Inter-simple sequence repeat-restriction fragment length polymorphisms for DNA fingerprinting.
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Animals↗
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Results for “simple sequence repeat”
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Use of (CA)n polymorphisms to determine the origin of blood cells after allogeneic canine marrow grafting.
We have used a polymerase chain reaction-based assay measuring polymorphic (CA)n repeats, a class of simple sequence repeats, to assess the success of allogeneic canine marrow transplants. Results were compared with those obtained with karyotype analysis of dividing cells in recipients that were sex mismatched with their marrow donors. Twenty recipients were conditioned for transplantation of genotypically DLA-identical littermate marrow by 450 cGy of total-body irradiation. In 2 recipients, results could not be compared, since either only cytogenetic or dinucleotide (CA)n marker data existed. Both dogs had autologous marrow recovery. In 15 of the remaining 18 recipients, complete agreement was found between the results obtained with dinucleotide (CA)n markers, cytogenetic studies, and granulocyte changes after transplantation. Seven of the 15 showed eventual autologous recovery, 6 displayed mixtures of host and donor cells, and 2 showed donor-type hematopoiesis. Two of the 18 dogs showed mixed chimerism with (CA)n markers and autologous recovery by cytogenetics, findings that may be related to differences in cells analyzed by the two techniques--i.e., all nucleated cells by (CA)n markers versus dividing cells by cytogenetics. In one additional recipient, results of marrow cytogenetics, granulocyte changes, and (CA)n markers were consistent with a successful allograft, while peripheral blood cytogenetics suggested autologous recovery, possibly the result of erroneous blood sampling. Polymerase chain reaction-based testing for dinucleotide repeat (CA)n polymorphisms, originally developed for genetic mapping in the dog, is useful and reliable when compared with cytogenetic studies, in assessing the success of allogeneic marrow transplants in dogs.
Animals↗
Development and transferability of apricot and grape EST microsatellite markers across taxa.
EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.
Base Sequence↗
Mono-nucleotide repeats (MNRs): a neglected polymorphism for generating high density genetic maps in silico.
Short, tandemly repeated DNA motifs, termed SSRs (simple sequence repeats) are widely distributed throughout eukaryotic genomes and exhibit a high degree of polymorphism. The availability of size-based methods for genotyping SSRs has made them the markers of choice for genetic linkage studies in all higher eukaryotes. These genotyping methods are not efficiently applicable to mononucleotide repeats (MNRs). Consequently, MNRs, although highly frequent in the genome, have generally been ignored as genetic markers. In contrast to single nucleotide polymorphisms (SNPs), SSRs can be identified in silico once the genomic sequence or segment of interest is available, without requiring any additional information. This makes possible ad-hoc saturation of a target chromosomal region with informative markers. In this context, MNRs appear to have much to offer by increasing the degree of marker saturation that can be obtained. By using the human genome sequence as a model, computational analysis demonstrates that MNRs in the size of 9-15 bp are highly abundant, with an average appearance every 2.9 kb, exceeding di- and tri-nucleotide SSRs frequencies by two- and five-fold, respectively. In order to enable practical, high throughput MNR genotyping, a rapid method was developed, based on sizing of fluorescent-labeled primer extension products. Genotyping of 16 arbitrarily chosen non-coding MNR sites along human chromosome 22 revealed that almost two-thirds (63%) of them were polymorphic, having 2-5 alleles per locus, with 20% of the polymorphic MNRs having more than two alleles. Thus, MNRs have potential for in silico saturation of sequenced eukaryote genomes with informative genetic markers.
Chromosome Mapping↗
Organization of immunoglobulin heavy chain constant and joining region genes in the channel catfish.
