AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat

Amplified fragment length polymorphism (AFLP) markers were used to enrich the map of the wheat chromosomal region containing the Thinopyrum-derived Lr19 leaf rust resistance gene. The region closest to Lr19 was targeted through the use of deletion and recombinant lines of the translocated segment. One of the AFLP bands thus identified was converted into a sequence-tagged-site (STS) marker. This assay generated a 130-bp PCR fragment in all Lr19-carrying lines tested, except for one deletion mutant, while non-carrier template failed to amplify any product. This sequence represents the first marker to map on the distal side of Lr19 on chromosome 7el₁. The conversion process of AFLP fragments to STS markers was technically difficult, mainly because of the presence of contaminating fragments. Various approaches were taken to reduce the frequency of false positives and to identify the correct clone. We were able to formulate a general verification strategy prior to clone sequencing. Various other factors causing problems with converting AFLP bands to an STS assays are also discussed. Received: 15 September 2000 / Accepted: 5 January 2001     

DNA methylation and AFLP marker distribution in the soybean genome

Amplified fragment length polymorphisms (AFLPs) have become important markers for genetic mapping because of their ability to reliably detect variation at a large number of loci. We report here the dissimilar distribution of two types of AFLP markers generated using restriction enzymes with varying sensitivities to cytosine methylation in the soybean genome. Initially, AFLP markers were placed on a scaffold map of 165 RFLP markers mapped in 42 recombinant inbred (F_6:7) lines. These markers were selected from a map of over 500 RFLPs analyzed in 300 recombinant inbred (F_6:7) lines generated by crossing BSR101×PI437.654. The randomness of AFLP marker map position was tested using a Poisson-model distribution. We found that AFLP markers generated using EcoRI/MseI deviated significantly from a random distribution, with 34% of the markers displaying dense clustering. In contrast to the EcoRI/MseI AFLP markers, PstI/MseI-generated AFLP markers did not cluster and were under represented in the EcoRI/MseI marker clusters. The restriction enzyme PstI is notably sensitive to cytosine methylation, and these results suggest that this sensitivity affected the distribution of the AFLP markers generated using this enzyme in the soybean genome. The common presence of one EcoRI/MseI AFLP cluster per linkage group and the infrequent presence of markers sensitive to methylation in these clusters are consistent with the low recombination frequency and the high level of cytosine methylation observed in the heterochromatic regions surrounding centromeres. Thus, the dense EcoRI/MseI AFLP marker clusters may be revealing structural features of the soybean genome, including the genetic locations of centromeres. Received: 5 November 1998 / Accepted: 20 February 1999     

An integrated high-density RFLP-AFLP map of tomato based on two Lycopersicon esculentum×L. pennellii F2 populations

 Two independent F₂ populations of Lycopersicon esculentum×L. pennellii which have previously been investigated in RFLP mapping studies were used for construction of a highly saturated integrated AFLP map. This map spanned 1482 cM and contained 67 RFLP markers, 1078 AFLP markers obtained with 22 EcoRI+MseI primer combinations and 97 AFLP markers obtained with five PstI+MseI primer combinations, 231 AFLP markers being common to both populations. The EcoRI+MseI AFLP markers were not evenly distributed over the chromosomes. Around the centromeric region, 848 EcoRI+ MseI AFLP markers were clustered and covered a genetic distance of 199 cM, corresponding to one EcoRI+ MseI AFLP marker per 0.23 cM; on the distal parts 1283 cM were covered by 230 EcoRI+MseI AFLP markers, corresponding to one marker per 5.6 cM. The PstI/MseI AFLP markers showed a more even distribution with 16 PstI/MseI AFLP markers covering a genetic distance of 199 cM around the centromeric regions and 81 PstI/MseI AFLP markers covering a genetic distance of 1283 cM on the more distal parts, corresponding to one marker per 12 and 16 cM respectively. In both populations a large number of loci showed a significant skewed segregation, but only chromosome 10 loci showed skewness that was similar for both populations. This ultra-dense molecular-marker map provides good perspectives for genetic and breeding purposes and map-based cloning. Received: 3 September 1998 / Accepted: 27 October 1998     

An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F₂ population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F₂ population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper. Received: 20 October 1999 / Accepted: 3 July 2000     

A high-density genetic map of Sorghum bicolor (L.) Moench based on 2926 AFLP®, RFLP and SSR markers

