Analysis of Genetic Diversity and Relationships in a Tunisian Fig （Ficus carica） Germplasm Collection by Random Amplified Microsatellite Polymorphisms

The random amplified mirosatellite polymorphism method was performed in a set of Tunisian fig landraces using eighteen primer combinations. A total of sixty three random amplified microsatellite polymorphism （RAMPO） markers were scored and used either to assess the genetic diversity in these cultivars or to detect cases of mislabeling. Opportunely, data proved that the designed procedure constitutes an attractive and fast method with low costs and prevents radio exposure. As a result, we have identified the primer combinations that are the most efficient to detect genetic polymorphism in this crop. Therefore, the derived unweighted pair-group method with arithmetic averages （UPGMA） dendrogram illustrates the genetic divergence among the landraces studied and exhibits a typically continuous variation. Moreover, no evident correlation between the sexes of trees was observed. In addition, using these markers, discrimination between landraces has been achieved. Thus, random amplified mirosatellite polymor- phism is proved to be powerful for characterizing the local fig germplasm.     

DNA polymorphism in the stem nematode Ditylenchus dipsaci: development of diagnostic markers for normal and giant races.

DNA polymorphism in the Ditylenchus dipsaci complex was investigated using amplified fragment length polymorphism (AFLP) to determine the relationships among populations growing mainly on Vicia faba and to develop diagnostic markers. Twenty-two populations of D. dipsaci originating from different geographical areas and one population of Ditylenchus myceliophagus were used. AFLP proved to be a powerful method to reveal intraspecific polymorphism even within the giant type. The analysis showed a clear distinction between the giant and normal populations, with genetic distances similar to those observed between normal populations and D. myceliophagus or giant populations and D. myceliophagus, strengthening the hypothesis that these two nematode types could be considered distinct species. Two specific AFLP markers differentiating the two types were converted into sequenced characterized amplified region (SCAR) markers. Used in a multiplex PCR, the SCAR primers proved to be a rapid and efficient tool to identify the giant and the normal types of D. dipsaci.     

Genetic distribution of Bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP)

Retrotransposons are present in high copy number in many plant genomes. They show a considerable degree of sequence heterogeneity and insertional polymorphism, both within and between species. We describe here a polymerase chain reaction (PCR)-based method which exploits this polymorphism for the generation of molecular markers in barley. The method produces amplified fragments containing a Bare–1-like retrotransposon long terminal repeat (LTR) sequence at one end and a flanking host restriction site at the other. The level of polymorphism is higher than that revealed by amplified fragment length polymorphism (AFLP) in barley. Segregation data for 55 fragments, which were polymorphic in a doubled haploid barley population, were analysed alongside an existing framework of some 400 other markers. The markers showed a widespread distribution over the seven linkage groups, which is consistent with the distribution of the Bare–1 class of retrotransposons in the barley genome based on in situ hybridisation data. The potential applicability of this method to the mapping of other multicopy sequences in plants is discussed.     

A new and versatile method for the successful conversion of AFLP markers into simple single locus markers

Genetic markers can efficiently be obtained by using amplified fragment length polymorphism (AFLP) fingerprinting because no prior information on DNA sequence is required. However, the conversion of AFLP markers from complex fingerprints into simple single locus assays is perceived as problematic because DNA sequence information is required for the design of new locus-specific PCR primers. In addition, single locus polymorphism (SNP) information is required to design an allele-specific assay. This paper describes a new and versatile method for the conversion of AFLP markers into simple assays. The protocol presented in this paper offers solutions for frequently occurring pitfalls and describes a procedure for the identification of the SNP responsible for the AFLP. By following this approach, a high success rate for the conversion of AFLP markers into locus-specific markers was obtained.     

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.     

Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat.

