Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

基于转录组测序的枫香EST-SSR引物开发及有效性评价

枫香是广西重要的乡土树种之一，具有较高的用材、观赏及药用价值。为验证枫香SSR引物的实际应用效果，该研究基于转录组测序技术，检测枫香SSR位点并设计引物，通过PCR扩增和聚丙烯酰胺凝胶电泳筛选出具有较高多态性的枫香EST-SSR引物，并对1个枫香天然群体进行遗传多样性分析。结果表明：(1)共发掘到23 777个SSR位点，单核苷酸重复类型SSR位点占总位点比例最高(46.54%),在重复次数上5～12次之间的SSR位点占比最高(72.36%)。(2)共开发出262对SSR引物，有效扩增率为53.1%,最终筛选出扩增稳定、条带清晰的引物18对。(3)多态性检测结果显示所有位点均具有多态性，天然群体遗传多样性结果显示该天然群体中等位基因数量(Na)、有效等位基因数量(Ne)、Shannon多样性指数(I)、观测杂合度(Ho)变化范围分别为2～4、1.112 8～2.609 6、0.208 9～1.112 7和0.275 9～1.000 0,平均值分别为2.333 3、1.957 4、0.708 5和0.722 6。综上认为，枫香中占优势的SSR位点重复类型和重复基序与其他物种基本相同，所开...     

基于磁珠富集法开发并筛选西川红景天的微卫星标记

利用FIASCO磁珠富集法,开发和筛选青藏高原特有珍贵植物西川红景天(Rhodiola alsia)多态性微卫星标记。结果表明:用(AG)15和(AC)15两种微卫星标记探针构建富集文库,共获得阳性克隆约2500个;从中随机挑取1200检测,发现具有多态性的阳性克隆达400个;随机挑取200多态性的阳性克隆进行测序,从中获得105个SSR位点,用在线软件primer3-2.3.4成功设计得SSR引物105对;其中45对可以成功扩增,而13对所扩增的片段在相距较远的4个自然居群的24个个体中显示较高的遗传多态性。用4个居群的80个个体检测这13对引物发现,平均等位基因数(A)约为9.192,观测杂合度(Ho)和期望杂合度(He)均值分别约为0.712和0.734,如此高的多态性已经满足后期研究的需要;数个位点在某些居群中显著偏离哈迪温伯格平衡(P0.01),这可能是实际研究的居群并不能达到哈迪—温伯格定律所假设的无限大等理想状态所致。结合此前基于表达序列标签(Expressed Sequence Tag,EST)序列开发的SSR多态性位点,该研究结果为今后利用SSR位点开展西川红景天的居群遗传结构分析和其他研究提供了一组有效工具。     

Development of 28 EST‐SSR markers based on transcriptome sequences of Gobiobotia filifer and cross‐species amplification

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.     

Molecular mapping of a quantitative trait locus for aluminum tolerance in wheat cultivar Atlas 66

Genetic improvement of aluminum (Al) tolerance is one of the cost-effective solutions to improve wheat (Triticum aestivum) productivity in acidic soils. The objectives of the present study were to identify quantitative trait loci (QTL) for Al-tolerance and associated PCR-based markers for marker-assisted breeding utilizing cultivar Atlas 66. A population of recombinant inbred lines (RILs) from the cross Atlas 66/Century was screened for Al-tolerance by measuring root-growth rate during Al treatment in hydroponics and root response to hematoxylin stain of Al treatment. After 797 pairs of SSR primers were screened for polymorphisms between the parents, 131 pairs were selected for bulk segregant analysis (BSA). A QTL analysis based on SSR markers revealed one QTL on the distal region of chromosome arm 4DL where a malate transporter gene was mapped. This major QTL accounted for nearly 50% of the phenotypic variation for Al-tolerance. The SSR markers Xgdm125 and Xwmc331 were the flanking markers for the QTL and have the potential to be used for high-throughput, marker-assisted selection in wheat-breeding programs.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.     

SSR-based linkage map with new markers using an intraspecific population of common wheat

