Genome skimming and plastid microsatellite profiling of alder trees (<Emphasis Type="Italic">Alnus</Emphasis> spp., Betulaceae): phylogenetic and phylogeographical prospects

Alders (Alnus spp.) represent keystone species trees of riparian and mountainous habitats of the northern hemisphere. Previous genetic studies have suggested a complex intrageneric diversification with numerous events of interspecific hybridization and polyploidization. Here, we first aim to test the present taxonomical treatment of Alnus by generating phylogenetic hypotheses based on plastid and nuclear data obtained from species belonging to the three main alder subgenera (Alnus, Alnobetula, and Clethropsis). A genome-skimming strategy was used to assemble the complete plastome and the nuclear ribosomal DNA cluster of 22 Eurasian and American alder individuals. Phylogenies based on these data strongly support an early diverging subgenus Alnobetula, while members of the subgenus Clethropsis do not constitute a monophyletic clade and are embedded within the subgenus Alnus. Incongruent topologies also sustain reticulate evolution within this group. Our results thus suggest considering the subgenera Clethropsis and Alnus within the same taxonomical unit. Our second aim is to test for the utility of highly variable plastid markers (microsatellites) to investigate the phylogeographic patterns of Eurasian alder species. Fifty-two polymorphic plastid microsatellite markers were developed and tested on 33 populations of the subgenus Alnus in western Eurasia. On average, 4.3 alleles per locus were revealed in 131 individuals of Alnus glutinosa, allowing the identification of 30 chlorotypes (multiloci profiles). Strong phylogeographic signals and recurrent cytoplasmic captures between co-occurring species are revealed, demonstrating that our plastid microsatellite profiling method is suitable for tracing the post-glacial spread of maternal lineages among alder species. All these results finally support the use of nuclear genomic regions for species identification and of plastid markers for phylogeographic aspects and origin certification in genetic resource management.     

Molecular taxonomy of Hyrcanian Alnus using nuclear ribosomal ITS and chloroplast trnH-psbA DNA barcode markers

The taxonomy and phylogeny of Hyrcanian Alnus (eight taxa) were investigated using sequence data from the nuclear ribosomal ITS and the chloroplast trnH-psbA intergenic spacer. The mean nucleotide compositions of the ITS region were completely equal for two main Hyrcanian taxa, and the ITS1 region had fewer variable sites than the ITS2 region. Two relatively distinct types of ITS2 were identified for two main clades of Hyrcanian Alnus, the A. subcoradata complex and A. glutinosa. Two recently described species, A. dolichocarpa and A. djavanshirii, did not show any diagnostic sites and had a similar pattern with A. subcordata. Three recognized subspecies of A. glutinosa were distributed in the A. incana complex. In the analysis of trnH-psbA sequence data, the three subgenera of Alnus were poorly resolved relative to one another. Alnus glutinosa had minimal sequence divergence from A. incana and A. tenuifolia, and A. subcordata had a minimum distance from A. cordata and A. orientalis. A maximum pairwise distance also was observed between Hyrcanian species (A. glutinosa and A. subcordata) with A. pendula and A. sieboldiana, respectively. Ultimately, the molecular phylogeny of Alnus based on two DNA barcode markers was not congruent with recent morphological classifications, so additional DNA markers should be explored for identifying Alder taxa in the Hyrcanian forest.     

Distribution of spore-positive and spore-negative nodules in stands ofAlnus glutinosa andAlnus incana in Finland

Summary The distribution of spore positive (Sp⁺) and spore negative (Sp⁻) nodules on the two native alder species (A. incana andA. glutinosa) in Finland was investigated. Nodules were collected throughout the country from different ecosystems (forests, swamps, lake- sea- and riversides, old pastures and fields as well as from alder plantations). OnA. incana Sp⁺ nodules predominated, whereas onA. glutinosa the vast majority of the nodules were of the Sp⁻ type. Sp⁺ nodules onA. glutinosa were found only at sites where the two alder species grew close together. This distribution pattern indicates an association of nodule type with alder species, the reasons for which are discussed. Indications of saprophytic growth in the Sp⁻ strain were also found.     

