trout: westslope cutthroat trout (WCT) Oncorhynchus clarki lewisi, Yellowstone cutthroat trout (YCT) Oncorhynchus clarki bouvieri, coastal cutthroat trout (CCT) Oncorhynchus clarki clarki, Lahontan cutthroat trout (LCT) Oncorhynchus clarki henshawi, and their hybrids. Several of the markers were linked to help strengthen hybrid determinations, and sex-specific species-specific SNP assays were also developed.     

Regional divergence and mosaic spatial distribution of two closely related damselfly species (Enallagma hageni and Enallagma ebrium)

North American Enallagma damselflies radiated during the Pleistocene, and species differ mainly by reproductive structures. Although morphologically very different, Enallagma hageni and Enallagma ebrium are genetically very similar. Partitioning of genetic variation (AFLP), isolation by distance and clustering analyses indicate that these morphospecies are locally differentiated genetically. Spatial analyses show that they are rarely sympatric at local sites, and their distributions form a mosaic of patches where one is clearly dominant over hundreds of square kilometers. However, these morphospecies are also not genetically more similar when they are sympatric, indicating that hybridization is probably not occurring. Given that these morphospecies are ecologically equivalent, strong assortative mating, reproductive interference and fast post-glacial recolonization may explain the origin and maintenance of these distributional patches across eastern North America. By limiting opportunities for gene flow, reproductive interference may play an unsuspected role in accelerating genetic differentiation in the early phases of nonecological speciation.     

Spatial scale of local adaptation and population genetic structure in a miniature succulent, Argyroderma pearsonii

Explicit understanding of the spatial scale of evolutionary processes is required in order to set targets for their effective conservation. Here, we explore the spatial context of neutral and adaptive divergence in the species-rich Knersvlakte region of South Africa. Specifically, we aimed to assess the importance of erosional drainage basins as spatial units of evolutionary process. We used amplified fragment length polymorphism (AFLP) and reciprocal transplants to investigate genetic differentiation in Argyroderma pearsonii, sampled from sparse and dense quartz habitats within each of three drainage basins. This design allowed assessment of differentiation at two distinct spatial scales; between habitats within basins, and between basins. We found near-perfect concordance between genetic clusters and basin occupancy, suggesting restricted interbasin gene flow. In addition, transplants reveal adaptive divergence between basins on the dense quartz habitat. We have shown that neutral and adaptive differentiation occurs between basins, but not between habitats within basins, suggesting that conservation plans aimed at conserving multiple interconnected drainage basins will capture an important axis of evolutionary process on the Knersvlakte.     

A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.) varieties

A sample set of registered perennial ryegrass varieties was used to compare how morphological characterisation and AFLP^® (AFLP^® is a registered trademark of Keygene N.V.) and STS molecular markers described variety relationships. All the varieties were confirmed as morphologically distinct, and both the STS and AFLP markers exposed sufficient genetic diversity to differentiate these registered ryegrass varieties. Distances obtained by each of the approaches were compared, with special attention given to the coincidences and divergences between the methods. When correlations between morphological, AFLP and STS distances were calculated and the corresponding scatter-plots constructed, the variety relationships appeared to be rather inconsistent across the methods, especially between morphology and the molecular markers. However, some consistencies were found for closely related material. An implication could be that these molecular-marker techniques, while not yet suited to certain operations in the traditional registration of new varieties, could be suitable methods for investigating disputable distinctness situations or possible EDV (EDV= essentially derived variety. An EDV is a variety being clearly distinct from, but conforming in the expression of the essential characteristics of, an ’initial variety’ (IV) from which it is found to have been predominantly derived) relationships, subject to establishing standardised protocols and statistical techniques. Some suggestions for such a protocol, including a statistical test for distinctness, are given.     

A first-generation genetic linkage map of the European flat oyster Ostrea edulis (L.) based on AFLP and microsatellite markers

This study presents the first genetic linkage map for the European flat oyster Ostrea edulis . Two hundred and forty-six AFLP and 20 microsatellite markers were genotyped in a three-generation pedigree comprising two grandparents, two parents and 92 progeny. Chi-square goodness-of-fit tests revealed high segregation distortion, which was significant for 32.8% of markers. Sixteen microsatellites and 235 AFLPs (170 type 1:1 AFLPs and 65 type 3:1 AFLPs) were used to build sex-specific linkage maps using crimap software. The first parental map (P₁) consisted of 104 markers grouped in nine linkage groups, and spanned 471.2 cM with an average spacing of 4.86 cM. The second parental map (P₂) consisted of 117 markers grouped in 10 linkage groups (which equals the haploid chromosome number), and covered 450.0 cM with an average spacing of 4.21 cM. The estimated coverage of the genome was 82.4% for the P₁ map and 84.2% for the P₂ map. Eight linkage groups that were probably homologous between the two parents contained the same microsatellites and 3:1 AFLPs (segregating through both parents). Distorted markers were not randomly distributed across the genome and tended to cluster in a few linkage groups. Sex-specific differences in recombination rates were evident. This first-generation genetic linkage map for O. edulis represents a major step towards the mapping of QTL such as resistance to bonamiasis, a parasitosis that has drastically decreased populations of flat oysters since the 1960s.     

