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German Spangenberg - One of the best experts on this subject based on the ideXlab platform.
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Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume White Clover (Trifolium repens)
Molecular Biology Reports, 2021Co-Authors: Rafael Narancio, John Mason, Ulrik John, Paula Giraldo, German SpangenbergAbstract:Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in White Clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in White Clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in White Clover. Two genes, Pyruvate dehydrogenase ( PDH ), and an ATP-dependent protease , identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in White Clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in White Clover, and provide resources for future copy number studies in this species.
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selection of optimal reference genes for quantitative rt pcr transcript abundance analysis in White Clover trifolium repens l
Functional Plant Biology, 2018Co-Authors: Rafael Narancio, Ulrik P John, John Mason, German SpangenbergAbstract:Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Relative quantification by qRT-PCR requires the use of a stably expressed reference gene. There are many ‘housekeeping’ genes reported in different plant species that are used as reference genes. However, it is important that the steady-state mRNA levels of these housekeeping genes are confirmed across different conditions and tissues in each species studied. Prior to this study, no comprehensive work had been performed in identifying optimal reference genes in White Clover (Trifolium repens L.). To identify suitable reference genes in White Clover, we analysed the transcript abundance stability of seven candidate genes in two organs (leaves and stolons) across two treatments (water-limited and well-watered). ΔCt, NormFinder and ANOVA tests were carried out to evaluate the mRNA level stability of candidate reference genes. According to the ΔCt results, the genes with the most stable mRNA levels were EF1α and ACT11. When stability among groups was evaluated by NormFinder, UBQ was the most stable across all organs and treatments. By multiple criteria, EF1α, followed by ACT11 and UBQ, was the most stably-expressed gene across organs and treatments, and each of these are recommended as reference genes for transcript abundance studies in White Clover.
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development and validation of protocols for product stewardship in transgenic White Clover trifolium repens l detection of the amv cp and npt2 transgenes in pollen honey and honey bees
Crop & Pasture Science, 2015Co-Authors: Stephen Panter, K F Smith, A. Mouradov, German SpangenbergAbstract:There are no current commercial releases of genetically modified White Clover, but several research groups are working on traits such as virus resistance, stress tolerance and bloat safety that are likely to provide large economic benefits for livestock farmers. However, White Clover pollen is a common constituent of honey produced by bees foraging White Clover flowers. Therefore, there is a need to develop tools to detect the presence of genetically modified pollen in White Clover honey. The results presented in this paper describe the development and application of PCR-based techniques to detect the Alfalfa mosaic virus coat protein gene (AMV CP) and the neomycin phosphotransferase 2 selectable marker gene (npt2) in genetically modified White Clover pollen, whether this pollen is collected fresh, from honey bees that have been foraging White Clover, or from honey. Further research and development will be required to develop ‘field-ready’ tools for the detection and quantification of these transgenes in pollen and honey products. However, this paper demonstrates prospects and principles in pollen and honey from honeybees foraging transgenic White Clover.
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biosynthesis of proanthocyanidins in White Clover flowers cross talk within the flavonoid pathway
Plant Physiology, 2012Co-Authors: Shamila Abeynayake, A. Mouradov, S. Rochfort, Stephen Panter, Ross L Chapman, Tracie Webster, German SpangenbergAbstract:Proanthocyanidins and anthocyanins are produced by closely related branches of the flavonoid pathway and utilize the same metabolic intermediates. Previous studies have shown a flexible mechanism of flux diversion at the branch-point between the anthocyanin and proanthocyanidin pathways, but the molecular basis for this mechanism is poorly understood. Floral tissues in White Clover plants (Trifolium repens) produce both proanthocyanidins and anthocyanins. This makes White Clover amenable to studies of proanthocyanidin and anthocyanin biosynthesis and possible interactions within the flavonoid pathway. Results of this study show that the anthocyanin and proanthocyanidin pathways are spatially colocalized within epidermal cells of petals and temporally overlap in partially open flowers. A correlation between spatiotemporal patterns of anthocyanin and proanthocyanidin biosynthesis with expression profiles of putative flavonoid-related genes indicates that these pathways may recruit different isoforms of flavonoid biosynthetic enzymes. Furthermore, in transgenic White Clover plants with down-regulated expression of the anthocyanidin reductase gene, levels of flavan 3-ols, anthocyanins, and flavonol glycosides and the expression levels of a range of genes encoding putative flavonoid biosynthetic enzymes and transcription factors were altered. This is consistent with the hypothesis that flux through the flavonoid pathway may be at least partially regulated by the availability of intermediates.
