Assessment of AFLP marker behaviour in enriching STS radiation hybrid maps.

Radiation hybrid (RH) mapping provides a powerful tool to build high-resolution maps of genomes. Here, we demonstrate the use of the AFLP technique for high-throughput typing of RH cell lines. Cattle were used as the model species because an RH panel was available to investigate the behaviour of AFLP markers within the microsatellite- and STS-based maps of this species. A total of 747 AFLP markers were typed on the TM112 RH radiation panel and 651 of these were assigned by two-point analysis to the 29 bovine autosomes and sex chromosomes. AFLP markers were added to the 1222 microsatellite and STS markers that were included in earlier RH maps. Multipoint maps were constructed for seven example chromosomes, which retained 248 microsatellite and STS markers, and added 123 AFLP markers at LOD 4. The addition of the AFLP markers increased the number of markers by 42.1% and the map length by 10.4%. The AFLP markers showed lower retention frequency (RF) values than the STS markers. The comparison of RF values in AFLP markers and their corresponding AFLP-derived STSs demonstrated that the lower RF values were due to the lower detection sensitivity of the AFLP technique. Despite these differences, AFLP and AFLP-derived STS markers mapped to identical or similar positions. These results demonstrate that it is possible to merge AFLP and microsatellite markers in the same map. The application of AFLP technology could permit the rapid construction of RH maps in species for which extensive genome information and large numbers of SNP and microsatellite markers are not available.

A high-density, integrated genetic linkage map of lettuce (Lactuca spp.).

An integrated map for lettuce comprising of 2,744 markers was developed from seven intra- and inter-specific mapping populations. A total of 560 markers that segregated in two or more populations were used to align the individual maps. 2,073 AFLP, 152 RFLP, 130 SSR, and 360 RAPD as well as 29 other markers were assigned to nine chromosomal linkage groups that spanned a total of 1,505 cM and ranged from 136 to 238 cM. The maximum interval between markers in the integrated map is 43 cM and the mean interval is 0.7 cM. The majority of markers segregated close to Mendelian expectations in the intra-specific crosses. In the two L. saligna x L. sativa inter-specific crosses, a total of 155 and 116 markers in 13 regions exhibited significant segregation distortion. Data visualization tools were developed to curate, display and query the data. The integrated map provides a framework for mapping ESTs in one core mapping population relative to phenotypes that segregate in other populations. It also provides large numbers of markers for marker assisted selection, candidate gene identification, and studies of genome evolution in the Compositae.

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs.

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers.

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.

A note on the measurement of genetic diversity within genebank accessions of lettuce (Lactuca sativa L.) using AFLP markers.

This paper discusses a statistical approach for measuring genetic diversity within genebank accessions of a self-fertilising species. This approach is applied to lettuce (Lactuca sativa L.), using AFLP marker data on a set of 1,390 accessions, representing six different lettuce types. Knowledge of the within-accession genetic diversity is important for decisions about the way accessions have to be maintained by genebanks. It is argued that if the within-accession diversity is small, as can be expected for a self-fertilising species like L. sativa, the best approach is to sample as many accessions as possible with only two plants per accession and estimate the within-accession diversity by the proportion of accessions of which the individuals are different.

Application of AFLP technology to radiation hybrid mapping.

We have investigated the use of AFLP technology as a tool for the high throughput enrichment of Radiation Hybrid (RH) maps. The 3000 rad TM112 bovine RH panel was assayed with 37 EcoRI/TaqI AFLP primer combinations. The number of selective nucleotides used during PCR was increased to seven, to reduce the complexity of the AFLP profile and minimise the overlap between hamster and bovine bands co-amplified from hybrid cell clones. Seven-hundred-forty-seven bovine AFLP bands were amplified that could be distinguished following electrophoresis. Repeatability was tested within and between laboratories on independent template preparations and an error rate of 1.3% found. Two-point linkage analysis clustered 428 AFLP fragments in 39 linkage groups of at least 4 markers. Multi-point maps were constructed for 5 sample linkage groups. The study demonstrated that the AFLP approach could be used to rapidly screen for the most informative clones during panel construction and to increase the number of markers on RH maps, which could be useful for joining linkage groups formed by other markers. The use of AFLP markers as anchor points between existing RH maps and other physical maps, such as BAC contigs, is also discussed.

Discrimination between selected lines of pigs using AFLP markers.