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa

The genetic diversity of Frankia populations in soil and in root nodules of sympatrically grown Alnus taxa was evaluated by rep-polymerase chain reaction (PCR) and nifH gene sequence analyses. Rep-PCR analyses of uncultured Frankia populations in root nodules of 12 Alnus taxa (n?=?10 nodules each) growing sympatrically in the Morton Arboretum near Chicago revealed identical patterns for nodules from each Alnus taxon, including replicate trees of the same host taxon, and low diversity overall with only three profiles retrieved. One profile was retrieved from all nodules of nine taxa (Alnus incana subsp. incana, Alnus japonica, Alnus glutinosa, Alnus incana subsp. tenuifolia, Alnus incana subsp. rugosa, Alnus rhombifolia, Alnus mandshurica, Alnus maritima, and Alnus serrulata), the second was found in all nodules of two plant taxa (A. incana subsp. hirsuta and A. glutinosa var. pyramidalis), and the third was unique for all Frankia populations in nodules of A. incana subsp. rugosa var. americana. Comparative sequence analyses of nifH gene fragments in nodules representing these three profiles assigned these frankiae to different subgroups within the Alnus host infection group. None of these sequences, however, represented frankiae detectable in soil as determined by sequence analysis of 73 clones from a Frankia-specific nifH gene clone library. Additional analyses of nodule populations from selected alders growing on different soils demonstrated the presence of different Frankia populations in nodules for each soil, with populations showing identical sequences in nodules from the same soil, but differences between plant taxa. These results suggest that soil environmental conditions and host plant genotype both have a role in the selection of Frankia strains by a host plant for root nodule formation, and that this selection is not merely a function of the abundance of a Frankia strain in soil.     

Growth responses and N and P use efficiency of three Alnus species as affected by arbuscular-mycorrhizal colonisation

In this study it was determined how different species ofAlnus (A. cordata, A. incana and A. glutinosa) responded tocolonisation by arbuscular mycorrhizal (AM) fungi (Glomusmosseae or Glomus intraradices) with regard togrowth and their ability to acquire and utilise nitrogen and phosphorus.Non-mycorrhizal plants but with phosphorus added, were used as control. InA. glutinosa the application of 75 ppm P hadsimilar effect on growth and P acquisition as did AM. Nevertheless,A.cordata and A. incana grew poorly when suppliedwith 75 ppm of P and required AM symbiosis for optimum growth andNand P uptake. The percentage increases in shoot dry biomass in AM colonised ascompared with P-fertilised plants were 441 (A. cordata)and644 (A. incana) whilst AM-colonised A.glutinosa matched P-fertilised plants. Plant shoot N/P ratioincreased in response to AM-colonisation indicating that mycorrhizal effects onN uptake are greater than on P uptake. Information concerning the directinfluence of AM on N acquisition and nutrient use efficiency byAlnus species is important. AM-colonisation provides anexcellent biological mechanism by which Alnus plantsbecamemore efficient P-users. That Alnus sp. are highlymycorrhizal-dependent plants was apparent because AM-colonisation was criticalfor growth of A. incana and A.cordata. In this respect, for maximizing the efficient uptake anduseof N and P, under the growth conditions provided, Alnusplants need to be mycorrhizal. AM symbiosis seems decisive for the successfulestablishment of Alnus sp. in revegetation strategies. Thelow N and P availability in soils where Alnus species areuseful candidates in any recolonisation and reclamation process emphasises theneed to investigate systems by which N and P uptake byAlnus plants can be enhanced.     

Study of the contribution of the shoot and/or root ofAlnus sp. in the compatibility between the host and a Sp+ Frankia strain using a grafting technique

Two alder species,Alnus glutinosa (L.) Gaertn. andAlnus incana (L) Moench, were inoculated with a Sp⁺ Frankia homogenate obtained fromA. incana root nodules. This inoculum formed effective nodules on the original host plant and ineffective nodules onA. glutinosa. Grafts between the two alder species were made to determine which part of the plant is involved in this phenomenon. The results obtained indicate that the compatibility between Alnus andFrankia is restricted to the root system.     

Isolation and characterization of 19 novel microsatellite loci in Rhododendron aureum and Rhododendron brachycarpum (Ericaceae)

Nineteen polymorphic microsatellite loci are described for Rhododendron aureum Georgi (Ericaceae), an endangered species in Korea, and the closely related Rhododendron brachycarpum. The sequences containing repeat motifs were identified using low-depth next generation sequencing and 148 microsatellite loci were examined for amplification success and the detection of polymorphisms. All 19 loci were polymorphic and the number of alleles for these markers varied from 4 to 25, with an average of 15 alleles per locus. Three R. aureum loci showed departure from Hardy–Weinberg equilibrium (HWE) and one R. brachycarpum locus deviated significantly from HWE. These microsatellite loci will be useful for investigating the genetic diversity and population structure of both species.     

A set of microsatellite markers for population genetics of leopard cat (Prionailurus bengalensis) and cross-species amplification in other felids

We describe the isolation and characterization of novel microsatellite loci from the leopard cat, Prionailurus bengalensis Kerr, 1792 (Family Felidae). Using Illumina HiSeq2500 sequencing technology, we sequenced the leopard cat genome and identified 1.5 million loci of simple sequence repeats with di- to deca-nucleotide motifs. We developed twelve polymorphic markers with tetra-nucleotide motif types after screening 35 loci for amplification and polymorphism. The observed and expected heterozygosities of the markers were 0.438 and 0.423, respectively. The number of alleles per locus ranged from 2 to 7, with a mean polymorphism information content of 0.383. Eleven loci were at Hardy-Weinberg equilibrium and no linkage disequilibrium was detected among any pairs of loci. We tested cross-species amplification of these markers across five other felids (Panthera tigris, P. pardus, P. onca, Acinonyx jubatus, and Felis catus). All loci were transferable to at least one other feline species and four amplified all five species. The microsatellite markers developed in this study will be valuable for estimating ecological parameters of populations and to establish conservation and management strategies for feline species.     