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validation of in silico predicted genic snps in White Clover trifolium repens l an outbreeding allopolyploid species
Molecular Genetics and Genomics, 2007Co-Authors: Noel O I Cogan, Michelle C Drayton, Rebecca C Ponting, A C Vecchies, N R Bannan, Tim Sawbridge, K F Smith, German Spangenberg, John W ForsterAbstract:White Clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to White Clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in White Clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.
Yan Zhang - One of the best experts on this subject based on the ideXlab platform.
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metabolic pathways regulated by chitosan contributing to drought resistance in White Clover
Journal of Proteome Research, 2017Co-Authors: Yan Zhang, Xinquan Zhang, Emily Merewitz, Yan Peng, Linkai Huang, Yanhong YanAbstract:Increased endogenous chitosan (CTS) could be associated with improved drought resistance in White Clover (Trifolium repens). Plants were pretreated with or without 1 mg/mL CTS and then were subjected to optimal or water-limited condition in controlled growth chambers for 6 days. Phenotypic and physiological results indicated that exogenous CTS significantly improved drought resistance of White Clover. Metabolome results showed that exogenous CTS induced a significant increase in endogenous CTS content during dehydration accompanied by the maintenance of greater accumulation of sugars, sugar alcohols, amino acids, organic acids, and other metabolites (ascorbate, glutathione, flavonoids, putrescine, and spermidine). These compounds are associated with osmotic adjustment, antioxidant defense, stress signaling, and energy metabolism under stress condition. Similarly, transcriptome revealed that many genes in relation to amino acid and carbohydrate metabolism, energy production and conversion, and ascorbate–gl...
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genome wide identification of microsatellites in White Clover trifolium repens l using fiasco and phpssrminer
Plant Methods, 2008Co-Authors: Yan Zhang, Patrick X Zhao, Joseph H Bouton, Maria J MonterosAbstract:Background Allotetraploid White Clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in White Clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for White Clover.
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genome mapping of White Clover trifolium repens l and comparative analysis within the trifolieae using cross species ssr markers
Theoretical and Applied Genetics, 2007Co-Authors: Yan Zhang, Mary K Sledge, Joe H BoutonAbstract: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.
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genome mapping of White Clover trifolium repens l and comparative analysis within the trifolieae using cross species ssr markers
Theoretical and Applied Genetics, 2007Co-Authors: Yan Zhang, Mary K Sledge, Joe H BoutonAbstract: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:10.1007/s00122-007-0523-3) contains supplementary material, which is available to authorized users.
C Guy - One of the best experts on this subject based on the ideXlab platform.
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comparison of perennial ryegrass lolium perenne l ploidy and White Clover trifolium repens l inclusion for herbage production utilization and nutritive value
Grass and Forage Science, 2018Co-Authors: C Guy, D Hennessy, T J Gilliland, F Coughlan, B Mcclearn, M Dineen, Brian C MccarthyAbstract:Increasing herbage dry‐matter (DM) production and utilization on‐farm improves efficiency and sustainability. The objective of this study was to investigate the effect of perennial ryegrass, Lolium perenne L., ploidy and White Clover, Trifolium repens L., inclusion on herbage DM production and utilization in an intensive animal grazing system, with high nitrogen (N) inputs (250 kg N ha⁻¹) and a high stocking rate (2.75 livestock units ha⁻¹). The study was a 2 × 2 factorial design, consisting of diploid and tetraploid cultivars sown as grass‐only and grass‐White Clover to give four sward treatments (diploid‐only, tetraploid‐only, diploid‐White Clover, tetraploid‐White Clover). These were evaluated at each grazing occasion (8–10 per year) for 3 years (2014–2016). Tetraploid‐White and grass‐White Clover swards had a lower sward density (−9 and −8 kg DM cm⁻¹, respectively) and perennial ryegrass tiller density (−820 and −1,436 tillers m⁻², respectively) leading to lower postgrazing sward heights (−0.15 and −0.41 cm, respectively), and maintained a superior nutritive value when compared with diploid‐only and grass‐only swards respectively. White Clover inclusion resulted in higher productivity characterized by increased herbage DM production (+1,468 kg DM ha⁻¹), improved nutritive value and increased utilization across the three grazing seasons. Varying sward composition with perennial ryegrass ploidy led to improved nutritive value and favourable grazing characteristics in tetraploid swards.
Jorgen Eriksen - One of the best experts on this subject based on the ideXlab platform.