Amplified fragment length polymorphism (AFLP) analysis of the genetic structure of the zebra mussel, Dreissena polymorpha, in the Mississippi River

1. We predicted that zebra mussel, Dreissena polymorpha (Pallas), genetic structure in the Mississippi River would follow a model of invasive species genetics, which predicts low genetic structure among populations of recently established species. This prediction was upheld in our previous genetic study using allozymes, however, one locus yielded anomalous results. 2. We employed amplified fragment length polymorphism (AFLP) analysis as a neutral marker to assess the amount of genetic structure within and among populations, and as a test of expected population structure from both invasion genetic theory, and the results from our previous study. 3. There was greater spatial differentiation, as measured by F_st, observed using AFLP's than for allozymes (P < 0.001). There was no evidence that AFLP variation conformed to an isolation by distance model, and genetic relationships of populations, as measured by AFLP markers, were not similar to those detected in our allozyme survey. 4. The lack of concordance between these two genetic marker systems probably reflects their differential responses to drift, migration, and selection occurring during this rapid invasion. Strong population structure is counter to predictions that populations of invasive species will not be differentiated, as with observations based on allozyme markers. Therefore, newly established species may require genetic surveys using multiple marker systems to evaluate population structure.     

Parallel evolution of ecomorphological traits in the European whitefish Coregonus lavaretus (L.) species complex during postglacial times

The extensive phenotypic polymorphism in the European whitefish has triggered evolutionary research in order to disentangle mechanisms underlying diversification. To illuminate the ecological distinctiveness in polymorphic whitefish, and evaluate taxonomic designations, we studied nine Norwegian lakes in three watercourses, which each harboured pairs of divergent whitefish morphs. We compared the morphology and life history of these morphs, documented the extent of genetic differentiation between them, and contrasted the niche use of sympatric morphs along both the habitat and resource axes. In all cases, sympatric morphs differed in the number of gill rakers, a highly heritable trait related to trophic utilization. Individual growth rate, age and size at maturity, diet and habitat use also differed between morphs within lakes, but were remarkably similar across lakes within the same morph. Microsatellite analyses confirmed for all but one pair that sympatric morphs were significantly genetically different, and that similar morphs from different lakes likely have a polyphyletic origin. These results are most compatible with the process of parallel evolution through recurrent postglacial divergence into pelagic and benthic niches in each of these lakes. We propose that sparsely and densely rakered whitefish sympatric pairs may be a likely case of ecological speciation, mediated in oligotrophic lakes with few trophic competitors.     

Isolation of microsatellite markers for Pelophylax nigromaculata and a tentative application in detecting interspecific introgression

Twenty-seven microsatellite markers were obtained from the black-spotted frog, Pelophylax nigromaculata, using the FIASCO (Fast Isolation by AFLP of Sequences containing repeats) protocol. Genotyping of 60 individuals showed that the number of alleles ranged from 4 to 25 with an average of 12.78 alleles per locus. The observed and expected heterozygosities ranged from 0.137 to 0.956 and 0.594 to 0.948, with averages of 0.531 and 0.809, respectively. Many of these loci were successfully cross-amplified in five other ranid species. The introgression between P. nigromaculata and Pelophylax plancyi was assessed using 5 microsatellite loci on 159 individuals collected from Liaoning, northeast China. Two distinct genetic clusters (pure species) as well as seven unassigned individuals were identified by factorial correspondence analysis (FCA), NewHybrids and Structure. The unassigned individuals, which are likely the product of interspecific hybridization, indicate the usefulness of these microsatellite loci for future studies of hybridization and introgression between these two ranid species.     

Genetic structure of a hybrid zone between two violets,Viola rossii Hemsl. and V. bissetii Maxim.: dominance of F1 individuals in a narrow contact range

The genetic composition of a hybrid zone can provide insight into the evolution of diversification in plants. We carried out morphological and amplified fragment length polymorphism analyses to investigate the genetic composition of a hybrid zone between two violets, Viola bissetii Hemsl. and Viola rossii Maxim. Our aim was to clarify the formation and maintenance of hybrids between these Viola species. We found that most hybrid individuals (V. bissetii × V. rossii) were of the F₁ generation, with a few of the F₂ generation. We found no backcrosses. The scarcity of post‐F₁ hybrids indicates that a species barrier is established between the parental species. The F₁‐dominated hybrid zone occupied only a narrow, intermediate ecotone between the parental habitats, suggesting that selection by environmental factors against hybrids may help to maintain the current conditions in this hybrid zone.     

