“Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved in vernalization response successfully discriminated genotypes in absence of allelic sequence information, and allowed to determine allele segregation in VrnA. Here we introduce the concept of “blind” mapping based on HRM as a powerful, fast and cheap method to map any DNA sequence polymorphisms without prior knowledge of allelic sequences in the key grassland species perennial ryegrass (Lolium perenne L.).     

Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa

Background  

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass (Lolium perenne). We report on the exploitation of a comprehensive EST collection in L. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding.     

An SSR-based genetic linkage map for perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

A simple sequence repeat (SSR)-based linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross reference population. A total of 309 unique perennial ryegrass SSR (LPSSR) primer pairs showing efficient amplification were evaluated for genetic polymorphism, with 31% detecting segregating alleles. Ninety-three loci have been assigned to positions on seven linkage groups. The majority of the mapped loci are derived from cloned sequences containing (CA)_n-type dinucleotide SSR arrays. A small number (7%) of primer pairs amplified fragments that mapped to more than one locus. The SSR locus data has been integrated with selected data for RFLP, AFLP and other loci mapped in the same population to produce a composite map containing 258 loci. The SSR loci cover 54% of the genetic map and show significant clustering around putative centromeric regions. BLASTN and BLASTX analysis of the sequences flanking mapped SSRs indicated that a majority (84%) are derived from non-genic sequences, with a small proportion corresponding to either known repetitive DNA sequence families or predicted genes. The mapped LPSSR loci provide the basis for linkage group assignment across multiple mapping populations.     

Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.)

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.), with the aim of developing a comprehensive set of loci for trait mapping and cultivar identification. Two libraries were constructed showing greater than 50% enrichment for a variety of SSR-motif types. Sequence characterisation of 1853 clones identified 859 SSR-containing clones, of which 718 were unique. Truncation of flanking sequences limited potential primer design to 366 clones. One-hundred selected SSR primer pairs were evaluated for amplification and genetic polymorphism across a panel of diverse genotypes. The efficiency of amplification was 81%. A relatively high level of SSR polymorphism was detected (67%), with a range of 2–7 alleles per locus. Mendelian segregation of alleles detected by selected SSR-locus primer pairs was demonstrated in the F₁ progeny of a pair cross. Cross-species amplification was detected in a number of related pasture and turfgrass species, with high levels of transfer to other Lolium species and members of the related genus Festuca. The identity of putative SSR ortholoci in these related species was confirmed by DNA sequence analysis. These loci constitute a valuable resource of ideal markers for the molecular breeding of ryegrasses and fescues. Received: 8 May 2000 / Accepted: 13 June 2000     

Development and mapping of a public reference set of SSR markers in Lolium perenne L.

We report on the characterization and mapping of 76 simple sequence repeat (SSR) markers for Lolium perenne. These markers are publicly available or obtained either from genomic libraries enriched for SSR motifs or L. perenne expressed sequence tag (EST) clones. Four L. perenne mapping populations were used to map the SSR markers. A consensus linkage map of the four mapping populations containing 65 of the SSR markers is presented, together with primer information and a quality score indicating the usefulness of the SSR marker in different populations. The SSR markers identified all seven L. perenne linkage groups.     

华仁杏EST-SSR标记引物设计与开发

随着生物科技的进步,ESTs（表达序列标签）已经成为开发SSR（简单重复序列）标记的重要资源。本文利用NCBI公共数据库下载蔷薇科EST序列22 458条,使用SSRHunter1.3软件进行了SSR搜索,从中获得22 527条SSR,应用Primer5.0软件设计并经由Oligo7.0软件检测,共得到61对EST-SSR引物。利用这些引物对8个华仁杏品种进行了PCR扩增及检测,得到10对能产生清晰多态性条带的EST-SSR标记,标明了10对引物的序列,为进一步开展华仁杏SSR分子标记辅助育种研究奠定了基础。     

Genome mapping of white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and comparative analysis within the Trifolieae using cross-species SSR markers

Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume used extensively as forage for livestock, is an important target for marker-assisted breeding. A genetic linkage map of white clover was constructed using simple sequence repeat (SSR) markers based on sequences from several Trifolieae species, including white clover, red clover (T. pratense L.), Medicago truncatula (Gaertn.) and soybean (Glycine max L.). An F₁ population consisting of 179 individuals, from a cross between two highly heterozygous genotypes, GA43 and Southern Regional Virus Resistant, was used for genetic mapping. A total of 1,571 SSR markers were screened for amplification and polymorphism using DNA from two parents and 14 F₁s of the mapping population. The map consists of 415 loci amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight homoeologous chromosome pairs of allotetraploid white clover were detected. Map length was estimated at 1,877 cM with 87% genome coverage. Map density was approximately 5 cM per locus. Segregation distortion was detected in six segments of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of map locations of markers originating from white clover, red clover, and alfalfa (M. sativa L.) revealed putative macro-colinearity between the three Trifolieae species. This map can be used to link quantitative trait loci with SSR markers, and accelerate the improvement of white clover by marker-assisted selection and breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of simple sequence repeat markers in the hexaploid forage grass timothy (<Emphasis Type="Italic">Phleum pratense</Emphasis> L.)

