Comparative AFLP mapping in two hexaploid oat populations

Amplified fragment length polymorphisms (AFLPs) can be used to quickly develop linkage maps in plant species and are especially useful for crops with large genomes like oat (Avena sativa L., 2n=6x=42). High reproducibility and consistency are crucial if AFLP linkage maps are employed for comparative mapping. We mapped AFLP markers in combination with restriction fragment length polymorphism (RFLP) markers in two recombinant inbred populations of hexaploid oat in two laboratories to test the consistency of AFLP markers in a polyploid crop. Eight primer combinations produced 102 and 121 scoreable AFLP markers in the respective populations. In a population from the cross Kanota×Ogle, AFLP markers were placed onto a RFLP reference map consisting of 32 linkage groups. Nineteen linkage groups from another population from the cross Kanota×Marion were assigned to the reference map using AFLP and RFLP markers homologous to those used in the Kanota× Ogle cross. Reproducibility of AFLP assays was high in both laboratories and between laboratories. The AFLP markers were well-distributed across the genome in both populations. Many AFLP markers tended to extend the distance between adjacent RFLP markers in linkage analysis. Of the 27 polymorphic AFLPs common in both populations, 20 mapped to homologous linkage groups, 4 were unlinked in at least one population, and 3 mapped to different linkage groups in the two crosses. We believe that 1 of the 3 markers that mapped to a different linkage group in the two populations mapped to homoeologous linkage groups. The linkage map of hexaploid oat is not yet complete, and genomic rearrangements such as translocations exist among cultivars and are likely to account for the remaining two non-syntenous mapping results. AFLPs provide not only a fast and powerful tool for mapping but could be useful in characterizing genomic structural variations among germplasms in hexaploid oat. Received: 17 December 1999 / Accepted: 28 July 2000     

DNA cloning and sequence analysis of chicken AFLP

Amplified fragment length polymorphisms (AFLP) have been shown to be useful for linkage mapping in chickens and other domestic animals. It is often desirable to convert AFLP bands to sequence-tagged site (STS) markers, in particular, so that AFLP-based linkage information can be integrated with recombinant DNA clone-based maps. Sixteen chicken AFLP bands were excised from gels, re-amplified, cloned and analysed. All inserts proved to be EcoRI-TaqI fragments, which suggests that unlabelled TaqI-TaqI AFLP fragments do not amplify well, and therefore do not significantly contaminate AFLP bands. For eight of the AFLP, the cloned fragment was used to probe blots of AFLP reaction fingerprints, confirming that the predominant DNA clone indeed contained the polymorphic fragment. Flanking regions of selected AFLP fragments were isolated using Vectorette cloning. The results obtained suggest that the these chicken AFLP most commonly arise from sequence polymorphism at or near the TaqI site.     

Primary genetic linkage maps of the ascidian, Ciona intestinalis

For whole-genome analysis in a basal chordate (protochordate), we used F1 pseudo-testcross mapping strategy and amplified fragment length polymorphism (AFLP) markers to construct primary linkage maps of the ascidian tunicate Ciona intestinalis. Two genetic maps consisted of 14 linkage groups, in agreement with the haploid chromosome number, and contained 276 and 125 AFLP loci derived from crosses between British and Neapolitan individuals. The two maps covered 4218.9 and 2086.9 cM, respectively, with an average marker interval of 16.1 and 18.9 cM. We observed a high recombinant ratio, ranging from 25 to 49 kb/cM, which can explain the high degree of polymorphism in this species. Some AFLP markers were converted to sequence tagged sites (STSs) by sequence determination, in order to create anchor markers for the fragmental physical map. Our recombination tools provide basic knowledge of genetic status and whole genome organization, and genetic markers to assist positional cloning in C. intestinalis.     

A physical amplified fragment-length polymorphism map of Arabidopsis.

We have positioned amplified fragment-length polymorphism (AFLP) markers directly on the genome sequence of a complex organism, Arabidopsis, by combining gel-based AFLP analysis with in silico restriction fragment analysis using the published genome sequence. For placement of the markers, we used information on restriction fragment size, four selective nucleotides, and the rough genetic position of the markers as deduced from the analysis of a limited number of Columbia (Col)/Landsberg (Ler) recombinant inbred lines. This approach allows for exact physical positioning of markers as opposed to the statistical localization resulting from traditional genetic mapping procedures. In addition, it is fast because no extensive segregation analysis is needed. In principle, the method can be applied to all organisms for which a complete or nearly complete genome sequence is available. We have located 1,267 AFLP Col/Ler markers resulting from 256 SacI+2, MseI+2 primer combinations to a physical position on the Arabidopsis genome. The positioning was verified by sequence analysis of 70 markers and by segregation analysis of two leaf-form mutants. Approximately 50% of the mapped Col/Ler AFLP markers can be used for segregation analysis in Col/C24, Col/Wassilewskija, or Col/Cape Verde Islands crosses. We present data on one such cross: the localization of a viviparous-like mutant segregating in a Col/C24 cross.     