The role of hybridization, polyploidization and glaciation in the origin and evolution of the apomictic Ranunculus cassubicus complex

The Ranunculus cassubicus complex, comprising diploids and polyploids, is a good model for studying the role of hybridization and polyploidy in the origin of apomixis. Results from amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses performed on 448 individuals were combined with evidence from morphology, isozymes, karyology and distribution. Our results indicated a unique hybrid origin for the apomictic hexaploid R. carpaticola from north-western Slovakia, involving two sexual parents: autotetraploid R. cassubicifolius from the northern pre-Alps, and diploid R. carpaticola from central Slovakia. The hybrids were intermediate to the parents, but unique alleles have resulted from genomic reorganisation in the allopolyploids, which might also have triggered apomixis. Their distribution patterns and estimated ages suggest that hybridization may be correlated with the last glacial period. Hybridization seems to be the major origination for apomicts in the R. cassubicus complex. Polyploidy creates novel sexual genotypes and acts as a springboard for the production of new hybrids, but it only results in a combination with hybridization in apomixis. In turn, asexuality has permitted the perpetuation and establishment of ecologically divergent hybrid genotypes.     

Clarification of the genetic component of hybrids between Phyllodoce caerulea and Phyllodoce aleutica (Ericaceae) in Hokkaido,northern Japan

Natural hybridization provides great opportunities to understand the interaction of genetics and ecology in determining species boundaries. We examined the genetic relationships of Phyllodoce taxa and revealed that most hybrids were fertile F₁s and an extremely small number of backcross and no F₂ plants were established in natural conditions. Because this trend was irrespective of regions, we conclude that negative endogenous selection may act after the germination of F₁ seeds and prevent the establishment of later‐generation hybrids. Based on these results, we discuss the ecological and evolutionary significance of natural hybridization in Phyllodoce taxa.     

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.     

A PCR-RFLP assay for identification and detection of Pythium myriotylum, causal agent of the cocoyam root rot disease

Aim: Development of a PCR‐RFLP assay that could reliably distinguish strains of Pythium myriotylum that are pathogenic to cocoyam from nonpathogens, as well as in planta detection of the pathogen. Methods and Results: Sequences of the internal transcribed spacer regions of nuclear ribosomal DNA (rDNA‐ITS) containing ITS1 and ITS2 of P. myriotylum isolates from cocoyam and other hosts were aligned and a restriction map was generated. rDNA‐ITS alignment report revealed a new single nucleotide polymorphism (SNP; thymine/cytosine) downstream to previously published SNP (guanine/adenine) between isolates of P. myriotylum that are pathogenic to cocoyam and nonpathogenic strains. This new SNP is within the restriction site of the endonuclease AarI. Based on this SNP, a PCR‐RFLP assay was developed for specific detection of P. myriotylum. The PCR amplicons of all isolates of P. myriotylum that infect cocoyam were cleaved by AarI, resulting to two bands (600/400 bp); but those from other hosts showed a single band (1000 bp), confirming the presence and specificity of the AarI restriction site. Also, the assay was effective in in planta detection of the pathogen on infected cocoyam roots without prior isolation of a pure culture. Conclusion: A PCR‐RFLP method was developed that differentiates isolates of P. myriotylum that are pathogenic to cocoyam from nonpathogens as well as from other fungi commonly found in the cocoyam rhizosphere. Significance and Impact of the Study: Early and rapid detection of the pathogen could be of great importance in certifying planting materials as disease‐free, enhancing sustainable management practices and limiting economic losses.     

A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding

Medicinal plants of the Panax genus belonging to Araliaceae family are well-known, rare plants used as tonics in traditional Chinese medicine and have been described in the Chinese Pharmacopoeia. Because of the high price and the huge human demand, these commercial products often contain adulterants. In this study, 377 sequences from four species were analyzed. Single nucleotide polymorphisms (SNPs) were detected and patterns of intragenomic variation in internal transcribed spacer 2 (ITS2) from the four Panax species were studied. Intraspecific variations were analyzed based on three typical DNA barcodings (ITS2, matK and psbA-trnH). Results from this study revealed that intraspecific genetic distances in Panax ginseng and Panax quinquefolius were quite low (0–0.002) and the multi-copy ITS2 could be considered a single locus in the genomes of these two species. Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Considering the mixed powder of P. ginseng and P. quinquefolius, double peaks could be clearly examined at SNP positions and the height of the peaks could indicate the mixed ratio roughly. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species and the mixed powder P. ginseng and P. quinquefolius.     