To develop simple sequence repeat (SSR) markers for the hexaploid forage grass timothy (Phleum pratense L.), we used four SSR-enriched genomic libraries to isolate 1,331 SSR-containing clones. All four libraries contained a high percentage of perfect clones, ranging from 78.1% to 91.6%. From these clones, we developed 355 SSR markers when tested from 502 SSR primer pairs. Using all 355 SSR markers we tested one screening panel consisting of eight timothy clones to detect the level of polymorphism and identify a set of loci suitable for framework mapping. The SSR markers detected 90.4% polymorphism between the parents of a pseudo-testcross F₁ population. These SSR markers will provide an ideal marker system to assist with gene targeting, QTL (quantitative trait locus) mapping, and marker-assisted selection in timothy.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Development and Characterization of EST-SSR Markers in the Eastern Oyster <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) in the eastern oyster (Crassostrea virginica). ESTs of the eastern oyster were downloaded from GenBank and screened for SSRs with at least eight units of dinucleotide or five units of tri-, tetra-, penta-, and hexa-nucleotide repeats. The screening of 9101 ESTs identified 127 (1.4%) SSR-containing sequences. Primers were designed for 88 SSR-containing ESTs with good and sufficient flanking sequences. Polymerase chain reaction (PCR) amplification was successful for 71 primer pairs, including 19 (27%) pairs that amplified fragments longer than expected sizes, probably due to introns. Sixty-six pairs that produced fragments shorter than 800 bp were screened for polymorphism in five oysters from three populations via polyacrylamide gels, and 53 of them (80%) were polymorphic. Fifty-three polymorphic SSRs were labeled and genotyped in 30 oysters from three populations via an automated sequencer. Five of the SSRs amplified more than two fragments per oyster, suggesting locus duplication. The remaining 48 SSRs had 2 alleles per individual, including 11 with null alleles. In the 30 oysters analyzed, the SSRs had an average of 9.3 alleles per locus, ranging from 2 to 24. Forty-three loci segregated in a family with 100 progeny, with nine showing significant deviation from Mendelian ratios (three after Bonferroni correction). Seventy percent of the loci were successfully amplified in C. rhizophorae and 34% in C. gigas. This study demonstrates that ESTs are valuable resources for the development of SSR markers in the eastern oyster, and EST-derived SSRs are more transferable across species than genomic SSRs.     

Molecular cloning and genetic mapping of perennial ryegrass casein protein kinase 2 α-subunit genes

The α-subunit of the casein protein kinase CK2 has been implicated in both light-regulated and circadian rhythm-controlled plant gene expression, including control of the flowering time. Two putative CK2α genes of perennial ryegrass (Lolium perenne L.) have been obtained from a cDNA library constructed with mRNA isolated from cold-acclimated crown tissue. The genomic organisation of the two genes was determined by Southern hybridisation analysis. Primer designs to the Lpck2a-1 and Lpck2a-2 cDNA sequences permitted the amplification of genomic products containing large intron sequences. Amplicon sequence analysis detected single nucleotide polymorphisms (SNPs) within the p150/112 reference mapping population. Validated SNPs, within diagnostic restriction enzyme sites, were used to design cleaved amplified polymorphic sequence (CAPS) assays. The Lpck2a-1 CAPS marker was assigned to perennial ryegrass linkage group (LG) 4 and the Lpck2a-2 CAPS marker was assigned to LG2. The location of the Lpck2a-1 gene locus supports the previous conclusion of conserved synteny between perennial ryegrass LG4, the Triticeae homoeologous group 5L chromosomes and the corresponding segment of rice chromosome 3. Allelic variation at the Lpck2a-1 and Lpck2a-2 gene loci was correlated with phenotypic variation for heading date and winter survival, respectively. SNP polymorphism may be used for the further study of the role of CK2α genes in the initiation of reproductive development and winter hardiness in grasses.     

