Efficient development of dinucleotide microsatellite markers in Norway spruce (Picea abies Karst.) through dot-blot selection (original) (raw)
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Tetranucleotide and Dinucleotide Microsatellite Markers for Red Spruce (Picea rubens
Three library enrichment protocols were used to develop tetra-and dinucleotide microsatellite markers for red spruce (Picea rubens). Two enrichment methods based on a single round of magnetic beads-based subtractive hybridization showed limited success. A third method with two successive rounds of subtractive hybridization had a high enrichment success but was found to increase the rate of redundancy and chimerical sequences among the clone sequences. Several single locus tetra-and dinucleotide markers were discovered and can be useful in mapping studies. All newly developed markers except one were characterized by a high frequency of null alleles. One new and one previously described microsatellite marker were identified as informative, reliable and characterized by a low null allele frequency, and were suitable for paternity/parentage and population genetic analyses in red spruce.
Theoretical and Applied Genetics, 2002
Trinucleotide microsatellites have proven to be the markers of choice in human genetic analysis because they are easier to genotype than dinucleotides. Their development can be more time-consuming due to their lower abundance in the genome. We isolated trinucleotide microsatellites in Norway spruce (Picea abies K.) using an enrichment procedure for the genomiclibrary construction. Here we report on the characterisation of 85 ATC microsatellite-containing clones, from which 39 markers were developed. Many of the clones showed the occurrence of tandem repeats of higher order than the trinucleotide ones, often resembling minisatellite repeats. The sequencing of a sample of the alleles at one of the loci revealed size homoplasy due to base substitutions within the microsatellite region. The presence of ATC motifs within repetitive sequence families was observed. We found a significant relationship between the level of polymorphism and the length of the microsatellite. The levels of variability for ATC trinucleotide markers were lower than those for dinucleotides, both when tested on all loci in a set of six individuals and on a subset of loci in four natural populations. This difference is most likely attributable to lower mutation rates for trinucleotide than for dinucleotide loci. The availability of markers with different mutation rates allows one to select the proper marker set to investigate population processes on different time scales.
Molecular Ecology Notes, 2005
The spruce ( Picea ) species are ecologically and economically important in Canada. Highly informative markers with high multiplex ratios are needed to assist spruce genomics, genetics, and breeding programs. Selectively amplified microsatellite polymorphic loci (SAMPL) markers are highly suitable for these programs. We have developed, optimized, and characterized a set of 10 new SAMPL primers in combination with 16 Mse I primers and resolved a large number of polymorphic SAMPL markers in spruce. The SAMPL primers were designed from the compound microsatellite repeats found in Norway spruce ( Picea abies ) and white spruce ( Picea glauca ). A total of 6313 polymorphic SAMPL makers were produced by 160 SAMPL-Mse I primers combinations in eight progeny of a spruce mapping population.
PloS one, 2014
Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies. A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce...
Molecular Ecology Notes, 2004
Few microsatellite markers have been specifically developed for Picea sitchensis. In January 2004 the appearance of over 10 000 sequences of expressed regions of DNA from P. glauca in GenBank presented an opportunity for the development of additional microsatellite markers in Sitka spruce. Mono‐ and dinucleotide repeat sequences were located in these sequences and primers were designed around these regions. Amplification was attempted in Sitka material from a broad geographical range and the level of polymorphism was assessed. Primers were also tested in progeny of a controlled cross. Nine polymorphic loci that demonstrated Mendelian inheritance in Sitka were discovered in this study.
Tree Genetics & Genomes, 2005
In order to analyze the large-scale structure of the genome of Norway spruce (Picea abies Karst.), a pseudo-testcross genetic linkage map was built using markers of six different types, belonging to the low (amplified fragment length polymorphisms, simple sequence repeats) or high (sequence-specific amplified polymorphisms, inter-retrotransposon amplified polymorphisms) copy-number fraction of the genome, and including expressed region-derived markers (expressed sequence tag polymorphisms). Twenty seven and 23 linkage groups of at least four markers were obtained for the female and the male parent maps, respectively. A subset of these linkage groups coalesced into 13 bi-parental linkage groups through markers shared between the two maps. This map was used to investigate the frequency of each marker type over chromosomes and the distribution of marker types relative to each other, using autocorrelation techniques. Our results show that, while the composition of chromosomes is homogeneous, low-and high-copy-number markers tend to occupy separate regions of the linkage groups, and that expressed sequences are preferentially associated with microsatellites and separated from retrotransposons. These results indicate that the spatial structure of Norway spruce chromosomes is not homogeneous.
Annals of Forest Science, 2006
Four populations of Norway spruce (Picea abies Karst.) were screened using nine nuclear microsatellite markers (three trinucleotides and six dinucleotides) and four chloroplast markers (all mononucleotides). Marker classes were compared for their variability, mutation rate and ability to detect differentiation between stands. Dinucleotide markers proved to be the most variable group and chloroplast stretches the least variable, with differences in mutation rate between the former and the latter spanning over two orders of magnitude. Variability correlated to the number of repeats but not to the absolute length of the microsatellite region. The different marker classes were combined with two different measures of genetic distance in order to investigate the performance of markers and evolutionary models for the study of genetic variation in natural populations of Norway spruce. Weir and Cockeram's F ST generally performed better in this clear-cut, four-population model study. Chloroplast haplotypes turned out to be the most sensitive marker system, being able to differentiate populations and to detect differences in genetic variability between sub-regions.
Microsatellite DNA as shared genetic markers among conifer species
Canadian Journal of Forest Research, 1999
Polymerase chain reaction (PCR) primer pairs for 21 simple sequence repeat (SSR) loci in Pinus strobus L. and 6 in Pinus radiata D. Don. were evaluated to determine whether SSR marker amplification could be achieved in 10 other conifer species. Eighty percent of SSR primer pairs for (AC) n loci that were polymorphic in P. strobus also amplified SSR loci in two other soft pines of the subgenus Strobus but not in seven hard pines of the subgenus Pinus, nor in Picea glauca (Moench) Voss or Pseudotsuga menziesii (Mirb.) Franco. The six P. strobus SSR primer pairs that did amplify loci from conifers other than soft pines were those that were specific to loci monomorphic within P. strobus. These six loci were also monomorphic within seven other species tested, but four of the loci were polymorphic among species. A comparison of allelic variation among the three soft pine species found only 25 shared alleles among a total of 122 alleles at eight loci. Primer pairs for dinucleotide SSR loci that were polymorphic in Pinus radiata also specifically amplified loci from various other hard pines but not from the soft pines or from the other conifers tested.