Triplet-repeat microsatellites shared among hard and soft pines

Vascular plant species have shown a low level of microsatellite conservation compared to many animal species. Finding trans-specific microsatellites for plants may be improved by using a priori knowledge of genome organization. Fifteen triplet-repeat microsatellites from hard pine (Pinus taeda L.) were tested for trans-specific amplification across seven hard pines (P. palustris Mill., P. echinata Mill., P. radiata D. Don., P. patula Schiede et Deppe, P. halepensis Mill., P. kesiya Royle), a soft pine (P. strobus L.), and Picea rubens Sargent. Seven of 15 microsatellites had trans-specific amplification in both hard and soft pine subgenera. Two P. taeda microsatellites had conserved flanking regions and repeat motifs in all seven hard pines, soft pine P. strobus, and P. rubens. Perfect triplet-repeat P. taeda microsatellites appear to be better candidates for trans-specific polymorphism than compound microsatellites. Not all perfect triplet-repeat microsatellites were conserved, but all conserved microsatellites had perfect repeat motifs. Persistent microsatellites PtTX2123 and PtTX3020 had highly conserved flanking regions and a conserved repeat motif composition with variable repeat unit numbers. Using trinucleotide microsatellites improved trans-specific microsatellite recovery among hard and soft pine 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.     

Comparative sequencing of a microsatellite locus reveals size homoplasy within and between european oak species (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Microsatellites (simple sequence repeats [SSRs]) are highly variable molecular markers that are a rich and readily assayed source of variation for population genetic studies. Cross-amplification between closely related species is possible when there are no (or few) sequence differences in the primer binding sites. The occurrence of nonhomologous fragments of the same size (size homoplasy) is a contraint of microsatellites. Size homoplasy can be caused by insertions/deletions (indels) in SSR flanking regions. We found that size variation in locus ssrQZAG9 is due to different repeat numbers of the SSR motifs but also to indels in SSR flanking regions. Indels were found within species belonging to sectionsRobur andCerris of genusQuercus and also between species of the 2 sections. In sectionRobur (Quercis robur L.,Quercus petraea [Matt.] Liebl.,Quercus pubescens Willd.), we detected rare alleles with an indel of 57 bp or 62 bp followed by a smaller indel of 12 bp in the SSR flanking regions. These alleles show a size range overlapping with that of alleles amplified inQuercus cerris L. (sectionCerris). Multiple alignments with sequences of sectionRobur revealed the same SSR repeat motif but multiple indels in SSR flanking regions inQ. cerris. We discuss the effects of size homoplasy of SSR loci for the study of interspecific gene flow and on estimates of population differentiation.     

Genome size and base composition of five<Emphasis Type="Italic"> Pinus</Emphasis> species from the Balkan region

The 2C DNA content and base composition of five Pinus (2n=24) species and two Pinus subspecies from the Balkan region have been estimated by flow cytometry. P. heldreichii (five populations) and P. peuce (one population) were assessed for the first time, as also were subspecies of P. nigra (three populations—two of subspecies nigra and one of subspecies dalmatica) along with P. sylvestris, and P. mugo from the same region. The 2C DNA values of these Pinus ranged from 42.5 pg to 54.9 pg (41.7–53.8×10⁹ bp), and the base composition was quite stable (about 39.5% GC). Significant differences were observed between two subspecies of P. nigra and even between two populations of subsp. nigra. The two other species (P. sylvestris and P. mugo) had 2C values of 42.5 pg and 42.8 pg, respectively, while that of P. peuce was 54.9 pg. These genome sizes are in accordance with published values except for P. sylvestris, which was 20% below estimates made by other authors.Communicated by M. Beckert     

Geography determines genetic relationships between species of mountain pine (Pinus mugo complex) in western Europe