Amplified fragment length polymorphic (AFLP) markers were used to discriminate between lines of pigs, divergently selected over seven generations for components of efficient lean growth rate. A total of 270 animals with 30 animals per line were genotyped for 239 polymorphic AFLP markers. Canonical variate analysis identified linear combinations of the AFLP marker scores that grouped animals by selection line with no overlap between selection lines. Cluster analysis of AFLP marker scores identified 10 groups of animals with 226 of the 270 animals clustered into nine groups, each consisting of animals from only one selection line. AFLP marker genotyping, using the EcoRI and TaqI restriction enzymes, provided an effective means of discriminating between animals of different selection lines that have arisen from one base population.

QTLs for resistance to powdery mildew in pepper under natural and artificial infections.

Epidemics of powdery mildew due to Leveillula taurica is an increasing problem in pepper production areas, particularly in coastal regions or greenhouse cultivation. The highly resistant genitor 'H3' was submitted to genetic analysis and QTL mapping in order to promote the introgression of its oligogenic resistance into large and sweet-fruited cultivars. The doubled-haploid progeny from the cross 'H3' (resistant) by 'Vania' (susceptible) was tested for resistance under both natural field infection and artificial inoculation tests, and QTL detection was compared for those two methods. Seven genomic regions including additive QTLs and epistatic interactions were detected, explaining altogether the major part of genotypic variance. Two genomic regions were common to both the evaluation methods, whereas other QTLs were method-specific, reflecting the environment dependence of powdery mildew epidemics. Orthologies with tomato genomic regions carrying resistance genes to L. taurica and Oidium lycopersicum were revealed by comparative mapping with pepper. Tight linkages between the detected QTLs and virus resistance or fruit color traits in pepper were also shown, which adds to the agronomic importance of these regions in pepper breeding programs.

A genetic linkage map of Guinea yam ( Dioscorea rotundata Poir.) based on AFLP markers.

A genetic linkage map of the tetraploid white yam ( Dioscorea rotundata Poir.) was constructed based on 341 co-dominantly scored amplified fragment length polymorphism (AFLP) markers segregating in an intraspecific F(1) cross. The F(1) mapping population was produced by crossing a landrace cultivar TDr 93-1 as female parent to a breeding line TDr 87/00211 as the male parent. The marker segregation data were split into maternal and paternal data sets, and separate genetic linkage maps were constructed since the mapping population was an F(1) cross between two presumed heterozygous parents. The markers segregated like a diploid cross-pollinator population suggesting that the D. rotundata genome is an allo-tetraploid (2n = 4 x = 40). The maternal map comprised 155 markers mapped on 12 linkage groups with a total map length of 891 cM. Three linkage groups consisted of maternal parent markers only. The paternal map consisted of 157 markers mapped on 13 linkage groups with a total map length of 852 cM. Three and one quantitative trait loci (QTLs) with effects on resistance to Yam Mosaic Virus (YMV) were identified on the maternal and paternal linkage maps, respectively. Prospects for detecting more QTLs and using marker-assisted selection in white yam breeding appear good, but this is subject to the identification of additional molecular markers to cover more of the genome.

A genetic linkage map of water yam ( Dioscorea alata L.) based on AFLP markers and QTL analysis for anthracnose resistance.

A genetic linkage map of the tetraploid water yam ( Dioscorea alata L.) genome was constructed based on 469 co-dominantly scored amplified fragment length polymorphism (AFLP) markers segregating in an intraspecific F(1) cross. The F(1) was obtained by crossing two improved breeding lines, TDa 95/00328 as female parent and TDa 87/01091 as male parent. Since the mapping population was an F(1) cross between presumed heterozygous parents, marker segregation data from both parents were initially split into maternal and paternal data sets, and separate genetic linkage maps were constructed. Later, data analysis showed that this was not necessary and thus the combined markers from both parents were used to construct a genetic linkage map. The 469 markers were mapped on 20 linkage groups with a total map length of 1,233 cM and a mean marker spacing of 2.62 cM. The markers segregated like a diploid cross-pollinator population suggesting that the water yam genome is allo-tetraploid (2n = 4 x = 40). QTL mapping revealed one AFLP marker E-14/M52-307 located on linkage group 2 that was associated with anthracnose resistance, explaining 10% of the total phenotypic variance. This map covers 65% of the yam genome and is the first linkage map reported for D. alata. The map provides a tool for further genetic analysis of traits of agronomic importance and for using marker-assisted selection in D. alata breeding programmes. QTL mapping opens new avenues for accumulating anthracnose resistance genes in preferred D. alata cultivars.

An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.

Towards an expanded and integrated linkage map of cucumber (Cucumis sativus L.).