Simple sequence repeats (SSRs) are valuable molecular markers in many plant species. In common wheat (Triticum aestivum L.), which is characteristic of its large genomes and alloploidy, SSRs are one of the most useful markers. To increase SSR marker sources and construct an SSR-based linkage map of appropriate density, we tried to develop new SSR markers from SSR-enriched genomic libraries and the public database. SSRs having (GA)n and (GT)n motifs were isolated from enriched libraries, and di- and tri-nucleotide repeats were mined from expressed sequence tags (ESTs) and DNA sequences of Triticum species in the public database. Of the 1,147 primer pairs designed, 842 primers gave accurate amplification products, and 478 primers showed polymorphism among the nine wheat lines examined. Using a doubled haploid (DH) population from an intraspecific cross between Kitamoe and Münstertaler (KM), we constructed an SSR-based linkage map that consisted of 464 loci: 185 loci from genomic libraries, 65 loci from the sequence database including ESTs, 213 loci from the SSR markers already reported, and 1 locus of morphological marker. Although newly developed SSR loci were distributed throughout all chromosomes, clustering of them around putative centromeric regions was found on several chromosomes. The total length of the KM map spanned 3,441 cM and corresponded to approximately 86% genome coverage. The KM map comprised of 23 linkage groups because two gaps of over 50 cM distance remained on chromosome 6A. This is a first report of SSR-based linkage map using single intraspecific population of common wheat. This mapping result suggests that it becomes possible to construct linkage maps with sufficient genome coverage using only SSR markers without RFLP markers, even in an intraspecific population of common wheat. Moreover, the new SSR markers will contribute to the enrichment of molecular marker resources in common wheat.     

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 validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

Mining and validating grape (<Emphasis Type="Italic">Vitis</Emphasis> L.) ESTs to develop EST-SSR markers for genotyping and mapping

Grape expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping. An integrated pipeline including several computational tools for SSR identification and functional annotation was developed to identify 6,447 EST-SSR sequences from a total collection of 215,609 grape ESTs retrieved from NCBI. The 6,447 EST-SSRs were further reduced to 1,701 non-redundant sequences via clustering analysis, and 1,037 of them were successfully designed with primer pairs flanking the SSR motifs. From them, 150 pairs of primers were randomly selected for PCR amplification, polymorphism and heterozygosity analysis in V. vinifera cvs. Riesling and Cabernet Sauvignon, and V. rotundifolia (muscadine grape) cvs. Summit and Noble, and 145 pairs of these primers yielded PCR products. Pairwise comparisons of loci between the parents Riesling and Cabernet Sauvignon showed that 72 were homozygous in both cultivars, while 70 loci were heterozygous in at least one cultivar of the two. Muscadine parents Noble and Summit had 90 homozygous SSR loci in both parents and contained 50 heterozygous loci in at least one of the two. These EST-SSR functional markers are a useful addition for grape genotyping and genome mapping.     

竹子叶绿体基因组SSR分子标记的开发及其应用

为探讨观赏竹叶片异质性的机理,根据麻竹(Dendrocalamus latiflorus)和绿竹(Bambusa oldhamii)叶绿体基因组序列开发SSR分子标记。结果表明,在麻竹和绿竹叶绿体基因组中分别存在87和86个SSR位点,其中三核苷酸重复类型最多,其次为单核苷酸重复类型。根据SSR位点设计21对引物,其中11对引物对6竹种能够扩增出稳定、清晰的条带,且具有多态性,引物有效率达到52.4%。聚类分析表明,6竹种可分为两大类群,与形态学分类结果基本一致。有4对引物在菲白竹(Pleioblastus fortunei)和白纹椎谷笹(Sasaella glabra f.albo-striata)的花叶中具有多态性,可作为区分观赏竹叶片异质性的分子标记。     

Isolation, characterization and mapping of simple sequence repeat markers in zoysiagrass (Zoysia spp.)

The genus Zoysia consists of 16 species that are naturally distributed on sea coasts and grasslands around the Pacific. Of these, Zoysia japonica, Zoysia matrella, and Zoysia tenuifolia are grown extensively as turfgrasses, and Z. japonica is also used as forage grass in Japan and other countries in East Asia. To develop simple sequence repeat (SSR) markers for zoysiagrass (Zoysia spp.), we used four SSR-enriched genomic libraries to isolate 1,163 unique SSR clones. All four libraries contained a high percentage of perfect clones, ranging from 67.1 to 96.0%, and compound clones occurred with higher frequencies in libraries A (28.6%) and D (11.6%). From these clones, we developed 1,044 SSR markers when we tested all 1,163 SSR primer pairs. Using all 1,044 SSR markers, we tested one screening panel consisting of eight Zoysia clones for testing PCR amplifications, from which five unrelated clones, among the eight, were used for polymorphism assessment, and found that the polymorphic information content ranged from 0 (monomorphic loci) to 0.88. Of the 1,044 SSR markers, 170 were segregated in our mapping population and we mapped 161 on existing amplified fragment length polymorphism-based linkage groups, using this mapping population. These SSR markers will provide an ideal marker system to assist with gene targeting, quantitative trait locus mapping, variety or species identification, and marker-assisted selection in Zoysia species.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

EST-SSR markers derived from Laminaria digitata and its transferable application in Saccharina japonica