Aim Our aims were to test whether morphological species of mountain pines were genetically supported in the western part of the distribution range of the Pinus mugo species complex (Pinus mugo Turra sensu lato), to resolve genetically homogeneous clusters of populations, to determine historical demographic processes, and to assess the potential hybridization of mountain pines with Scots pine, Pinus sylvestris L. Location Populations were sampled in the Iberian System, the Pyrenees, the French Mont Ventoux, Vosges and Jura mountains, the German Black Forest and throughout the Alps. This corresponded to a range‐wide sampling for mountain pine sensu stricto (Pinus uncinata Ram.) and to a sampling of the western parts of the ranges of dwarf mountain pine (Pinus mugo Turra sensu stricto) and bog pine/peatbog pine [Pinus rotundata Link/Pinus × pseudopumilio (Willk.) Beck]. Methods In total, 786 individuals of P. mugo sensu lato from 29 natural populations, and 85 individuals of P. sylvestris from four natural populations were genotyped at three chloroplast microsatellites (cpSSRs). Populations were characterized for standard genetic diversity statistics and signs of demographic expansion. Genetic structure was explored using analysis of molecular variance, differentiation statistics and Bayesian analysis of population structure (BAPS). Results One hundred haplotypes were identified in P. mugo sensu lato. There was a stronger differentiation between geographical regions than between morphologically identified taxa (P. mugo sensu stricto, P. uncinata and P. rotundata/P. ×pseudopumilio). Overall genetic differentiation was weak (G_ST = 0.070) and displayed a clear phylogeographic structure [N_ST = 0.263, N_ST > N_ST (permuted), P < 0.001]. BAPS identified a Pyrenean and an Alpine gene pool, along with several smaller genetic clusters corresponding to peripheral populations. Main conclusions The core regions of the Pyrenees and Alps were probably recolonized, respectively by P. uncinata and P. uncinata/P. mugo sensu stricto, from multiple glacial refugia that were well connected by pollen flow within the mountain chains. Pinus rotundata/P. × pseudopumilio populations from the Black Forest, Vosges and Jura mountains were probably recolonized from various glacial populations that kept their genetic distinctiveness despite late glacial and early Holocene expansion. Marginal P. uncinata populations from the Iberian System are compatible with elevational shifts and long‐term isolation. The causes of haplotype sharing between P. mugo sensu lato and P. sylvestris require further research.     

Single-copy,species-transferable microsatellite markers developed from loblolly pine ESTs

Microsatellites, or simple sequence repeats (SSRs), are usually regarded as the markers of choice in population genetics research because they exhibit high variability. The development cost of these markers is usually high. In addition, microsatellite primers developed for one species often do not cross-amplify in related species, requiring separate development for each species. However, microsatellites found in expressed sequence tags (ESTs) might better cross-amplify as they reside in or near conserved coding DNA. In this study, we identified 14 Pinus taeda (loblolly pine) EST-SSRs from public EST databases and tested for their cross-species transferability to P. contorta ssp. latifolia, P. ponderosa, and P. sylvestris. As part of our development of a P. contorta microsatellite set, we also compared their transferability to that of 99 traditional microsatellite markers developed in P. taeda and tested on P. contorta ssp. latifolia. Compared to traditional microsatellites, EST-SSRs had higher transfer rates across pine species; however, the level of polymorphism of microsatellites derived from ESTs was lower. Sequence analyses revealed that the frequencies of insertions/deletions and base substitutions were lower in EST-SSRs than in other types of microsatellites, confirming that EST-SSRs are more conserved than traditional SSRs. Our results also provide a battery of 23 polymorphic, robust microsatellite primer pairs for lodgepole pine.Communicated by O. Savolainen     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Microsatellites in Zea - variability,patterns of mutations,and use for evolutionary studies

To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees. Received: 10 February 2001 / Accepted: 21 May 2001     

Microsatellites as DNA markers in Sitka spruce

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.     