Linkage maps in cucumber (Cucumis sativus var. sativus L.) have been constructed using morphological traits, isozymes, restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs). The lack of polymorphism in cucumber has led to the construction of relatively unsaturated maps (13- to 80-point). We have added amplified fragment length polymorphism (AFLP) markers to existing narrow-based (within C. sativus) and wide-based (C. sativus x C. sativus var. hardwickii) maps. JOINMAP v. 2.0 was used to construct maps and to join these with historical maps from several previous studies. Our narrow- and wide-based merged maps contain 255 and 197 markers, respectively, including morphological traits, disease resistance loci, isozymes, RFLPs, RAPDs, and AFLPs. Condensation of total map distance occurred in merged maps compared to historic maps using many of the same markers. This phenomenon is most likely due to differences in map construction algorithms. The merged maps represent the best fit of the data used and are an important first step towards the construction of a comprehensive linkage map for cucumber. Identification of additional anchor markers between the narrow- and wide-based maps presented here may allow their future integration into a unified model.

A genetic map of cucumber composed of RAPDs, RFLPs, AFLPs, and loci conditioning resistance to papaya ringspot and zucchini yellow mosaic viruses.

The watermelon strain of papaya ringspot virus (PRSV-W) and zucchini yellow mosaic virus (ZYMV) are potyviruses that cause significant disease losses in cucumber. Resistances have been identified primarily in exotic germplasm that require transfer to elite cultivated backgrounds. To select more efficiently for virus resistances, we identified molecular markers tightly linked to PRSV-W and ZYMV resistances in cucumber. We generated F6 recombinant inbred lines (RILs) from a cross between Cucumis sativus L. 'Straight 8' and a line from 'Taichung Mou Gua', TMG1 (susceptible and resistant, respectively, to both viruses), and studied the segregations of amplified fragment length polymorphism (AFLP) markers, randomly amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), and resistances to PRSV-W and ZYMV. A 353-point map of cucumber was generated, delineating 12 linkage groups at LOD 3.5. Linkage arrangements among RFLPs were consistent with previously published maps; however linkages among RAPDs in our map did not agree with a previously published map. Resistances to PRSV-W and ZYMV were tightly linked (2.2 cM) and mapped to the end of one linkage group. One AFLP cosegregated with resistance to ZYMV.

The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids.

Mi-1, a Lycopersicon peruvianum gene conferring resistance to the agricultural pests, root-knot nematodes, and introgressed into tomato, has been cloned using a selective restriction fragment amplification based strategy. Complementation analysis of a susceptible tomato line with a 100 kb cosmid array yielded a single cosmid clone capable of conferring resistance both to the root-knot nematode Meloidogyne incognita and to an unrelated pathogen, the potato aphid Macrosiphum euphorbiae. This resistance was stable. The Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat-containing type of plant resistance genes.

Identification of AFLP molecular markers for resistance against Melampsora larici-populina in Populus.

We have identified AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus defined this resistance. This locus, which we have designated Melampsora resistance (Mer), confers resistance against E1, E2, and E3, three different races of Melampsora larici-populina. In order to identify molecular markers linked to the Mer locus we decided to combine two different techniques: (1) the high-density marker technology, AFLP, which allows the analysis of thousands of markers in a relatively short time, and (2) the Bulked Segregant Analysis (BSA), a method which facilitates the identification of markers that are tightly linked to the locus of interest. We analyzed approximately 11,500 selectively amplified DNA fragments using 144 primer combinations and identified three markers tightly linked to the Mer locus. The markers can be useful in current breeding programs and are the basis for future cloning of the resistance gene.

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers.

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21-GP179 interval. Two of those could not be separated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.

AFLP: a new technique for DNA fingerprinting.

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.

Transient occurrence of extrachromosomal DNA of an Arabidopsis thaliana transposon-like element, Tat1.

Analysis of 11 genomic clones containing the S-adenosylmethionine synthetase 1 gene (sam1) of Arabidopsis thaliana revealed the presence of a 431-base-pair (bp) insertion in the 3' end of sam1 in one of these clones. The inserted sequence, called Tat1, shows structural features of a transposon. It is flanked by a 5-bp duplication of the target site DNA and has 13-bp inverted repeats at its termini. Two highly homologous elements situated in a different genomic context were isolated from a genomic library. Genomic Southern analysis indicates that there are at least four copies of Tat1 present in the A. thaliana ecotype Columbia genome. Different hybridization patterns are observed with DNAs derived from different ecotypes of Arabidopsis thaliana, indicating that the element has moved since the divergence of these ecotypes. In two populations of A. thaliana, linear extrachromosomal Tat1-homologous DNA has been observed. The presented data are consistent with the hypothesis that Tat1 is an active transposable element.

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana.

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.

An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein.

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.