Application of AFLP markers to genome mapping in poultry

The amplified fragment length polymorphism (AFLP) technique has been used to enhance marker density in the East Lansing reference chicken genome map, using a backcross family derived from a Red Jungle Fowl by White Leghorn mating with White Leghorn as the recurrent parent. To date, 204 AFLP markers have been added, expanding overall map coverage by about 25%. To the limits of our resolution, AFLP markers are distributed relatively evenly across the EL reference map. AFLP are about 60% as frequent in a cross within White Leghorns (line 7(2) x 6(3)) in comparison to the more divergent reference map population. Based on apparent identity of size, about 40% of the 7(2) x 6(3) cross AFLP fragments were also polymorphic in the reference map cross. Primer pairs in which one primer contains 3' extensions of three selective nucleotides and the other has two selective nucleotides successfully generated AFLP from chicken DNA, but such pairs appeared to amplify only a subset of those fragments to which they have an exact sequence match. Three different restriction enzymes with 4 bp recognition sites (TaqI, HinP1I and MspI) were found to work well with EcoRI as the rarer of the two AFLP restriction enzymes used, with HinP1I being the most effective of the three. AFLP markers are likely to provide an economical method with which to enhance framework linkage maps of chicken and probably other avian genomes.     

AFLP Linkage Map of the Oomycete Phytophthora infestans

Here we present the first comprehensive genetic linkage map of the heterothallic oomycetous plant pathogen Phytophthora infestans. The map is based on polymorphic DNA markers generated by the DNA fingerprinting technique AFLP (Vos et al., 1995, Nucleic Acids Res. 23: 4407-4414). AFLP fingerprints were made from single zoospore progeny and 73 F1 progeny from two field isolates of P. infestans. The parental isolates appeared to be homokaryotic and diploid, their AFLP patterns were mitotically stable, and segregation ratios in the F1 progeny were largely Mendelian. In addition to 183 AFLP markers, 7 RFLP markers and the mating type locus were mapped. The linkage map comprises 10 major and 7 minor linkage groups covering a total of 827 cM. The major linkage groups are composed of markers derived from both parents, whereas the minor linkage groups contain markers from either the A1 or the A2 mating type parent. Non-Mendelian segregation ratios were found for the mating type locus and for 13 AFLP markers, all of which are located on the same linkage group as the mating type locus. Copyright 1997 Academic Press     

Conversion of AFLP markers to high-throughput markers in a complex polyploid,sugarcane

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications.     

Construction of an AFLP genetic map with nearly complete genome coverage in Pinus taeda

  De novo construction of complete genetic linkage maps requires large mapping populations, large numbers of genetic markers, and efficient algorithms for ordering markers and evaluating order confidence. We constructed a complete genetic map of an individual loblolly pine (Pinus taeda L.) using amplified fragment length polymorphism (AFLP) markers segregating in haploid megagametophytes and PGRI mapping software. We generated 521 polymorphic fragments from 21 AFLP primer pairs. A total of 508 fragments mapped to 12 linkage groups, which is equal to the Pinus haploid chromosome number. Bootstrap locus order matrices and recombination matrices generated by PGRI were used to select 184 framework markers that could be ordered confidently. Order support was also evaluated using log likelihood criteria in MAPMAKER. Optimal marker orders from PGRI and MAPMAKER were identical, but the implied reliability of orders differed greatly. The framework map provides nearly complete coverage of the genome, estimated at approximately 1700 cM in length using a modified estimator. This map should provide a useful framework for merging existing loblolly pine maps and adding multiallelic markers as they become available. Map coverage with dominant markers in both linkage phases will make the map useful for subsequent quantitative trait locus mapping in families derived by self-pollination. Received: 7 August 1998 / Accepted: 27 October 1998     

Linkage maps for Arabidopsis lyrata subsp. lyrata and Arabidopsis lyrata subsp. petraea combining anonymous and Arabidopsis thaliana-derived markers.