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medium term response of microbial community to rhizodeposits of White Clover and ryegrass and tracing of active processes induced by 13c and 15n labelled exudates
Soil Biology & Biochemistry, 2014Co-Authors: Gedrimė Kuslienė, Jim Rasmussen, Yakov Kuzyakov, Jorgen EriksenAbstract:Abstract Rhizodeposition affects the microbial community in the rhizosphere, and microbial composition and activity may therefore differ in soil depending on plant species. We hypothesised that these differences increase over the plant growth period because roots occupy larger soil volumes and release more rhizodeposits. We tested how such medium-term responses of the microbial community can be explained by the short-term utilisation of root exudates. To test this we analysed 15 N incorporation into microbial biomass, phospholipid fatty acid (PLFA) composition and 13 C incorporation into the PLFAs of specific microbial groups in soil under White Clover ( Trifolium repens L.) and ryegrass ( Lolium perenne L.) following leaf-labelling with 13 C-bicarbonate and 15 N-urea. In this way microbial N and 15 N and the composition of PLFAs reflect the medium-term (two months) response of microorganisms to rhizodeposits, whereas the 13 C-label of the PLFAs reflects the short-term (one week) utilisation of root exudates following labelling of shoots. In the medium term, microbial biomass N and 15 N were greater under the ryegrass, whereas total PLFA was higher under White Clover. The relative abundance of fungi and actinomycetes was unaffected by plant species, but pool of Gram-negative and Gram-positive bacteria was greater under White Clover at the 10 percent significance level. In the short term, microorganisms more actively utilised fresh exudates ( 13 C-labelled) of ryegrass than of White Clover. We expected ryegrass exudates initially to be incorporated into bacterial PLFA and into fungi over time, but surprisingly fungi had the highest utilisation of ryegrass-derived C over the week. At 0–5 cm soil depth, White Clover exudates were utilised only by bacteria, whereas fungi dominated at 5–15 cm. This reflects differences in the quality of White Clover exudates or differences in the microbial community composition at the two depths. We conclude that despite clear short-term differences in microbial response to the exudates of White Clover and ryegrass, this is only to a limited extent transferred into medium-term defects on the composition of the microbial communities under the two plant species. Hence, our study showed that different short-term C utilisation patterns may lead to similar medium-term responses of the microbial community.
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complementary effects of red Clover inclusion in ryegrass White Clover swards for grazing and cutting
Grass and Forage Science, 2014Co-Authors: Jorgen Eriksen, Margrethe Askegaard, Karen SoegaardAbstract:Increasing plant species diversity in grasslands may improve productivity and stability of yields. In a field experiment, we investigated the herbage dry-matter (DM) yield and crude protein content of two-species swards of perennial ryegrass–White Clover (Lolium perenne L.–Trifolium repens L.) and three-species swards of ryegrass–White Clover with red Clover (Trifolium pratense L.). Five different managements represented cutting, grazing and combinations thereof, with different slurry fertilization treatments in 1- to 4-year-old swards. The three-species mixture out-yielded the two-species mixture in years 1 and 2. Across all 4 years, yields were 8–10% higher in cut swards. Inclusion of red Clover increased the yields of Clover across the 4 years by 51% without fertilizer and by 90% when fertilized. Responses to slurry fertilization were similar in both mixtures and were mainly independent of sward age. There was a complementary effect over the season and across managements. Red Clover dominated in the first and third cuts; White Clover dominated in the second and fourth cuts. Red Clover dominated in cut swards and White Clover in grazed swards. Future prospects of the inclusion of red Clover in sown swards are discussed. These may include higher nitrogen-use efficiency in ruminants, increased soil fertility and improved sward flexibility to cope with changing managements. The findings also suggest positive yield effects of alternating between cutting and grazing within the season or between years.
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spatial and temporal variation in n transfer in grass White Clover mixtures at three northern european field sites
Soil Biology & Biochemistry, 2013Co-Authors: Thorey Gylfadottir, Jim Rasmussen, Ralf Loges, Jorgen Eriksen, Aslaug HelgadottirAbstract:Abstract The N flow dynamics in grass–Clover mixtures are not well understood. Spatial distributions and temporal differences in inter- and intra-species N transfer were investigated at field sites in Iceland, Germany, and Denmark, with three different managements at the Danish site. Both grass and White Clover were leaf-labeled four times during a full year and the short-term N transfer to neighboring grass and White Clover was determined in 10 cm intervals up to 1 m from the labeled plant. In addition 15N-injection of inorganic N at 5, 15, and 25 cm soil depth was carried out at the Danish site to compare spatial and temporal N transfer patterns to soil inorganic N uptake. The short-term N transfer from White Clover to the closest companion grass reached levels of more than 50% of N from labeled White Clover late in the growing season, thus questioning whether longer-term root turnover processes are dominating N transfer. The horizontal N transfer to grass exceeded 50 cm from the labeled plant at one site. The study showed that the competitive ability of White Clover is as important for N dynamics in grass–White Clover mixtures as that of the companion grass. Intra-species N transfer showed that both grass and White Clover have reuptake of deposited N corresponding to at least 1% of the N found in leaf biomass. The study also showed that N transfer cannot be explained simply by competition for soil inorganic N, which indicates transfer via organic N forms.