Development and characterization of 11 microsatellite markers in a widespread Neotropical seasonally dry forest tree species,Geoffroea spinosa Jacq. (Leguminosae)

Eleven dinucleotide microsatellites were developed in Geoffroea spinosa (Leguminosae), a widespread tree of the seasonally dry Neotropical forests, and characterized on six populations from Peru, Argentina and Paraguay. Four of them amplified on the Peruvian populations only, probably because of mutations in the microsatellite flanking regions in the other populations. Ten microsatellites were found polymorphic, with within population gene diversities ranging from 0.17 to 0.95, and a number of alleles varying from seven to 19. A significant overall genetic differentiation was also found (θ = 0.212; P < 0.01).     

Preference and performance of the sawfly Diprion pini on host and non-host plants of the genus Pinus

The sawfly, Diprion pini L., is a pest of Pinus in Europe and is mainly found on P. sylvestris L. and P. nigra laricio Poiret. The relative importance of female oviposition capacity and behaviour, egg development, and larval survival on a new host plant was measured on 11 pine species. Five were natural host plants and six non-host plants, five of which are not indigenous to Europe. Oviposition choice tests showed that females discriminated between the pine species. Egg and larval development also differed between pine species. However, the female choice was not linked with hatching rate and larval development. Results of biological tests clearly indicated that there were different response patterns of D. pini life stages in relation to pine species, and these patterns were the same with insects of four different origins. We discuss the importance of each potential barrier to colonisation of a new host.     

Multiplex panels of polymorphic microsatellite loci for two cryptic bat species of the genus Pipistrellus,developed by cross‐species amplification within the family Vespertilionidae

The paper presents multiplex panels of polymorphic microsatellites for two closely related cryptic species Pipistrellus pipistrellus and Pipistrellus pygmaeus. We tested the cross‐species amplification of 34 microsatellite loci, originally developed for five vespertilionid bat species. Ten and nine polymorphic loci in P. pipistrellus (mean number of alleles per locus = 10.5) and P. pygmaeus (8.1), respectively, in three multiplex polymerase chain reactions per species were amplified. All loci can be analysed in a single fragment analysis and can be used as markers to the study of evolution and the ecology of structured populations of socially living bats.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Development and characterization of SSR markers in Ribes species

Eleven microsatellite loci were identified and characterized in blackcurrant (Ribes nigrum L.) and related species. An enriched library was constructed and screened with simple sequence repeat (SSR) oligonucleotides. Positive clones were sequenced and primers were designed flanking the repeat motifs. These 11 microsatellites produce amplification products polymorphic across a range of Ribes germplasm, predominantly from the Eucoreosma section of the genus. The number of alleles varied from 2 to 18 with levels of diversity ranging from 0.18 to 0.91.     

Isolation and characterization of microsatellites in Echinochloa (L.) Beauv. spp.

Five primer pairs were developed that amplify microsatellite loci in three agronomically important Echinochloa (L.) Beauv. species: E. colona (L.) Link, E. crus‐galli (L.) Beauv. and E. crus‐pavonis (Kunth) Schultes. The microsatellites were tested on 24 individuals representing three species collected in rice fields from different geographical regions and revealed 3–7 alleles per microsatellite. Gene diversity [1 ? Σp_ij²] for four polymorphic loci within E. crus‐galli ranged from 0.12 to 0.61. Alleles at a fifth locus were useful in discriminating the species. The microsatellites should provide useful markers for intraspecific diversity studies and aid classification of species within this complex genus.     

Characterization of sequence polymorphisms from microsatellite flanking regions in <Emphasis Type="Italic">Vitis</Emphasis> spp