The public availability of numerous expressed sequence tag (EST) enables EST-based SSR (simple sequence repeat) markers to be widely used for genetics and breeding studies. In the present study, EST-SSR markers were developed from ESTs of Laminaria digitata and were transferred to the non-congeneric species Saccharina japonica. Among the 2,668 non-redundant ESTs, 83 (3.1%) ESTs containing SSR were identified totally, with an average of one SSR per 13.6 kb. Analysis of SSR motifs revealed that the trinucleotide and tetranucleotide were major motifs, accounted for 44.58% and 16.87%, respectively. Based on the 83 ESTs containing SSR, we designed 45 pairs of primers in the flanking regions of the SSR, of which 13 pairs showed polymorphism in a wild S. japonica population, and the mean alleles per locus was 3.6 (ranging from 2 to 6). The observed (Ho) and expected (He) heterozygosities of these EST-SSRs were 0.234–0.632 and 0.260–0.635, respectively. All loci were in Hardy–Weinberg equilibrium in the wild population and no linkage disequilibrium was detected among loci. The obtained EST-SSR markers can facilitate and promote related research such as ecological investigation, genetic diversity assessment and breeding practice of S. japonica as well.     

CONSTRUCTION OF A GENETIC LINKAGE MAP FOR PORPHYRA HAITANENSIS (BANGIALES,RHODOPHYTA) BASED ON SEQUENCE‐RELATED AMPLIFIED POLYMORPHISM AND SIMPLE SEQUENCE REPEAT MARKERS1

Molecular markers and molecular genetic maps are prerequisites for molecular breeding in any plant species. A comprehensive genetic linkage map for cultivated Porphyra haitanensis T. J. Chang et B. F. Zheng has not yet been developed. In this study, 157 double haploid (DH) lines [derived from a YSIII (wildtype) × RTPM (red‐type artificial pigmentation mutant) cross] were used as a mapping population in P. haitanensis. A total of 60 pairs of sequence‐related amplified polymorphism (SRAP) primers and 39 pairs of simple sequence repeat (SSR) primers were used to detect polymorphisms between the two parents. Fifteen SRAP and 16 SSR polymorphic primer pairs were selected to analyze the DH population. A linkage genetic map comprising 67 SRAP markers and 20 SSR markers in five linkage groups, with a total length of 830.6 cM and an average of 10.13 cM between markers, was constructed. The markers were distributed evenly in all linkage groups without clustering. The linkage groups comprised 12–23 markers ranging in length from 134.2 to 197.3 cM. The estimated genome length of P. haitanensis was 942.4 cM, with 88.1% coverage. This is the first report of a comprehensive genetic map in P. haitanensis. The map presented here will provide a basis for the development of high‐density genetic linkage maps and lay the foundation for molecular breeding work in P. haitanensis.     

Linkage disequilibrium in European elite maize germplasm investigated with SSRs

Information about the extent and genomic distribution of linkage disequilibrium (LD) is of fundamental importance for association mapping. The main objectives of this study were to (1) investigate genetic diversity within germplasm groups of elite European maize (Zea mays L.) inbred lines, (2) examine the population structure of elite European maize germplasm, and (3) determine the extent and genomic distribution of LD between pairs of simple sequence repeat (SSR) markers. We examined genetic diversity and LD in a cross section of European and US elite breeding material comprising 147 inbred lines genotyped with 100 SSR markers. For gene diversity within each group, significant (P<0.05) differences existed among the groups. The LD was significant (P<0.05) for 49% of the SSR marker pairs in the 80 flint lines and for 56% of the SSR marker pairs in the 57 dent lines. The ratio of linked to unlinked loci in LD was 1.1 for both germplasm groups. The high incidence of LD suggests that the extent of LD between SSR markers should allow the detection of marker-phenotype associations in a genome scan. However, our results also indicate that a high proportion of the observed LD is generated by forces, such as relatedness, population stratification, and genetic drift, which cause a high risk of detecting false positives in association mapping.     

Development of polymorphic EST-derived SSR markers for the shrimp, Fenneropenaeus chinensis

In our study, we evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Fenneropenaeus chinensis. Characteristics of 11 EST-SSR loci were investigated using 34 F. chinensis individuals. The number of alleles per locus ranged from 4 to 9. The observed heterozygosity ranged from 0.118 to 0.826, while the expected heterozygosity ranged from 0.623 to 0.847. Two loci (T2 and T12) conformed to the Hardy–Weinberg equilibrium. There was no LD observed between all pairs of EST-SSRs loci. The PIC values of 11 microsatellite loci were higher than 0.5. These loci and markers will be useful for population genetics and systemic evolution of F. chinensis.     

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.