Using AFLP technology and a recombinant inbred line population derived from the sorghum cross of BTx623 × IS3620C, a high-density genetic map of the sorghum genome was constructed. The 1713 cM map encompassed 2926 loci distributed on ten linkage groups; 2454 of those loci are AFLP products generated from either the EcoRI/MseI or PstI/MseI enzyme combinations. Among the non-AFLP markers, 136 are SSRs previously mapped in sorghum, and 203 are cDNA and genomic clones from rice, barley, oat, and maize. This latter group of markers has been mapped in various grass species and, as such, can serve as reference markers in comparative mapping. Of the nearly 3000 markers mapped, 692 comprised a LOD 3.0 framework map on which the remaining markers were placed with lower resolution (LOD <3.0). By comparing the map positions of the common grass markers in all sorghum maps reported to date, it was determined that these reference markers were essentially collinear in all published maps. Some clustering of the EcoRI/MseI AFLP markers was observed, possibly in centromeric regions. In general, however, the AFLP markers filled most of the gaps left by the RFLP/SSR markers demonstrating that AFLP technology is effective in providing excellent genome coverage. A web site, http://SorghumGenome.tamu.edu, has been created to provide all the necessary information to facilitate the use of this map and the 2590 PCR-based markers. Finally, we discuss how the information contained in this map is being integrated into a sorghum physical map for map-based gene isolation, comparative genome analysis, and as a source of sequence-ready clones for genome sequencing projects.     

Two high-density AFLP® linkage maps of Zea mays L.: analysis of distribution of AFLP markers

This study demonstrates the relative ease of generating high-density linkage maps using the AFLP® technology. Two high-density AFLP linkage maps of Zea mays L. were generated based on: (1) a B73 × Mo17 recombinant inbred population and (2) a D32 × D145 immortalized F₂ population. Although AFLP technology is in essence a mono-allelic marker system, markers can be scored quantitatively and used to deduce zygosity. AFLP markers were generated using the enzyme combinations EcoRI/MseI and PstI/MseI. A total of 1539 and 1355 AFLP markers have been mapped in the two populations, respectively. Among the mapped PstI/MseIAFLP markers we have included fragments bounded by a methylated PstI site (^mAFLP markers). Mapping these ^mAFLP markers shows that the presence of C-methylation segregates in perfect accordance with the primary target sequence, leading to Mendelian inheritance. Simultaneous mapping of PstI/MseIAFLP and PstI/MseI ^mAFLP markers allowed us to identify a number of epi-alleles, showing allelic variation in the CpNpG methylation only. However, their frequency in maize is low. Map comparison shows that, despite some rearrangements, most of the AFLP markers that are common in both populations, map at similar positions. This would indicate that AFLP markers are predominantly single-locus markers. Changes in map order occur mainly in marker-dense regions. These marker-dense regions, representing clusters of mainly EcoRI/MseI AFLP and PstI/MseI ^mAFLP markers, co- localize well with the putative centromeric regions of the maize chromosomes. In contrast, PstI/MseImarkers are more uniformly distributed over the genome.     

DNA fingerprinting based on microsatellite-anchored fragment length polymorphisms,and isolation of sequence-specific PCR markers in lupin (Lupinus angustifolius L.)

We report a method of microsatellite-anchored fragment length polymorphisms for DNA fingerprinting. The method combines the concept of AFLP and the microsatellite-anchor primer technique. Genomic DNA was digested by one restriction enzyme MseI. One AFLP adaptor (MseI adaptor) was ligated onto the restriction fragments. DNA fingerprints were produced by PCR using one microsatellite-anchor primer in combination with one MseI-primer. The method allows co-amplification of over 100 DNA fragments containing microsatellite motifs per PCR. Polymorphisms detected from lupin by this method included those arising from variation in the number of microsatellite repeat units targeted by the microsatellite-anchor primers, from variation on the annealing sites for the SSR-anchor primers, from insertions/deletions outside the SSR region, and from variation in restriction sites. The first three types of polymorphisms were readily converted into sequence-specific PCR markers suitable for marker-assisted breeding.     