Arabidopsis lyrata, a close relative of the model plant Arabidopsis thaliana, is 1 of a few plant species for which the genome is to be entirely sequenced, which promises to yield important insights into genome evolution. Only 2 sparse linkage maps have been published, and these were based solely on markers derived from the A. thaliana genome. Because the genome of A. lyrata is practically twice as large as that of A. thaliana, the extent of map coverage of the A. lyrata genome remains uncertain. In this study, a 2-way pseudo-testcross strategy was used to construct genetic linkage maps of A. lyrata subsp. petraea and A. lyrata subsp. lyrata, using simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers from the A. thaliana genome, and anonymous amplified fragment length polymorphism (AFLP) markers that could potentially uncover regions unique to the A. lyrata genome. The SSR and CAPS markers largely confirmed the relationships between linkage groups in A. lyrata and A. thaliana. AFLP markers slightly increased the coverage of the A. lyrata maps, but mostly increased marker density on the linkage groups. We noted a much lower level of polymorphism and a greater segregation distortion in A. lyrata subsp. lyrata markers. The implications of these findings for the sequencing of the A. lyrata genome are discussed.     

AFLP Linkage Map of the OomycetePhytophthora infestans

Here we present the first comprehensive genetic linkage map of the heterothallic oomycetous plant pathogenPhytophthora infestans.The map is based on polymorphic DNA markers generated by the DNA fingerprinting technique AFLP (Voset al.,1995,Nucleic Acids Res.23:4407–4414). AFLP fingerprints were made from single zoospore progeny and 73 F1 progeny from two field isolates ofP. infestans.The parental isolates appeared to be homokaryotic and diploid, their AFLP patterns were mitotically stable, and segregation ratios in the F1 progeny were largely Mendelian. In addition to 183 AFLP markers, 7 RFLP markers and the mating type locus were mapped. The linkage map comprises 10 major and 7 minor linkage groups covering a total of 827 cM. The major linkage groups are composed of markers derived from both parents, whereas the minor linkage groups contain markers from either the A1 or the A2 mating type parent. Non-Mendelian segregation ratios were found for the mating type locus and for 13 AFLP markers, all of which are located on the same linkage group as the mating type locus.     

Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing. Received: 11 April 1996 / Accepted: 14 June 1996     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

AFLP标记的特点及其在昆虫学研究中的应用

扩增片段长度多态性（AFLP）是一种新兴的很有效的分子遗传标记方法, 它通过对基因组DNA限制性内切酶酶切片段进行选择性扩增而揭示多态性,具有快速、经济简便、不需要预先知道模板DNA的信息、模板需要量少、重复性高、结果可靠及具有很高的信息含量等优点。AFLP也具有缺点,主要是标记是显性的,同其他显性标记一样,不能区分杂合体和纯合体,因而不能更好地估算种群遗传的变异,对种群遗传结构的分析不能提供更多的统计信息;AFLP技术较复杂,而且经常使用放射性同位素,对模板DNA质量要求也较高。为了克服AFLP的这些缺点,人们又在其基础上发展了其他相关技术,例如AFRP、SAMPL、DALP和TE-AFLP等。目前AFLP在昆虫方面的应用还不是很多,处于初级阶段,主要应用在生态型鉴定、种群遗传分析、连锁图谱构建等方面,相信随着其技术的发展完善,必将会越来越多地应用于昆虫学的研究中。     

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.     

Development of an AFLP based linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and construction of a Ler/Cvi recombinant inbred line population

An amplified fragment polymorphism (AFLP) based linkage map has been generated for a new Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred line (RIL) population. A total of 321 molecular PCR based markers and the erecta mutation were mapped. AFLP markers were also analysed in the Landsberg erecta/Columbia (Ler/Col) RIL population ( Lister & Dean 1993) and 395 AFLP markers have been integrated into the previous Arabidopsis molecular map of 122 RFLPs, CAPSs and SSLPs. This enabled the evaluation of the efficiency and robustness of AFLP technology for linkage analyses in Arabidopsis. AFLP markers were found throughout the linkage map. The two RIL maps could be integrated through 49 common markers which all mapped at similar positions. Comparison of both maps led to the conclusion that segregating bands from a common parent can be compared between different populations, and that AFLP bands of similar molecular size, amplified with the same primer combination in two different ecotypes, are likely to correspond to the same locus. AFLPs were found clustering around the centromeric regions, and the authors have established the map position of the centromere of chromosome 3 by a quantitative analysis of AFLP bands using trisomic plants. AFLP markers were also used to estimate the polymorphism rate among the three ecotypes. The larger polymorphism rate found between Ler and Cvi compared to Ler and Col will mean that the new RIL population will provide a useful material to map DNA polymorphisms and quantitative trait loci.     

