Simple Sequence Repeats Repeat Research Articles

Clonal structure and recruitment in British wild cherry ( Prunus avium L.)

The extent of clonal reproduction may significantly influence the genetic structure and reproductive ability of wild cherry ( Prunus avium L.) populations. In this study we used simple sequence repeat (SSR) variation to examine the relative frequency and distribution of vegetatively derived wild cherry in two ancient woodlands subject to contrasting management regimes. The probability of identical genotypes arising through sexual reproduction was determined to be negligible, both statistically and through screening a large population of known full-siblings. Only 246 genotypes were identified in the 551 trees examined from the natural populations. Thus, asexual recruitment accounted for a significant proportion of trees. Forty-five genets with multiple clonal trees (clonal groups) were identified. Clonal trees accounted for approximately 48% and 65% of all trees in the managed and unmanaged sites, respectively. Wild cherry trees were distributed in an aggregated manner, but to a stronger degree in the unmanaged woodland which was characterised by a disrupted canopy layer created by storm damage. However, clonal groups were generally non-exclusive in their distribution and displayed a ‘guerrilla’ strategy where further single and multi-ramet genotypes commonly shared the territory of the dominant clone. The clonal groups at the managed site were more exclusive than those in the unmanaged site. Evidence of somatic mutation occurring within clonal groups (i.e. where neighbouring trees matched at all except one locus and the disparity between groups was restricted to a single SSR repeat unit) was also observed, adding further complexity to the clonal structure of wild cherry. Natural disturbance and woodland management appear to influence clonal structure at this study site. Clonal genets in the unmanaged site contained significantly higher numbers of significantly smaller ramets and, on average, covered less than half the area of clonal groups at the managed site. Recruitment, both asexual and sexual, was also markedly higher at the unmanaged site and disproportionately high in years following major winter storms and attendant disturbance of the canopy. Whilst high levels of clonality were observed in both sites it is likely that self-incompatibility and the ‘guerrilla’ strategy of clonal growth adopted by wild cherry enable relatively high levels of genetic diversity to be maintained. Our study suggests that a policy of non-management, especially following winter storms, may help maintain genetic diversity through increased levels of both sexual and asexual recruitment in wild cherry.

Usefulness of SSR Derived from Tetraploid Gossypium spp. for Analyses of Diploid Gossypium spp.

This investigation is the first attempt to utilize tetraploid species-derived primers to generate markers for mapping in diploid cotton species. About 113 individual F2 plants, developed from the cross between Gossypium nelsoni and G. australe, were used to construct a linkage map. One allotetraploid (ADj) G. hirsutum (TM-1) plant and its putative diploid ancestors, G. arboreum (A2) and G. raimondii (D5), were also included for comparative analysis. We developed a high-throughput multi-mix PCR assay for simple sequence repeat (SSR) marker separation using fluorescent-labeled capillary electrophoresis. Of a total of 205 tetraploid-derived SSR primers, 83% amplified in all four diploid species, indicating that flanking primer sequences are conserved in the diploid and tetraploid ADj genome of cotton. DNA sequence information of 18 randomly selected clones revealed that all of the clones contained SSR repeat motifs in the diploid species, suggesting that SSR markers including the flanking primer sequences have been conserved at the inter-specific level in genomic DNA across years of evolution. About 85% of the amplified primers showed polymorphism among the diploid genome species. Forty percent of SSRs in the diploid G. nelsoni and 35% in G. australe amplified the same size DNA fragment as in the tetraploid, (TM-1). This is further evidence that many of the tetraploid SSRs are conserved across diploid and tetraploid species. G. nelsoni compared with G. australe had more conserved genomic regions in common with G. hirsutum. From 73 markers scored in the F2 population, 50 exhibited codominance and 23 dominance. There were 47 markers linked across 10 linkage groups covering a total map distance of 754 cM with an average of 16 cM per marker. One linkage group (lg-3) with six loci has been assigned to chromosome 20. Comparative analysis of DNA sequences between G. hirsutum and its two putative parental diploid species indicated that changes within a few selected clones have occurred during evolution as follows: (1) by increasing or decreasing the number of the repeat motif, (2) by single nucleotide mutations occurring within or outside of the repeat motif, (3) by inter conversion of a simple to a compound SSR motif, and (4) by inversion of the repeat regions. Tetraploid-derived microsatellites should be useful in comparative genetic mapping of both tetraploid and diploid Gossypium species for evolutionary studies and introgression of agriculturally important traits from exotic diploid and tetraploid germplasm sources.

SSRPrimer and SSR Taxonomy Tree: Biome SSR discovery

Simple sequence repeat (SSR) molecular genetic markers have become important tools for a broad range of applications such as genome mapping and genetic diversity studies. SSRs are readily identified within DNA sequence data and PCR primers can be designed for their amplification. These PCR primers frequently cross amplify within related species. We report a web-based tool, SSR Primer, that integrates SPUTNIK, an SSR repeat finder, with Primer3, a primer design program, within one pipeline. On submission of multiple FASTA formatted sequences, the script screens each sequence for SSRs using SPUTNIK. Results are then parsed to Primer3 for locus specific primer design. We have applied this tool for the discovery of SSRs within the complete GenBank database, and have designed PCR amplification primers for over 13 million SSRs. The SSR Taxonomy Tree server provides web-based searching and browsing of species and taxa for the visualisation and download of these SSR amplification primers. These tools are available at .