A channel catfish genomic lambda library was screened with CH and JH probes which were derived from our earlier sequence analyses on different full-length heavy chain cDNA clones. One clone, designated C7, contained a genomic insert of about 18 kb and hybridized with specific probes for each of the four domains of the known C region gene as well as with different oligonucleotides specific for JH gene segments. Southern blot hybridization analysis identified a cluster of JH gene segments which are closely linked to the CH gene. Sequence analysis of the CH-proximal JH element, located about 1.9 kb upstream from the CH1 domain, showed that this element contains 5'-recombination signals typical of JH elements defined in higher vertebrates, i.e. a nonamer, a 24 bp spacer, and a heptamer. The coding region of this JH element was identical to that contained in the variable region sequence of a cDNA clone previously reported. Sequence analysis of the catfish JH-CH intron suggests that several sequences are present which appear similar to important transcriptional regulatory elements found within JH-CH introns of higher vertebrates. These features include sequences similar to higher vertebrate enhancer elements and regulatory octamers. An additional feature reminiscent of some higher vertebrate heavy chain switch regions is a repetitive sequence area composed of tandemly repeated simple sequences. Lastly, several restriction length polymorphisms were identified and mapped within a 1 kb region located immediately upstream from the JH cluster. This finding suggests that polymorphisms within the IgH locus should be useful in the analyses of channel catfish populations. These combined studies provide further evidence that the genomic organization of heavy chain genes in bony fish shares common organizational features with those known from higher vertebrates.
Amino Acid Sequence↗
Cloned microsatellite repeats differ between 4-base restriction endonucleases.
Simple sequence repeat (SSR) loci are an important marker type for population genetic studies despite the limitation that development of novel loci requires construction and screening of genomic DNA libraries. The common practice of size fractioning genomic DNA before cloning could lead to differential representation of SSR loci within genomic libraries. In addition, linkage mapping studies have shown that small numbers of SSR markers are not randomly distributed within the genomes from which they are isolated. From attempts to clone five SSR repeat sequences in two wild plant species we show that the numbers and repeat type of potential SSR markers depend on the restriction endonuclease used to sample the genome when constructing DNA libraries. This observation is consistent with unequal sampling of the genome by different restriction enzymes. However, as a group the five SSR repeat sequences are not associated with a given restriction enzyme, suggesting they are not clumped within the genome. Use of multiple restriction enzymes to construct DNA libraries may help ensure that cloned SSR loci are drawn from diverse locations in the genome, helping to meet the assumption of randomly located marker loci required for population genetic inferences.
Arabidopsis↗
The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions.
The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.
Animals↗
Mapping maize microsatellites and polymerase chain reaction confirmation of the targeted repeats using a CT primer.
Microsatellites, also called simple sequence repeats (SSRs), have yielded an important class of DNA markers most notable for mapping mammalian genomes. To study the occurrence of microsatellites and their inheritance in maize, a search was made of 280 maize GenBank sequences. Six SSRs were chosen and unique flanking primers were designed for polymerase chain reaction (PCR) amplification. Eight different maize inbreds were studied with these six primer pairs and a mean of 3.5 polymorphic patterns occurred within the expected size range. For five of these putative microsatellites, the segregation in a maize restriction fragment length polymorphism mapping population was analyzed. Four of the microsatellites cosegregated with the Adh1, Gpc1, Pdk1, and Tpi genes from which the primer sequences were derived. The fifth primer pair (MZEGPA1) showed segregating polymorphisms, but the products were larger than expected. To verify the existence of the original SSRs in the segregating PCR products, a CT primer, containing a CT SSR and an arbitrary leader sequence, was used to reamplify these products. The four microsatellites that cosegregated with the original gene were reamplified as anticipated, whereas a suspicious 230-bp product obtained when using the MZEGPA1 primers could not be reamplified. Based on these results it is concluded that microsatellites can be a valuable tool for maize mapping.
Base Sequence↗
Double-stranded RNA as a not-self alarm signal: to evade, most viruses purine-load their RNAs, but some (HTLV-1, Epstein-Barr) pyrimidine-load.