Utilizing next generation sequencing to characterize microsatellite loci in a tropical aquatic plant species Cryptocoryne cordata var. cordata (Araceae)

Cryptocoryne cordata var. cordata (2n = 34) is an aquatic plant species distributed from the southern part of Peninsular Thailand through the Malay Peninsula. It propagates both sexually and asexually via stolons. The current study is aimed at developing nuclear microsatellite markers for the species using next generation sequencing (Roche 454 pyrosequencing) from genomic DNA. A total of 41,653 reads was generated, of which 3636 fragments contained at least one repeat motif. Seventy two primer sets in the flanking region of dinucleotide, trinucleotide and tetranucleotides repeat motifs were designed and tested for efficiency in polymerase chain reaction amplification. Using these primer sets, 11 new microsatellite marker loci were successfully amplified with unambiguous polymorphic alleles exhibited across 30 individuals examined. The number of alleles per locus ranged from 2 to 6, while observed and expected heterozygosity ranged from 0.8190 to 1.0000 and 0.5401 to 0.7548, respectively. Genotype frequencies at all loci departed significantly from Hardy–Weinberg equilibrium. Linkage disequilibrium was not detected between any pair of loci. Cross-species amplification was successful across a panel of ten Cryptocoryne species. The markers described in this study will be useful for evaluating genetic diversity within and between populations, levels of gene flow, and the population dynamics of clones. They will be of further value in the development of effective conservation programs for Cryptocoryne species.     

Genetic diversity and genetic structure of black alder (<Emphasis Type="Italic">Alnus glutinosa</Emphasis> [L.] Gaertn) in the Belgium-Luxembourg-France cross-border area

Due to its beneficial effects on river ecosystems, black alder (Alnus glutinosa) is one of the tree species selected for planting on riverbanks in the cross-border area encompassing Wallonia in Belgium, Lorraine in France, and Luxembourg. The preservation of this species, however, is threatened by an invasive pathogen that particularly targets and kills young alder individuals. The objectives of this study were to characterize the genetic diversity and the genetic structure of A. glutinosa at this local level with the aim of assisting the conservation and replanting strategies and to determine if a germplasm collection comprising individuals from the same cross-border area captures the diversity present in the region. Nuclear simple sequence repeat (SSR) and chloroplastic DNA (cpDNA) markers were used to analyze four local wild populations and the germplasm collection which is representative of two river catchments and six legal provenance regions. Three populations distant from the studied area were also included. A panel of 14 nuclear SSR loci revealed high allelic diversity and very low differentiation among wild populations (mean F _ST?=?0.014). The germplasm collection displayed a range of alleles that were representative of the different populations, and no significant differentiation between the germplasm collection and the local wild populations was observed, making this collection, as far as allelic diversity is concerned, suitable for providing trees for riverbank replanting programs. Using SSR markers, various statistical approaches consistently indicated the lack of a significant geographical structure at the level of the river catchments or provenance regions. In contrast, two cpDNA haplotypes were detected and displayed a cross-border geographically structured distribution that could be taken into account in defining new cross-border provenance regions.     

Not one but three: undetected invasive <Emphasis Type="Italic">Alnus</Emphasis> species in northwestern Patagonia confirmed with cpDNA and ITS sequences

Species of Alnus (alders) have become invaders in several parts of the world. Here we report the presence of three naturalized alien species: A. glutinosa, A. incana and A. rubra from several populations in nature reserves of northwestern Patagonia, an area of remarkably high biodiversity. Alnus glutinosa had been cited previously for Chile and southern Argentina, but A. incana and A. rubra are here reported for the first time. As we found morphological variation within and among the populations of these introduced species that makes their discrimination difficult, we used chloroplast (trnH-psbA) and nuclear ribosomal (ITS) DNA sequences to confirm their identifications from morphological characteristics. Results from nuclear and chloroplast sequence data confirm the morphological tentative identification of the three species and remark the utility of molecular information together with morphology for the detection of introduced species of taxonomically difficult groups. The invasive characteristics of these alien tree species are discussed in relation to the conservation of the nature reserves where they are found.     