Development of simple sequence repeat (SSR) markers and construction of an SSR-based linkage map in Italian ryegrass (Lolium multiflorum Lam.)

In order to develop simple sequence repeat (SSR) markers in Italian ryegrass, we constructed a genomic library enriched for (CA)n-containing SSR repeats. A total of 1,544 clones were sequenced, of which 1,044 (67.6%) contained SSR motifs, and 395 unique clones were chosen for primer design. Three hundred and fifty-seven of these clones amplified products of the expected size in both parents of a two-way pseudo-testcross F(1) mapping population, and 260 primer pairs detected genetic polymorphism in the F(1) population. Genetic loci detected by a total of 218 primer pairs were assigned to locations on seven linkage groups, representing the seven chromosomes of the haploid Italian ryegrass karyotype. The SSR markers covered 887.8 cM of the female map and 795.8 cM of the male map. The average distance between two flanking SSR markers was 3.2 cM. The SSR markers developed in this study will be useful in cultivar discrimination, linkage analysis, and marker-assisted selection of Italian ryegrass and closely related species.

Transferability of SSR markers from lychee (Litchi chinensisSonn.) to pulasan (Nephelium ramboutan-akeL.)

Introduction. Pulasan and lychee are from the same family and closely related, therefore the SSR markers are expected to be highly transferable between these two taxa. We investigated the transferability of 12 lychee (Litchi chinensis Sonn.) simple sequence repeat (SSR) loci to pulasan (Nephelium ramboutan-ake L.). Materials and methods. Genomic DNA was extracted from 20 accessions of pulasan for the PCR amplification of the SSR loci using 12 pairs of SSR primers derived from lychee. The PCR products were resolved on dena- turing polyacrylamide gels. Results. The percentage of SSR transferability from lychee to pulasan was 58.3% and the percentage of polymorphic SSR markers was 25%. Discussion. The moderate transferability and low polymorphism rates suggest the possibility of interrup- tions within the repeat motif and mutations in the flanking sequences of SSR repeat motifs. Our results did not reveal a high rate of transferability between lychee and pulasan. However, this study showed that the SSR markers developed in lychee are a good source of molecular markers for pulasan. Malaysia / Litchi chinensis / Nephelium ramboutan-ake / molecular biology / genetic markers / PCR / genetic polymorphism

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.)

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.

Microsatellite markers from sugarcane ( Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane ( Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG) n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera ( Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.

A simple sequence repeat-based linkage map of barley.

A total of 568 new simple sequence repeat (SSR)-based markers for barley have been developed from a combination of database sequences and small insert genomic libraries enriched for a range of short simple sequence repeats. Analysis of the SSRs on 16 barley cultivars revealed variable levels of informativeness but no obvious correlation was found with SSR repeat length, motif type, or map position. Of the 568 SSRs developed, 242 were genetically mapped, 216 with 37 previously published SSRs in a single doubled-haploid population derived from the F(1) of an interspecific cross between the cultivar Lina and Hordeum spontaneum Canada Park and 26 SSRs in two other mapping populations. A total of 27 SSRs amplified multiple loci. Centromeric clustering of markers was observed in the main mapping population; however, the clustering severity was reduced in intraspecific crosses, supporting the notion that the observed marker distribution was largely a genetical effect. The mapped SSRs provide a framework for rapidly assigning chromosomal designations and polarity in future mapping programs in barley and a convenient alternative to RFLP for aligning information derived from different populations. A list of the 242 primer pairs that amplify mapped SSRs from total barley genomic DNA is presented.

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.

Isolation and characterization of simple sequence repeat loci in the gray tree frog, Hyla chrysoscelis.

A gray tree frog (Hyla chrysoscelis) genomic library was constructed and characterized with regard to the incidence and complexity of simple sequence repeat (SSR) loci. The partial genomic library, containing approximately 10,000 clones with an average-sized insert of 350 bp, was screened with six SSR repeat oligonucleotides (AC, AG, ACG, AGC, AAC, and AAG). Screening identified 31 unique positive clones containing 41 SSR loci. Sequences of tandemly arrayed dinucleotide repeats were more common (36 of 41) than trinucleotide repeats. Twenty-six loci were identified using the AC dinucleotide probe, while 7 loci were identified using the AG dinucleotide probe. An additional 3 AT dinucleotide loci were serendipitously identified. The AT repeats generally comprised the longest dinucleotide repeat loci. The SSR repeat loci reported here should provide potent markers for identity, parentage, and short-lineage determinations in large-scale experiments using gray tree frogs.