For double-stranded RNA (dsRNA) to signal the presence of foreign (non-self) nucleic acid, self-RNA-self-RNA interactions should be minimized. Indeed, self-RNAs appear to have been fine-tuned over evolutionary time by the introduction of purines in clusters in the loop regions of stem-loop structures. This adaptation should militate against the "kissing" interactions which initiate formation of dsRNA. Our analyses of virus base compositions suggest that, to avoid triggering the host cell's dsRNA surveillance mechanism, most viruses purine-load their RNAs to resemble host RNAs ("stealth" strategy). However, some GC-rich latent viruses (HTLV-1, EBV) pyrimidine-load their RNAs. It is suggested that when virus production begins, these RNAs suddenly increase in concentration and impair host mRNA function by virtue of an excess of complementary "kissing" interactions ("surprise" strategy). Remarkably, the only mRNA expressed in the most fundamental form of EBV latency (the "EBNA-1 program") is purine-loaded. This apparent stealth strategy is reinforced by a simple sequence repeat which prefers purine-rich codons. During latent infection the EBNA-1 protein may evade recognition by cytotoxic T-cells, not by virtue of containing a simple sequence amino acid repeat as has been proposed, but by virtue of the encoding mRNA being purine-loaded to prevent interactions with host RNAs of either genic or non-genic origin.
Animals↗
A transcriptome-wide approach for rapid pathotype discrimination of Puccinia striiformis f. sp. tritici in north-western India.
Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici (Pst) remains a major constraint to wheat production in India due to the rapid evolution and frequent emergence of virulent pathotypes. Rapid and reliable discrimination of Pst pathotypes is essential for effective resistance deployment and surveillance. In the present study, transcriptome-wide simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were exploited to develop and validate molecular markers for pathotype-specific detection of Pst pathotypes prevalent in North India (110S119, 238S119, 46S119, 110S84 and 78S84). Microsatellite mining from 6103 core orthologous clusters comprising 51,127 transcripts mined 14,634 SSR loci, from which 93 primer pairs were synthesized. However, only three SSR markers exhibited polymorphism indicating limited discrimination potential of expressed sequence-derived (EST) SSRs for pathotype differentiation. In contrast, SNP discovery through stringent variant calling and filtration yielded 186 pathotype-specific homokaryotic SNPs, of which 56 high-confidence loci were selected for Kompetitive Allele-Specific PCR (KASP) assay development. A total of 48 KASP markers were synthesized and 14 demonstrated clear pathotype- or cluster-specific polymorphism representing substantially higher resolution than SSR markers. The high SNP-to-KASP conversion efficiency (~ 95%) and reproducible fluorescence-based clustering emphasize the robustness of KASP assay. Comparative evaluation revealed that SNP-based KASP markers provide superior discriminatory capacity for closely related Pst pathotypes and represent a promising complementary molecular approach for rapid identification of predominant Indian Pst pathotypes. The validated marker panel developed in this study can complement conventional virulence phenotyping and field pathogenomics approaches for surveillance of currently known pathotypes, while continued refinement may accommodate future changes in pathogen populations.
India↗
SSR marker development for analysis of the genetic diversity and identification of species and infraspecific ranks in the genus Phyllostachys.
Bamboo plants possess important ecological, economic, and cultural values. However, it is difficult to accurately identify them on the basis of their morphological traits alone. Here, based on the whole-genome data of moso bamboo (Phyllostachys edulis) and its 20 forms, we conducted preliminary identification and comparative analyses of simple sequence repeats (SSRs) to develop molecular markers. In total, 3,835,632 SSR loci were identified from 31,537.81 Mb of genomic sequences, among which dinucleotide SSRs were the most abundant. Most SSRs were located in intergenic regions, whereas relatively fewer were in genic regions. In addition, we found that SSR-containing genes involved in plant hormone signal transduction may be associated with the morphogenesis of moso bamboo, which was speculated to be related to differential gene expression patterns among different forms. Furthermore, 206 SSR primer pairs with polymorphisms were obtained to analyse the genetic diversity of moso bamboo and its forms, which exhibited moderate polymorphism. The proportion of genetic variation among species within the genus Phyllostachys was 58%, while that within species was 42%. Moso bamboo and its 20 forms had relatively close genetic relationships and low genetic differentiation, while 20 species of the genus Phyllostachys were clustered into three groups with distinct levels of genetic diversity. Finally, DNA fingerprints and molecular identity cards were constructed for 20 moso bamboo forms and 20 species of the genus Phyllostachys using core SSR markers. These results provide novel SSR markers for bamboo identification, germplasm conservation, and molecular marker-assisted breeding.