Seventy‐five EST‐linked Atlantic salmon (Salmo salar L.) microsatellite markers and their cross‐amplification in five salmonid species

An Atlantic salmon (Salmo salar L.) expressed sequence tag (EST) database consisting of 58 146 ESTs was screened for microsatellite sequences. Subsequent development of 75 polymorphic EST‐associated microsatellite markers in this species is described together with cross‐species amplification results of 133 gene‐associated tandem repeat markers in five salmonid species (Salmo trutta, Oncorhynchus mykiss, Salvelinus aplinus, Thymallus thymallus, Coregonus lavaretus). The number of alleles among EST‐linked microsatellites in Atlantic salmon ranged from two to 41 with an average of 12 alleles per locus. Cross‐species amplification resulted in detection of a total of 111 polymorphic locus‐species combinations (12–32 loci per species).     

Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Effects of Frankia on field performance of Alnus clones and seedlings

Field performance of tissue cultured clones and seedlings of Alnus viridis ssp. crispa, A. glutinosa, A. incana, and A. japonica was assessed five years after outplanting in central Ontario. Half the individuals were inoculated with a mixture of four Frankia isolates prior to planting. Inoculation produced significant increases (25% to 33%) in biomass production of two clones of A. glutinosa and one of A. incana. Woody biomass increments for the first five years, averaged across all clones and seedlings, were highest in A. japonica and A. incana (4.3 and 3.7 Mg ha^–1 yr^–1, respectively). Individual tree growth improved markedly in lower slope positions, but total plot biomass did not show similar gains in downslope positions owing to higher mortality and aphid (Paraprociphilus tessellatus) infestation. Aphids occurred in 22% of Frankia-inoculated individuals, and 15% of non-inoculated individuals. The fastest growing species, A. incana and A. japonica, were most susceptible to aphid attack. Growth of the best clones of A. glutinosa and A. incana exceeded seedling growth by 51% and 76%, respectively. The high growth variation in clones of the same species with similar geographic origins and the excellent performance of tissue cultured stock suggest that rapid genetic gains in an Alnus breeding program might be obtained by clonal propagation.     

Characterization of Alder Phytophthora Isolates from Wallonia and Development of SCAR Primers for their Specific Detection

Isolates of alder Phytophthora were collected in the southern part of Belgium on riverbanks planted with Alnus glutinosa and A. incana. They were compared with strains isolated in other European countries in terms of maximum temperature for growth, oogonia shape, pathogenicity on Alnus seedlings and genetic traits. Using both molecular techniques [random amplified polymorphic DNA (RAPD) and random amplified microsatellite (RAMS)], two groups of isolates were identified, the first group being further divided into two subgroups, Ia and Ib, using RAPD. Most of the Walloon alder Phytophthora isolates as well as the standard type from UK (formally designated P. alni subsp. alni) fell into group Ia. One isolate was classified in group Ib with the German and Dutch variants (P. alni subsp. multiformis), while three isolates were placed with the Swedish variant (P. alni subsp. uniformis) in group II. In terms of morphological properties, isolates from groups Ia and Ib developed colonies with a felt‐like appearance and usually produced numerous oogonia, varying from wavy to warty after 1 week (group Ia) or 2–3 weeks (Ib) in darkness. In contrast, colonies from group II isolates were generally irregular, and smooth oogonia were produced in low quantities after approximately 1 month in culture. A polymerase chain reaction (PCR) using sequence‐characterized amplification region (SCAR) primers derived from a polymorphic amplification product generated with a RAPD primer was developed for the specific detection of alder Phytophthora. The specificity and sensitivity of this test are discussed here.     

Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.     

What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles

Sixty-five microsatellite alleles amplified from ancestral citrus accessions classified in three separate genera were evaluated for sequence polymorphism to establish the basis of inter- and intra-allelic genetic variation, evaluate the extent of size homoplasy, and determine an appropriate model (stepwise or infinite allele) for analysis of citrus microsatellite alleles. Sequences for each locus were aligned and subsequently used to determine relationships between alleles of different taxa via parsimony. Interallelic size variation at each SSR locus examined was due to changes in repeat copy number with one exception. Sequencing these alleles uncovered new distinct point mutations in the microsatellite region and the region flanking the microsatellite. Several of the point mutations were found to be genus, species, or allele specific, and some mutations were informative about the inferred evolutionary relationships among alleles. Overall, homoplasy was observed in alleles from all three loci, where the core microsatellite repeat was changed causing alleles of the same size class to be identical in state but not identical by descent. Because nearly all changes in allele size (with one exception) were due to expansion or contraction of the repeat motif, this suggests that a stepwise mutation model, which assumes homoplasy may occur, would be the most appropriate for analyzing Citrus SSR data. The collected data indicate that microsatellites can be a useful tool for evaluating Citrus species and two related genera since repeat motifs were reasonably well retained. However, this work also demonstrated that the number of microsatellite alleles is clearly an underestimate of the number of sequence variants present.     

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, <Emphasis Type="Italic">Laticauda semifasciata</Emphasis> (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The cross-species amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.