Sequence databases could be efficiently exploited for development of DNA markers if it were known which gene regions reveal the most polymorphism when amplified by PCR. We developed PCR primer pairs that target specific regions of previously sequenced genes from Avena and Zea species. Primers were targeted to amplify 40 introns, 24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48 maize inbred lines (previously assayed for simple-sequence repeat (SSR) polymorphism) for amplification-product polymorphism. We also developed primers to target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22 hexaploid oat cultivars and 2 diploid Avena species for amplification-product polymorphism. In maize, 67% of promoter markers, 58% of intron markers, and 13% of exon markers exhibited amplification-product polymorphisms. Among polymorphic primer pairs in maize, genotype diversity was highest for SSR markers (0.60) followed by intron markers (0.46), exon markers (0.42), and promoter markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58% of intron markers revealed polymorphisms, but among the cultivars only, 21% of SSR markers and 50% of intron markers were polymorphic. Polymorphic-sequence-tagged sites for plant-breeding applications can be created easily by targeting noncoding gene regions.     

Conversion of AFLP bands into high-throughput DNA markers

The conversion of AFLP bands into polymorphic sequence-tagged-site (STS) markers is necessary for high-throughput genotype scoring. Technical hurdles that must be overcome arise from genome complexity (particularly sequence duplication), from the low-molecular-weight nature of the AFLP bands and from the location of the polymorphism within the AFLP band. We generated six STS markers from ten AFLP bands (four AFLPs were from co-dominant pairs of bands) in soybean (Glycine max). The markers were all linked to one of two loci, rhg1 on linkage group G and Rhg4 on linkage group A2, that confer resistance to the soybean cyst nematode (Heterodera glycines I.). When the polymorphic AFLP band sequence contained a duplicated sequence or could not be converted to a locus-specific STS marker, direct sequencing of BAC clones anchored to a physical map generated locus-specific flanking sequences at the polymorphic locus. When the polymorphism was adjacent to the restriction site used in the AFLP analysis, single primer extension was performed to reconstruct the polymorphism. The six converted AFLP markers represented 996 bp of sequence from alleles of each of two cultivars and identified eight insertions or deletions, two microsatellites and eight single-nucleotide polymorphisms (SNPs). The polymorphic sequences were used to design a non-electrophoretic, fluorometric assay (based on the TaqMan technology) and/or develop electrophoretic STS markers for high-throughput genotype determination during marker-assisted breeding for resistance to cyst nematode. We conclude that the converted AFLP markers contained polymorphism at a 10- to 20-fold higher frequency than expected for adapted soybean cultivars and that the efficiency of AFLP band conversion to STS can be improved using BAC libraries and physical maps. The method provides an efficient tool for SNP and STS discovery suitable for marker-assisted breeding and genomics.     

Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11.

We have shown that many of the Alu repeats found in the GenBank database are polymorphic and that this polymorphism can be detected by a simple technique, single-strand conformation polymorphism (SSCP) analysis, after polymerase chain reaction (PCR) amplification of each repeat from DNA of individuals. Here, we describe a method for collecting many anonymous Alu repeats and their flanks in a chromosome-specific phage library and cloning them into plasmids. The flanking single-copy sequences of each repeat in the plasmid were then determined, and 20mer to 30mer segments of these sequences were used as primers for the PCR-SSCP analysis. Many new polymorphic DNA markers on chromosome 11 were obtained with this method. These markers can also serve as sequence-tagged sites for physical mapping of the genome.     

Characterization and compilation of polymorphic simple sequence repeat (SSR) markers of peanut from public database

ABSTRACT: BACKGROUND: There are several reports describing thousands of SSR markers in the peanut (Arachis hypogaea L.) genome. There is a need to integrate various research reports of peanut DNA polymorphism into a single platform. Further, because of lack of uniformity in the labeling of these markers across the publications, there is some confusion on the identities of many markers. We describe below an effort to develop a central comprehensive database of polymorphic SSR markers in peanut. FINDINGS: We compiled 1,343 SSR markers as detecting polymorphism (14.5%) within a total of 9,274 markers. Amongst all polymorphic SSRs examined, we found that AG motif (36.5%) was the most abundant followed by AAG (12.1%), AAT (10.9%), and AT (10.3%).The mean length of SSR repeats in dinucleotide SSRs was significantly longer than that in trinucleotide SSRs. Dinucleotide SSRs showed higher polymorphism frequency for genomic SSRs when compared to trinucleotide SSRs, while for EST-SSRs, the frequency of polymorphic SSRs was higher in trinucleotide SSRs than in dinucleotide SSRs. The correlation of the length of SSR and the frequency of polymorphism revealed that the frequency of polymorphism was decreased as motif repeat number increased. CONCLUSIONS: The assembled polymorphic SSRs would enhance the density of the existing genetic maps of peanut, which could also be a useful source of DNA markers suitable for high-throughput QTL mapping and marker-assisted selection in peanut improvement and thus would be of value to breeders. KEYWORDS: SSR, motif, polymorphism, cultivated peanut.     