Microsatellite Repeats↗
High-resolution genetic analysis of the Sd-1 aphid resistance locus in Malus spp.
Aphids cause serious physical and economic damage to most major crops throughout the world through feeding damage, with consequent symptom development and virus transmission. The rosy leaf-curling aphid ( Dysaphis devecta Wlk.) is a pest of apple ( Malus spp.) which displays an exceptionally clear phenotype with respect to susceptible and resistant symptoms. The Sd-1 locus for resistance to D. devecta biotypes 1 and 2 is present in Cox's Orange Pippin and its progeny and had previously been mapped to the top of linkage group 7. Detailed fine mapping of the locus was initiated with AFLP bulked segregant analysis of both pedigree and segregating bulks, which identified three new marker loci. Preliminary marker order in the Sd-1 region was established through mapping in a family derived from Prima x Fiesta, with additional segregation analysis on a Fiesta x Golden Delicious family. Previous recombinant data was re-evaluated and corrected. Two co-segregating AFLP fragments were found to contain a common (GA)(23) repeat, from which a PCR-based simple sequence repeat (SSR) assay was developed. A high-resolution map around the Sd-1 region was established by analysing a large meta-population of Sd-1 recombinants using 759 additional individuals from different families. The Sd-1 gene has been located within a 1.3-cM interval flanked by the molecular markers SdSSRa and 2B12a and co-locates with the RFLP marker MC064. Allelism between Sd-1 and Sd-2 resistant sources was tested. Molecular markers tightly linked to Sd-1 were shown to be co-segregating with the Sd-2 locus, which indicated that Sd-1 and Sd-2 loci are at least tightly linked and, probably, allelic.
Journal Article↗
Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis.
Spontaneous neurologic mutations in the mouse provide powerful tools for the study of mammalian central nervous system development. The study of mouse neurologic mutants has led to a better understanding of the complex mechanisms involved in the development of the nervous system. Because few of these mutations have been identified, molecular probes distinguishing heterozygotes from homozygotes are generally unavailable. Further, most neurologic mouse mutants breed poorly as homozygotes, making it necessary to breed heterozygotes and select homozygous mutant progeny based on phenotype. The requirement for heterozygous breeding and the lack of molecular markers specific for the mutation have hampered developmental studies because the underlying neurologic perturbations occur before the mutant mice can be identified by phenotype. The recent identification and chromosomal assignment of simple sequence repeats (SSRs), repetitive sequences of DNA found at a high density throughout the mouse genome, provide the tools for mapping mutations in the mouse and for subsequent genotyping of potential mutants prior to phenotype onset. The SSRs are useful because these markers are polymorphic (for review see Weber, J.L., Human DNA polymorphisms based on length variations in simple-sequence tandem repeats. In: K.E. Davies and S.M. Tilghman (Eds.), Genetic and Physical Mapping. Genome Analysis, Vol. I, Cold Spring Harbor Laboratory Press, Plainview, NY, 1990, pp. 159-181 [16]), that is, the size of the individual SSRs differs among strains of mice. Following polymerase chain reaction (PCR) amplification of an SSR and separation of PCR products by polyacrylamide gel electrophoresis, one can easily visualize differences in the size of the PCR product between mouse strains. Many mutations in the mouse arose spontaneously on inbred strains and were subsequently backcrossed onto a different strain. After many generations of congenic backcrosses, the only DNA retained from the original mutant strain is composed of the mutant gene and closely linked regions. Thus, it is possible to cross the mutant strain to a different mouse strain and map the mutation by correlating mutant phenotype to SSRs the same size as the original mutant strain. We have mapped the tottering (tg), Purkinje cell degeneration (pcd), and nervous (nr) mutations using SSRs in backcrossed mouse strains. The SSRs distinguishing mutant from normal strains can then be used to genotype potential mutant pups before the onset of the mutant phenotype. The protocol described below can be adapted to almost any mutation congenically inbred for genotyping. Here we describe a method for selecting primers appropriate for genotyping potential mouse mutants and a rapid protocol for genotype screening. Even with SSRs distinguishing mutant from normal mice, genotyping several mice simultaneously can be a daunting task. This is primarily because the protocols available for preparing DNA for PCR amplification are time-consuming, requiring several purification steps including phenol extractions. Although kits are commercially available for DNA preparation without organic extractions, these kits tend to be expensive. The protocol described is a rapid, inexpensive method of determining the genotype of mice using PCR analysis of dried blood spots. The protocol only requires PCR primers distinguishing among alleles and is therefore ideal for the rapid identification of potential mutants for those mouse mutations which have been mapped using microsatellite markers. The DNA preparation protocol may also be used in rapid screening of potential transgenic mice.