Identification and mapping of AFLP markers linked to peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) resistance to the aphid vector of groundnut rosette disease

Groundnut rosette disease is the most destructive viral disease of peanut in Africa and can cause serious yield losses under favourable conditions. The development of disease-resistant cultivars is the most effective control strategy. Resistance to the aphid vector, Aphis craccivora, was identified in the breeding line ICG 12991 and is controlled by a single recessive gene. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) analysis were employed to identify DNA markers linked to aphid resistance and for the development of a partial genetic linkage map. A F_2:3 population was developed from a cross using the aphid-resistant parent ICG 12991. Genotyping was carried out in the F₂ generation and phenotyping in the F₃ generation. Results were used to assign individual F₂ lines as homozygous-resistant, homozygous-susceptible or segregating. A total of 308 AFLP (20 EcoRI+3/MseI+3, 144 MluI+3/MseI+3 and 144 PstI+3/MseI+3) primer combinations were used to identify markers associated with aphid resistance in the F_2:3 population. Twenty putative markers were identified, of which 12 mapped to five linkage groups covering a map distance of 139.4 cM. A single recessive gene was mapped on linkage group 1, 3.9 cM from a marker originating from the susceptible parent, that explained 76.1% of the phenotypic variation for aphid resistance. This study represents the first report on the identification of molecular markers closely linked to aphid resistance to groundnut rosette disease and the construction of the first partial genetic linkage map for cultivated peanut.     

Integrated map of AFLP, SSLP and RFLP markers using a recombinant inbred population of rice (Oryza sativa L.)

 A molecular map of rice consisting of 231 amplified fragment length polymorphisms (AFLPs), 212 restriction fragment length polymorphisms (RFLPs), 86 simple-sequence length polymorphisms (SSLPs), five isozyme loci, and two morphological mutant loci [phenol staining of grain (Ph), semi-dwarf habit (sd-1)] has been constructed using an F₁₁ recombinant inbred (RI) population. The mapping population consisted of 164 RI lines and was developed via single-seed descent from an intercross between the genetically divergent parents Milyang 23 (M) (tongil type) and Gihobyeo (G) ( japonica type). A subset of previously mapped RFLP and SSLP markers were used to construct the map framework. The AFLP markers were derived from ten EcoRI(+2) and MseI(+3) primer combinations. All marker types were well distributed throughout the 12 chromosomes. The integrated map covered 1814 cM, with an average interval size of 3.4 cM. The MG map is a cornerstone of the Korean Rice Genome Research Program (KRGRP) and is being continuously refined through the addition of partially sequenced cDNA markers derived from an immature-seed cDNA library developed in Korea, and microsatellite markers developed at Cornell. The population is also being used for quantitative trait locus (QTL) analysis and as the basis for marker-assisted variety development. Received: 24 June 1997 / Accepted: 25 November 1997     

Genetic mapping of a fusarium wilt resistance gene (Fom-2) in melon (Cucumis melo L.)

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis is one of the most devastating diseases in melon production worldwide. The most effective control measure available is the use of resistant varieties. Identifying molecular markers linked to resistance genes can serve as a valuable tool for the selection of resistant genotypes. Bulked segregant analysis was used to identify markers linked to the Fom-2 genes, which confers resistance to races 0 and 1 of the fungal pathogen. Pooled DNA from homozygous resistant or homozygous susceptible progeny of F₂ cross between MR-1 and AY was screened using 240 PstI/MseI and 200 EcoRI/MseI primer combinations to identify AFLP markers linked to Fom-2. Fifteen markers potentially linked to Fom-2 were identified, all with EcoRI/MseI primer pairs. These were mapped relative to Fom-2 in a backcross (BC) population of 60 progeny derived from MR-1 × AY with AY as recurrent parent. Two AFLP markers (ACT/CAT1 and AAC/CAT1) flanked the gene at 1.7 and 3.3 cM, respectively. Moreover, AFLP marker AGG/CCC and the previously identified RAPD marker 596-1 cosegregated with Fom-2. These two dominant markers were converted to co-dominant markers by designing specific PCR primers that produced product length polymorphisms between the parents. A survey of 45 melon genotypes from diverse geographic origins with the co-dominant markers demonstrated a high correlation between fragment size and the resistance phenotype. These markers may therefore be useful in marker-assisted breeding programs.     

Physical and genetic mapping of amplified fragment length polymorphisms and the leaf rust resistance Lr3 gene on chromosome 6BL of wheat

The Argentinian wheat cultivar Sinvalocho MA carries the Lr3 gene for leaf rust resistance on distal chromosome 6BL. In this cultivar, 33 spontaneous susceptible lines were isolated and cytogenetically characterized by C-banding. The analysis revealed deletions on chromosome 6BL in most lines. One line was nulli-6B, two lines were ditelo 6BS, two, three, and ten lines had long terminal deletions of 40, 30, and 20%, respectively, three lines showed very small terminal deletions, and one line had an intercalary deletion of 11%. Physical mapping of 55 amplified fragment length polymorphism (AFLP) markers detected differences between deletions and led to the division of 6BL into seven bins delimited by deletion breakpoints. The most distal bin, with a length smaller than 5% of 6BL, contained 22 AFLP markers and the Lr3 gene. Polymorphism for nine AFLPs between Sinvalocho MA and the rust leaf susceptible cultivar Gamma 6 was used to construct a linkage map of Lr3. This gene is at a genetic distance of 0.9 cM from a group of seven closely linked AFLPs. The location of the gene in a high recombinogenic region indicated a physical distance of approximately 1 Mb to the markers.     