RAPDs and noncoding chloroplast DNA reveal a single origin of the cultivated Allium fistulosum from A. altaicum (Alliaceae)

The origin of the crop species Allium fistulosum (bunching onion) and its relation to its wild relative A. altaicum were surveyed with a restriction fragment length polymorphism (RFLP) analysis of five noncoding cpDNA regions and with a random amplified polymorhic DNA (RAPD) analysis of nuclear DNA. Sixteen accessions of A. altaicum, 14 accessions of A. fistulosum, representing the morphological variability of the species, and five additional outgroup species from Allium section Cepa were included in this study. The RFLP analysis detected 14 phylogenetically informative character transformations, whereas RAPD revealed 126 polymorphic fragments. Generalized parsimony, neighbor-joining analysis of genetic distances, and a principal co-ordinate analysis were able to distinguish the two species, but only RAPD data allowed clarification of the interrelationship of the two taxa. The main results of this investigation were: (1) A. fistulosum is of monophyletic origin, and (2) A. fistulosum originated from an A. altaicum progenitor, making A. altaicum a paraphyletic species. Compared with A. altaicum the cultivated accessions of the bunching onion show less genetic variability, a phenomenon that often occurs in crop species due to the severe genetic bottleneck of domestication. Allium altaicum and A. fistulosum easily hybridize when grown together, and most garden-grown material is of recent hybrid origin.     

Evolutionary aspects of population structure for molecular and quantitative traits in the freshwater snail Radix balthica

Detecting the action of selection in natural populations can be achieved using the QST-FST comparison that relies on the estimation of FST with neutral markers, and QST using quantitative traits potentially under selection. QST higher than FST suggests the action of directional selection and thus potential local adaptation. In this article, we apply the QST-FST comparison to four populations of the hermaphroditic freshwater snail Radix balthica located in a floodplain habitat. In contrast to most studies published so far, we did not detect evidence of directional selection for local optima for any of the traits we measured: QST calculated using three different methods was never higher than FST. A strong inbreeding depression was also detected, indicating that outcrossing is probably predominant over selfing in the studied populations. Our results suggest that in this floodplain habitat, local adaptation of R. balthica populations may be hindered by genetic drift, and possibly altered by uneven gene flow linked to flood frequency.     

Pleistocene refugia and polytopic replacement of diploids by tetraploids in the Patagonian and Subantarctic plant Hypochaeris incana (Asteraceae, Cichorieae)

We report the phylogeographic pattern of the Patagonian and Subantarctic plant Hypochaeris incana endemic to southeastern South America. We applied amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) analysis to 28 and 32 populations, respectively, throughout its distributional range and assessed ploidy levels using flow cytometry. While cpDNA data suggest repeated or simultaneous parallel colonization of Patagonia and Tierra del Fuego by several haplotypes and/or hybridization, AFLPs reveal three clusters corresponding to geographic regions. The central and northern Patagonian clusters (∼38–51° S), which are closer to the outgroup, contain mainly tetraploid, isolated and highly differentiated populations with low genetic diversity. To the contrary, the southern Patagonian and Fuegian cluster (∼51–55° S) contains mainly diploid populations with high genetic diversity and connected by high levels of gene flow. The data suggest that H. incana originated at the diploid level in central or northern Patagonia, from where it migrated south. All three areas, northern, central and southern, have similar levels of rare and private AFLP bands, suggesting that all three served as refugia for H. incana during glacial times. In southern Patagonia and Tierra del Fuego, the species seems to have expanded its populational system in postglacial times, when the climate became warmer and more humid. In central and northern Patagonia, the populations seem to have become restricted to favourable sites with increasing temperature and decreasing moisture and there was a parallel replacement of diploids by tetraploids in local populations.     

Determination of common genetic variants in cytidine deaminase (CDA) gene in Indian ethnic population

Cytidine deaminase (CDA) is the major enzyme involved in metabolism of gemcitabine, a pyrimidine analog widely used for chemotherapy of solid tumors. While only low amounts of administered gemcitabine undergo intracellular phosphorylation into active forms and involve in antineoplastic activities, majority of it is rapidly inactivated by CDA and excreted to avoid drug toxicity. Knowledge of the genetic polymorphisms mildly effecting cellular activity of the enzyme CDA is therefore crucial to understanding drug-induced toxicities associated with gemcitabine. Functional significance and allele frequencies for common SNPs including 79A>C (*2) and 208G>A (*3) have been reported in various ethnic populations including Caucasian, African, Korean and Japanese. However, such studies have not been reported in any Indian sub-population. In the present study, conventional polymerase chain reaction (PCR) based amplification using gene specific primers and Sanger sequencing were performed to identify CDA variants in 50 healthy individuals from Indian sub-population. Established common variant 79A>C known to reduce CDA activity was observed at a frequency of 0.14 in the study cohort. In addition to other known variants, one novel variant, c.325−209T>C was detected at a frequency of 0.06. Genetic variants in CDA gene and their frequencies established in our study hold value in pharmacogenetics.