Animals↗
Patterns of CAG repeat interruptions in SCA1 and SCA2 genes in relation to repeat instability.
About 3% of the human genome is composed of simple sequence repeats and many of these sequences occur within genes. These repeats are often polymorphic in a normal population and their expansion in specific genes leads to a number of hereditary neurological diseases. Normal variants of disease-related genes contain either pure or interrupted repeats, and the postulated function of the interruptions is to prevent repeat expansions. Their structural role in the repeat tracts of genes and transcripts awaits detailed characterization. In this study, we have determined the SCA1 and SCA2 genotypes in a Polish population and found significant differences in allele spectra and frequencies from those reported for other populations. They are discussed in relation to the repeat expansion mechanism and disease incidence. We postulate that the dynamic mutation of the genes SCA1 (also ATX1 or ataxin 1) and SCA2 (also ATX2 or ataxin 2) may begin from the expansion of long pure repeat tracts without the prior loss of interruptions. A simple way of cost-effective allelotyping of CAG repeat regions in the SCA1 and SCA2genes is also shown. The reliable SSCP/duplex analysis presented here may be the method of choice for the systematic searching of genes for known and novel interrupted repeats.
Alleles↗
Development and validation of whole-genome SSR markers in sugar beet (Beta vulgaris L.).
Sugar beet (Beta vulgaris L.) is an important sugar and cash crop worldwide. To systematically characterize SSR (Simple Sequence Repeat) loci across sugar beet chromosomes and enable the precise identification of germplasm resources, this study conducted a genome-wide scan for SSR loci, analyzed their distribution patterns, and determined their genotypes using resequencing data from 123 sugar beet varieties. The results revealed an abundance of SSR loci in the sugar beet genome, with a total of 135, 379 identified, from which 135, 344 pairs of SSR primers were designed (135, 344 primer pairs successfully designed; 35 loci failed to meet design criteria). Specifically, 31, 748 primer pairs were designed based on SSRs located in unassigned scaffolds, and 103, 596 primer pairs from SSRs assigned to the nine chromosomes. Through bioinformatic analysis, we identified 28, 768 SSR primers located in multi-copy genes with PIC (Polymorphism Information Content) ≥ 0.5, and 2, 326 SSR markers located in single-copy genes residing in various genic regions (among which 543 had PIC ≥ 0.5, with the highest reaching 0.776). PCR (Polymerase Chain Reaction) validation confirmed 20 robust and polymorphic markers producing clear and reproducible bands. Among them, 10 SSR primers located in multi-copy genes exhibited three or more polymorphic types, and 10 markers located in single-copy genes displayed 2-3 polymorphic types. The most polymorphic marker, YCD-4-2, detected 11 polymorphic types across 48 varieties. Furthermore, to explore markers with potential functional significance, we annotated the genes harboring SSR markers located in single-copy genes. The results showed that 1, 264 SSRs located in single-copy genes were localized to 967 genes, which are significantly enriched in pathways related to carbohydrate metabolism, stress responses, and plant-pathogen interactions. The 20 validated markers and the 2, 326 SSRs located in single-copy genes provided in this study can be directly applied to fingerprinting of sugar beet varieties, seed purity testing, and marker-assisted selection, thus representing a practical resource for molecular breeding.
genome-wide↗
Intimate association of microsatellite repeats with retrotransposons and other dispersed repetitive elements in barley.