High resolution linkage maps of the model organism Petunia reveal substantial synteny decay with the related genome of tomato

Two linkage maps were constructed for the model plant Petunia. Mapping populations were obtained by crossing the wild species Petunia axillaris subsp. axillaris with Petunia inflata, and Petunia axillaris subsp. parodii with Petunia exserta. Both maps cover the seven chromosomes of Petunia, and span 970 centimorgans (cM) and 700 cM of the genomes, respectively. In total, 207 markers were mapped. Of these, 28 are multilocus amplified fragment length polymorphism (AFLP) markers and 179 are gene-derived markers. For the first time we report on the development and mapping of 83 Petunia microsatellites. The two maps retain the same marker order, but display significant differences of recombination frequencies at orthologous mapping intervals. A complex pattern of genomic rearrangements was detected with the related genome of tomato (Solanum lycopersicum), indicating that synteny between Petunia and other Solanaceae crops has been considerably disrupted. The newly developed markers will facilitate the genetic characterization of mutants and ecological studies on genetic diversity and speciation within the genus Petunia. The maps will provide a powerful tool to link genetic and genomic information and will be useful to support sequence assembly of the Petunia genome.     

Linkage Mapping Identifies the Sex Determining Region as a Single Locus in the Pennate Diatom Seminavis robusta

The pennate diatom Seminavis robusta, characterized by an archetypical diatom life cycle including a heterothallic mating system, is emerging as a model system for studying the molecular regulation of the diatom cell and life cycle. One of its main advantages compared with other diatom model systems is that sexual crosses can be made routinely, offering unprecedented possibilities for forward genetics. To date, nothing is known about the genetic basis of sex determination in diatoms. Here, we report on the construction of mating type-specific linkage maps for S. robusta, and use them to identify a single locus sex determination system in this diatom. We identified 13 mating type plus and 15 mating type minus linkage groups obtained from the analysis of 463 AFLP markers segregating in a full-sib family, covering 963.7 and 972.2 cM, respectively. Five linkage group pairs could be identified as putative homologues. The mating type phenotype mapped as a monogenic trait, disclosing the mating type plus as the heterogametic sex. This study provides the first evidence for a genetic sex determining mechanism in a diatom.     

Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping

By combining the amplified fragment length polymorphism (AFLP) technique with selective genotyping, we constructed a linkage map for rice and assigned each linkage group to a corresponding chromosome. The AFLP map, consisting of 202 AFLP markers, was generated from 74 recombinant inbred lines (RIL) which were selected from both extremes of the population (250 lines) with respect to the response to complete submergence. Map length was 1756 cM, with an average interval size of 8.5 cM. To assign linkage groups to chromosomes, we used 50 previously mapped AFLP markers as anchor markers distributed over the 12 chromosomes. Other AFLP markers were then assigned to specific chromosomes based on their linkage to anchor markers. This AFLP map is equivalent to the RFLP/AFLP map constructed previously as the anchors were in the same order in both maps. Furthermore, tests with two restriction fragment length polymorphism (RFLP) markers and two sequence-tagged site (STS) markers showed that they mapped in the expected positions. Using this AFLP map, a major gene for submergence tolerance was localized on chromosome 9. Quantitative trait loci (QTL) associated with submergence tolerance were detected on chromosomes 6, 7, 11, and 12. We conclude that the combination of AFLP mapping and selective genotyping provides a much faster and easier approach to QTL identification than the use of RFLP markers. Received: 20 December 1996 / Accepted: 21 January 1997     

Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to a SCAR marker for rapid selection

A Brassica juncea mapping population was generated and scored for seed coat colour. A combination of bulked segregant analysis and AFLP methodology was employed to identify markers linked to seed coat colour in B. juncea. AFLP analysis using 16 primer combinations revealed seven AFLP markers polymorphic between the parents and the bulks. Individual plants from the segregating population were analysed, and three AFLP markers were identified as being tightly linked to the seed coat colour trait and specific for brown-seeded individuals. Since AFLP markers are not adapted for large-scale application in plant breeding, our objective was to develop a fast, cheap and reliable PCR-based assay. Towards this goal, we employed PCR-walking technology to isolate sequences adjacent to the linked AFLP marker. Based on the sequence information of the cloned flanking sequence of marker AFLP8, primers were designed. Amplification using the locus-specific primers generated bands at 0.5 kb and 1.2 kb with the yellow-seeded parent and a 1.1-kb band with the brown-seeded parent. Thus, the dominant AFLP marker (AFLP8) was converted into a simple codominant SCAR (Sequence Characterized Amplified Region) marker and designated as SCM08. Scoring of this marker in a segregating population easily distinguished yellow- and brown-seeded B. juncea and also differentiated between homozygous (BB) and heterozygous (Bb) brown-seeded individuals. Thus, this marker will be useful for the development of yellow seed B. juncea cultivars and facilitate the map-based cloning of genes responsible for seed coat colour trait. Received: 2 October 1999 / Accepted: 11 November 1999     

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.