John W Forster - One of the best experts on this subject based on the ideXlab platform.
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validation of in silico predicted genic snps in White Clover trifolium repens l an outbreeding allopolyploid species
Molecular Genetics and Genomics, 2007Co-Authors: Noel O I Cogan, Michelle C Drayton, Rebecca C Ponting, A C Vecchies, N R Bannan, Tim Sawbridge, K F Smith, German Spangenberg, John W ForsterAbstract:White Clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to White Clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in White Clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.
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a microsatellite map of White Clover
Theoretical and Applied Genetics, 2004Co-Authors: Brent A Barrett, John W Forster, Tim Sawbridge, Andrew G Griffiths, M Schreiber, Nicholas W Ellison, C Mercer, J H Bouton, B Ong, German SpangenbergAbstract:The White Clover (Trifolium repens) nuclear genome (n=2x=16) is an important yet under-characterised genetic environment. We have developed simple sequence repeat (SSR) genetic markers for the White Clover genome by mining an expressed sequence tag (EST) database and by isolation from enriched genomic libraries. A total of 2,086 EST-derived SSRs (EST-SSRs) were identified among 26,480 database accessions. Evaluation of 792 EST-SSR primer pairs resulted in 566 usable EST-SSRs. Of these, 335 polymorphic EST-SSRs, used in concert with 30 genomic SSRs, detected 493 loci in the White Clover genome using 92 F1 progeny from a pair cross between two highly heterozygous genotypes—364/7 and 6525/5. Map length, as estimated using the joinmap algorithm, was 1,144 cM and spanned all 16 homologues. The R (red leaf) locus was mapped to linkage group B1 and is tightly linked to the microsatellite locus prs318c. The eight homoeologous pairs of linkage groups within the White Clover genome were identified using 96 homoeologous loci. Segregation distortion was detected in four areas (groups A1, D1, D2 and H2). Marker locus density varied among and within linkage groups. This is the first time EST-SSRs have been used to build a whole-genome functional map and to describe subgenome organisation in an allopolyploid species, and T. repens is the only Trifolieae species to date to be mapped exclusively with SSRs. This gene-based microsatellite map will enable the resolution of quantitative traits into Mendelian characters, the characterisation of syntenic relationships with other genomes and acceleration of White Clover improvement programmes.
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an ssr and aflp molecular marker based genetic map of White Clover trifolium repens l
Plant Science, 2003Co-Authors: Elizabeth Jones, L J Hughes, Michelle C Drayton, Michael T Abberton, T P T Michaelsonyeates, Charlotte Bowen, John W ForsterAbstract:A framework genetic map of White Clover (Trifolium repens L.) has been constructed using an F2 progeny set derived from the intercross of fourth and fifth generation inbred genotypes carrying a self-fertile mutation (Sf). White Clover SSR (TRSSR) and AFLP markers were used to derive a map with 135 markers (78 TRSSR loci and 57 AFLP loci) assigned to 18 linkage groups (LGs) with a total map length of 825 cm. Sixteen of these LGs are presumed to correspond to the 16 chromosomes of the White Clover karyotype. A limited number of multiple loci were detected. Substantial segregation distortion was observed for both mapped and unmapped loci, with a bias towards heterozygous types and a preponderance of distorted markers on certain LGs. This observation, along with a high degree of residual heterozygosity within the inbred parental genotypes, suggests that reduced individual fitness due to loss of heterosis is a major effect for White Clover and will limit the applicability of F2 or backcross mapping strategies for this species. A core set of map-assigned co-dominant, single locus SSR markers has been defined for whole genome scans of genetic variability in White Clover.
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development and characterisation of simple sequence repeat ssr markers for White Clover trifolium repens l
Theoretical and Applied Genetics, 2001Co-Authors: Roland Kolliker, Elizabeth Jones, Michelle C Drayton, M P Dupal, John W ForsterAbstract: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.