Identification and validation of a core set of informative genic SSR and SNP markers for assaying functional diversity in barley

A ‘core set’ of 28 simple sequence repeat (SSR) and 28 single nucleotide polymorphism (SNP) markers for barley was developed after screening six diverse genotypes (DGs) representing six countries (Afghanistan, Pakistan, Algeria, Egypt, Jordan and Syria) with 50 SSR and 50 SNP markers derived from expressed sequence tags (ESTs). The markers of the core set are single locus with very high quality amplifications, high polymorphism information content (PIC) and are distributed across the barley genome. PIC values for the selected SSR and SNP markers ranged between 0.32–0.72 (average 0.58) and 0.28–0.50 (average 0.42), respectively. To make the SNP genotyping cost effective, CAPS (cleaved amplified polymorphic sequence) and indel assays were developed for 23 markers and the remaining 5 SNP markers were optimized for pyrosequencing. A high coefficient of correlations (r = 0.96, P < 0.005) between the genetic similarity matrices of SSR and SNP genotyping data of the core set on diverse genotypes (DGs) and their similar groupings according to the geographical distribution in both SSR and SNP phenograms with high bootstrap values underline the utility and reliability of the core set. A comparative allelic and sequence diversity for SSR and SNP markers between the DGs and six elite parental genotypes (PGs) of mapping populations showed comparable diverse nature of two germplasm sets. However, unique SNPs and indels were observed in both germplasm sets providing more datapoints for analysing haplotypes in a better way for the corresponding SNP marker. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat

Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivum L.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1–7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.Communicated by R. Hagemann     

Parental genome composition and genetic classifications of derivatives from intergeneric crosses of Festuca mairei and Lolium perenne

Intergeneric hybridization between Festuca and Lolium has been a long-term goal of forage and turfgrass breeders to generate improved cultivars by combining stress tolerance of Festuca and rapid establishment of Lolium. However, wide-distance hybridizations usually result in the wild genome being eliminated from the hybrid due to incomplete chromosome pairing and crossovers. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to detect the parental genome composition of F₁ hybrids and backcross, generated from crosses between Festuca mairei St. Yves (Fm) and Lolium perenne L. (Lp). Each of the hybrids exhibited integration of Fm and Lp genomes with varying levels of Fm/Lp genome ratios. However, cluster and principle component analyses of the progeny consistently revealed four groups depending on the amount of genome introgression from both parents. The parental genome composition and classifications of intergeneric progeny would be useful for breeding material selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tall fescue EST-SSR markers with transferability across several grass species

Tall fescue (Festuca arundinacea Schreb.) is a major cool season forage and turf grass in the temperate regions of the world. It is also a close relative of other important forage and turf grasses, including meadow fescue and the cultivated ryegrass species. Until now, no SSR markers have been developed from the tall fescue genome. We designed 157 EST-SSR primer pairs from tall fescue ESTs and tested them on 11 genotypes representing seven grass species. Nearly 92% of the primer pairs produced characteristic simple sequence repeat (SSR) bands in at least one species. A large proportion of the primer pairs produced clear reproducible bands in other grass species, with most success in the close taxonomic relatives of tall fescue. A high level of marker polymorphism was observed in the outcrossing species tall fescue and ryegrass (66%). The marker polymorphism in the self-pollinated species rice and wheat was low (43% and 38%, respectively). These SSR markers were useful in the evaluation of genetic relationships among the Festuca and Lolium species. Sequencing of selected PCR bands revealed that the nucleotide sequences of the forage grass genotypes were highly conserved. The two cereal species, particularly rice, had significantly different nucleotide sequences compared to the forage grasses. Our results indicate that the tall fescue EST-SSR markers are valuable genetic markers for the Festuca and Lolium genera. These are also potentially useful markers for comparative genomics among several grass species.Electronic Supplementary Material Supplementary material is available for this article at .     

Tall fescue genomic SSR markers: development and transferability across multiple grass species

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT) _n repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Development of transcript-associated microsatellite markers for diversity and linkage mapping studies in hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

Data mining of gene sequences available from various projects dealing with the development of expressed sequence tags (ESTs) can contribute to the discovery of new microsatellite markers. Our aim was to develop new microsatellite markers in hop isolated from an enriched cDNA library and from coding GenBank sequences and to test their suitability in hop diversity studies and for construction of a linkage map. In a set of 614 coding GenBank sequences, 72 containing microsatellites were found (11.7%); the most frequent were trinucleotide repeats (54.0%) followed by dinucleotide repeats (34.5%). Additionally, 11 sequences containing microsatellites were isolated from an enriched cDNA library. A total of 34 primer pairs were designed, 29 based on GenBank sequences and five on sequences from the cDNA enriched library. Twenty-seven (79.4%) coding microsatellites were successfully amplified and used in diversity and linkage mapping studies. Eleven primer pairs amplified 12 coding microsatellite loci suitable for mapping and were placed on female and male linkage maps. We were able to extend previous simple sequence repeat (SSR) female, male and integral maps by 38.8, 25.8 and 40.0 cM, respectively. In the diversity study, 36 diverse hop genotypes were analyzed. Twenty-four coding microsatellites were polymorphic, 17 showing co-dominant behavior and 7 primer pairs amplifying three or more bands in some hop genotypes. Altogether, 143 microsatellite DNA fragments were amplified and they revealed a clear separation of hop genotypes according to geographical region, use or breeding history. In addition, a discussion and comparison of results with other plant coding/EST SSR studies is presented. Our results showed that these microsatellite markers can enhance hop diversity and linkage mapping studies and are a comparable marker system to non-coding SSRs.