Simple sequence repeat (SSR)-based genetic markers are being actively developed for the majority of crop plant species. In barley, characterization of 290 dinucleotide repeat-containing clones from SSR-enriched libraries has revealed that a high percentage are associated with cereal retrotransposon-like and other dispersed repetitive elements. Associations found were with BARE-1, WIS2-1A, PREM1 and the dispersed repetitive element R173. Additional similarities between different SSR clones, which have no matches in DNA sequence databases, indicate that this phenomenon is probably widespread in the barley genome. Sequence homologies to the non-coding regions of several cereal genes were also explained by homology to mobile genetic elements. The SSRs found can therefore be classified into two types: (1) those with unique sequences on either flank, and (2) those which are intimately associated with retro-transposons and other dispersed repetitive elements. As the cereal genome is thought to consist largely of this type of DNA, some random association would be expected. However, the conserved positions of the SSRs, relative to repetitive elements, indicate that they have arisen non-randomly. Furthermore, this class of SSRs can be classified into three subtypes: (1) those which are positioned 3' of a transposable element with unique sequence on the other flank, (2) those positioned 5' of a transposable element, and (3) those which have arisen from an internal sequence and so have transposable element sequence on both flanks. The first appear to be analogous to the class of SSRs in mammalian systems which are associated with Alu elements and SINEs (short interspersed elements) and which have been postulated to arise following integration of an extended and polyadenylated retro-transcript into the host genome, followed by mutation of the poly(A) tract and expansion into an SSR. For the second, we postulate that a proto-SSR (A-rich sequence) has acted as a 'landing pad' for transposable element insertion (rather than being the result of insertion), while the third includes those which have evolved as a component of an active transposable element which has spread throughout the genome during bursts of transposition activity. The implications of these associations for genome and SSR evolution in barley are discussed.
Base Sequence↗
TROLL--tandem repeat occurrence locator.
SUMMARY: Tandem Repeat Occurrence Locator (TROLL), is a light-weight Simple Sequence Repeat (SSR) finder based on a slight modification of the Aho-Corasick algorithm. It is fast and only requires a standard Personal Computer (PC) to operate. We report running times of 127 s to find all SSRs of length 20 bp or more on the complete Arabdopsis genome--approx. 130 Mbases divided in five chromosomes--using a PC Athlon 650 MHz with 256 MB of RAM. AVAILABILITY: TROLL is an open source project and is available at http://finder.sourceforge.net.
Algorithms↗
Cross-species transferability and mapping of genomic and cDNA SSRs in pines.
Two unigene datasets of Pinus taeda and Pinus pinaster were screened to detect di-, tri- and tetranucleotide repeated motifs using the SSRIT script. A total of 419 simple sequence repeats (SSRs) were identified, from which only 12.8% overlapped between the two sets. The position of the SSRs within their coding sequences were predicted using FrameD. Trinucleotides appeared to be the most abundant repeated motif (63 and 51% in P. taeda and P. pinaster, respectively) and tended to be found within translated regions (76% in both species), whereas dinucleotide repeats were preferentially found within the 5'- and 3'-untranslated regions (75 and 65%, respectively). Fifty-three primer pairs amplifying a single PCR fragment in the source species (mainly P. taeda), were tested for amplification in six other pine species. The amplification rate with other pine species was high and corresponded with the phylogenetic distance between species, varying from 64.6% in P. canariensis to 94.2% in P. radiata. Genomic SSRs were found to be less transferable; 58 of the 107 primer pairs (i.e. 54%) derived from P. radiata amplified a single fragment in P. pinaster. Nine cDNA-SSRs were located to their chromosomes in two P. pinaster linkage maps. The level of polymorphism of these cDNA-SSRs was compared to that of previously and newly developed genomic-SSRs. Overall, genomic SSRs tend to perform better in terms of heterozygosity and number of alleles. This study suggests that useful SSR markers can be developed from pine ESTs.
Base Sequence↗