Comparative analysis of the efficiency of intron-length polymorphism of β-tubulin genes and microsatellite loci for flax varieties genotyping

Efficacy of the β-tubulin introns lenght polymorphism method (TBP) was used for Ukrainian bredeed flax cultivars genotyping. For this purpose, TBP data were compared with data produced using the two most effective species-specific SSR markers. Both methods were used to evaluate intra- and intercultivar flax polymorphism. For each cultivar, PIC data (Polymorphism Information Content) and the range of allele lenghts, as well as the number of allele phenotypes, were calculated using TBP and SSR markers. The dendrograms, built using Nei and Li’s similarity coefficient, differ for SSR and TBP markers. Most flax cultivars of Ukrainian selection were genetically heterogeneous. The TBP method was highly efficient for differentiation of flax genotypes versus SSR analysis.     

Interspersed repetitive element polymerase chain reaction product mapping using a mouse interspecific backcross

We have developed a rapid method of generating and simultaneously mapping interrepeat polymerase chain reaction products using DNA from interspecific backcross animals derived from mating C57BL/6J and Mus spretus mice. This method is based on the high degree of B1, B2, and L1 dispersed repeat position polymorphism found between these two species of mouse. We have mapped 13 new loci to 9 different chromosomes and have found no evidence of clustering among these loci. The advantages of this approach are that no prior knowledge of sequence is required, a single PCR reaction generates many markers which can be mapped simultaneously, and only 50 ng of each backcross DNA (a finite resource) is required. We anticipate that many more markers remain to be characterized in this valuable new source of polymorphism.     

Application of single nucleotide polymorphism markers to chum salmon Oncorhynchus keta: discovery, genotyping and linkage phase resolution

This study describes (1) the application of new methods to the discovery of informative single nucleotide polymorphism (SNP) markers in chum salmon Oncorhynchus keta, (2) a method to resolve the linkage phase of closely linked SNPs and (3) a method to inexpensively genotype them. Finally, it demonstrates that these SNPs provide information that discriminates among O. keta populations from different geographical regions of the northern Pacific Ocean. These informative markers can be used in conjunction with mixed-stock analysis to learn about the spatial and temporal marine distributions of O. keta and the factors that influence the distributions.     

Detection of gene-anchored amplification polymorphism (GAAP) in the vicinity of plant mitochondrial genes

A simple, semi-automatable method was established for assessing polymorphism in plant mitochondrial genome. A set of 41 mitochondrial markers based on the published Arabidopsis thaliana sequence was developed in Brassicaceae using a gene-anchored amplification polymorphism (GAAP) strategy. PCR primers were selected based on conserved coding regions of mitochondrial genes and used to amplify the corresponding 5' and/or 3' non-coding flanking regions in order to maximise sequence variability between haplotypes. The variations in fragment size were analysed on a LiCor DNA sequencer, but the methodology is compatible with various sequencing systems using denaturing polyacrylamide gels. One advantage of the method is that GAAP products can be directly sequenced (without any cloning steps) through labelled M13 consensus sequences. Mitochondrial GAAP loci gave clear and simple patterns (one or two bands) that were easy to score and highly reproducible. Nearly all mitochondrial loci examined in A. thaliana were conserved within the Brassicaceae family, and half of the primers generated products when DNA from a distant species, Beta vulgaris (Chenopodiaceae), was used as template. The GAAP markers revealed low levels of polymorphism within species but exhibited a high level of polymorphism among genera and families. Our results showed some discrepancies with respect to the published mtDNA sequence of A. thaliana.     