Single nucleotide polymorphisms or SNPs are the most abundant form of genetic variation in the genome of plants and animals. Microsatellites are hypervariable regions of genome, while their flanking regions are assumed to be as conserved as the average of the genome. In the present study, flanking sequences of 10 microsatellite loci were compared in different cultivars of Vitis to determine the existing polymorphism. For every microsatellite, about 8 homozygous cultivars (regarding the microsatellite genotype) were chosen for sequencing. A total of 45 different varieties of Vitis and 91 sequences were analysed. Sequence polymorphisms were detected for all the microsatellite flanking regions studied, including single nucleotide polymorphisms (SNPs), insertions and deletions. The number of identified changes varied considerably among the loci with a frequency of one polymorphism every 41 nucleotides, being VVMD5 the most polymorphic one. A number of SNPs were used to design SNP markers, which were scored by dideoxy single base primer extension and capillary electrophoresis methodology. These SNP markers were employed to genotype 21 cultivars of Vitis vinifera and 4 varieties of other Vitis species. The utility of the markers developed as well as their utility for varietal identification and pedigree studies is discussed, using a similar study carried out with the 10 microsatellites as a reference.     

A critical evaluation of reproductive barriers between closely related species using DNA markers - a case study in Pinus

Former controlled crosses between twelve Pinus montana var. rostrata (Pinus mugo complex) and eight P. sylvestris clones revealed that only two P. sylvestris had efficiently fertilised P. montana. Two species-diagnostic chloroplast DNA markers were applied to verify the species purity of the parental clones. All maternal P. montana were unambiguously confirmed to belong to the P. mugo complex at both chloroplast DNA marker loci. Six P. sylvestris clones carried the `sylvestris' haplotypes. However, the same two P. sylvestris clones that had efficiently fertilised P. montana displayed the chloroplast haplotypes diagnostic to the P. mugo complex. The patterns of highly polymorphic cpDNA microsatellite markers in parents and offspring ruled out contamination by foreign pollen. We concluded that the two clones successful in the crosses represent fertile hybrids between the two species with P. mugo as the pollen donor. Consequently, DNA markers are proposed for verifying or falsifying the success of artificial fertilisation in general. The existence of crossing barriers between the two Pinus species, meaningful to the postulated natural hybridisation and the evolution of their populations in sympatric stands, was indicated and is newly discussed.     

<Emphasis Type="Italic">Prunus</Emphasis> microsatellite marker transferability across rosaceous crops

A total of 145 microsatellite primer pairs from Prunus DNA sequences were studied for transferability in a set of eight cultivars from nine rosaceous species (almond, peach, apricot, Japanese plum, European plum, cherry, apple, pear, and strawberry), 25 each of almond genomic, peach genomic, peach expressed sequence tags (EST), and Japanese plum genomic, 22 of almond EST, and 23 of apricot (13 EST and 10 genomic), all known to produce single-locus and polymorphic simple-sequence repeats in the species where they were developed. Most primer pairs (83.6%) amplified bands of the expected size range in other Prunus. Transferability, i.e., the proportion of microsatellites that amplified and were polymorphic, was also high in Prunus (63.9%). Almond and Japanese plum were the most variable among the diploid species (all but the hexaploid European plum) and peach the least polymorphic. Thirty-one microsatellites amplified and were polymorphic in all Prunus species studied, 12 of which, covering its whole genome, are proposed as the “universal Prunus set”. In contrast, only 16.3% were transferable in species of other Rosaceae genera (apple, pear, and strawberry). Polymorphic Prunus microsatellites also detected lower levels of variability in the non-congeneric species. No significant differences were detected in transferability and the ability to detect variability between microsatellites of EST and genomic origin.     

Polymorphic dinucleotide microsatellite loci in the sandfly Lutzomyia longipalpis (Diptera: Phlebotominae)

The sandfly Lutzomyia longipalpis, an important vector of visceral leishmaniasis in the New World, is believed to be a species complex. In an effort to better understand population dynamics and speciation in this vector we developed a panel of dinucleotide — (CA)_n— microsatellite loci using an enrichment technique. Eleven polymorphic loci that produced consistent allelic banding patterns were characterized using a laboratory population of L. longipalpis. These dinucleotide microsatellite loci were more polymorphic than trinucleotide microsatellites characterized in wild‐caught samples of two other sandfly species; the variability of these loci was unexpected because the laboratory flies were believed to be inbred.     

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

Microsatellite loci were isolated in Haliotis fulgens using a (CT)_n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)_n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.