High degree of transferability of 86 newly developed zebra finch EST-linked microsatellite markers in 8 bird species

High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3-51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.     

A suite of molecular markers for identifying species, detecting introgression and describing population structure in spadefoot toads (Spea spp.)

Two congeneric species of spadefoot toad, Spea multiplicata and Spea bombifrons, have been the focus of hybridization studies since the 1970s. Because complex hybrids are not readily distinguished phenotypically, genetic markers are needed to identify introgressed individuals. We therefore developed a set of molecular markers (amplified fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and single nucleotide polymorphism) for identifying pure-species, F1 hybrids and more complex introgressed types. To do so, we tested a series of markers across both species and known hybrids using populations in both allopatry and sympatry. We retained those markers that differentiated the two pure-species and also consistently identified known species hybrids. These markers are well suited for identifying hybrids between these species. Moreover, those markers that show variation within each species can be used in conjunction with existing molecular markers in studies of population structure and gene flow.     

Cloning of AFLP markers detected by fluorescence capillary electrophoresis

The available methods to isolate specific amplified fragment length polymorphism (AFLP) markers can be used only if markers are detected by radioactive labeling, silver staining, or ethidium bromide staining; these methods are useless if modern and automated genetic analyzers are used to detect AFLP markers by fluorescent labeling. We have developed a method that allows for isolation and cloning of specific AFLP markers obtained with a laser-induced fluorescence capillary electrophoresis system. This procedure has been tested on 5Arabidopsis thaliana polymorphic AFLP markers, and the nucleotide sequences obtained from these cloned markers were identified and located in theArabidopsis genome.     

Identifying disease polymorphisms from case–control genetic association data

In case-control association studies, it is typical to observe several associated polymorphisms in a gene region. Often the most significantly associated polymorphism is considered to be the disease polymorphism; however, it is not clear whether it is the disease polymorphism or there is more than one disease polymorphism in the gene region. Currently, there is no method that can handle these problems based on the linkage disequilibrium (LD) relationship between polymorphisms. To distinguish real disease polymorphisms from markers in LD, a method that can detect disease polymorphisms in a gene region has been developed. Relying on the LD between polymorphisms in controls, the proposed method utilizes model-based likelihood ratio tests to find disease polymorphisms. This method shows reliable Type I and Type II error rates when sample sizes are large enough, and works better with re-sequenced data. Applying this method to fine mapping using re-sequencing or dense genotyping data would provide important information regarding the genetic architecture of complex traits.     

Polymerase chain reaction-based genotyping for allotypic markers of immunoglobulin kappa shows allelic association of Km with kappa variable segment.

Allotypic markers of immunoglobulin kappa (Km) may be determined using a novel method of amplification of the constant segment (C kappa) (IGKC) by polymerase chain reaction (PCR) followed by restriction enzyme digestion. Restriction sites in the C kappa PCR product correlate with allotypic differences among Km(1), Km(1,2), and Km(3) alleles. An AccI site in the PCR product correlates with Km(3); and presence or absence of a MaeII site correlates with the Km(1) or Km(1,2) allele, respectively. Km allelic frequencies were determined in a Caucasian population and compared to genotypic frequencies of nearby polymorphic markers. Among unrelated individuals with rheumatoid arthritis (RA) and controls, there is no evidence of allelic association between CD8A and polymorphic markers of the immunoglobulin kappa region [a V kappa (IGKV) BglII polymorphism about 24 kb centromeric to C kappa, Km allotype, and a SacI polymorphism 3.5 kb telomeric to the C kappa segment]. Similarly, there is no allelic association of the SacI C kappa polymorphism with Km or with the BglII V kappa polymorphism. However, there is evidence of allelic association of V kappa B3 and Km, specifically between the V kappa BglII 2.2-kb allele and Km(3) and also between the V kappa 3.5-kb allele and Km(1,2). Therefore, Km typing by PCR-based methods suggests the presence of allelic association between polymorphisms within the coding region of the C kappa segment and the nearest V kappa segment.