A genetic map of an interspecific cross in Allium based on amplified fragment length polymorphism (AFLPTM) markers

Segregation of 692 polymorphic AFLP^TM (amplified fragment length polymorphism) fragments was determined in an F₂ of the interspecific cross A. roylei x A. cepa. Two different enzyme combinations were used, PstI/MseIand EcoRI/MseI; in the latter one extra selective nucleotide was added to the MseI primer. The map based on A. cepa markers consisted of eight linkage groups with 262 markers covering 694 cM of the expected 800 cM. The map based on A. roylei markers comprised 15 linkage groups with 243 markers and had a length of 626 cM. The two maps were not integrated, and 25% of the markers remained unlinked. One of the alliinase genes and a SCAR marker linked to the disease resistance gene to downy mildew are present on this map. Of the AFLP markers, 50—80% were polymorphic between A. cepa and A. roylei; the level of polymorphic markers between different A. cepa accessions was4-8%. Received: 28 August 1998 / Accepted: 31 March 1999     

A genetic linkage map of tef [Eragrostis tef (Zucc.) Trotter] based on amplified fragment length polymorphism

A genetic linkage map of tef was constructed with amplified fragment length polymorphism (AFLP) markers using F₅ recombinant inbred lines (RILs) derived by single seed descent from the intraspecific cross of ’Kaye Murri’×’Fesho’. A total of 192 EcoRI/MseI primer combinations were screened for parental polymorphism. Around three polymorphic fragments per primer combination were detected, indicating a low polymorphism level in tef. Fifty primer combinations were selected to assay the mapping population, and 226 loci segregated among 85 F₅ RILs. Most AFLP loci behaved as dominant markers (presence or absence of a band), but about 15% of the loci were codominant. Significant deviations from the expected Mendelian segregation ratio were observed for 26 loci. The genetic linkage map comprised 211 markers assembled into 25 linkage groups and covered 2,149 cM of genome. AFLP is an efficient marker system for mapping plant species with low polymorphism such as tef. This is the first genetic linkage map constructed for tef. It will facilitate the mapping of genes controlling agronomically important traits and cultivar improvement in tef. Received: 27 April 1998 / Accepted: 4 January 1999     

An integrated interspecific AFLP map of lettuce (Lactuca) based on two L. sativa × L. saligna F2 populations

AFLP markers were obtained with 12 EcoRI/ MseI primer combinations on two independent F₂ populations of Lactuca sativa ×Lactuca saligna. The polymorphism rates of the AFLP products between the two different L. saligna lines was 39%, between the two different L. sativa cultivars 13% and between the L. sativa and L. saligna parents on average 81%. In both F₂ populations segregation distortion was found, but only Chromosome 5 showed skewness that was similar for both populations. Two independent genetic maps of the two F₂ populations were constructed that could be integrated due to the high similarity in marker order and map distances of 124 markers common to both populations. The integrated map consisted of 476 AFLP markers and 12 SSRs on nine linkage groups spanning 854 cM. The AFLP markers on the integrated map were randomly distributed with an average spacing between markers of 1.8 cM and a maximal distance of 16 cM. Furthermore, the AFLP markers did not show severe clustering. This AFLP map provides good opportunities for use in QTL mapping and marker-assisted selection. Received: 13 July 2000 / Accepted: 19 January 2001     

AFLP-derived STS markers for the identification of sex in Asparagus officinalis L.

For a simple, rapid and PCR-based screening of sex in the cultivated asparagus (Asparagus officinalis L.), we developed five STS markers from previously mapped, low-copy, sex-linked AFLP markers. A male/female PCR assay was feasible with these STS markers either by direct amplification or by digestion with restriction enzymes. Similar to the AFLP markers from which they were derived, STS4150.1, STS4150.2, STS4150.3 and STS3156 did not give recombinants in five different populations. STS3660 could be scored codominantly, enabling the differentiation of XY from YY males in the screened F₂ mapping population. The use of the sex-linked STS markers should allow early identification of sex, thus accelerating the breeding process for new asparagus varieties. Further, 10 additional AFLP markers obtained with PstI/MseI primer combinations have been mapped on the L5 chromosome, bringing the total number of known AFLP and STS markers flanking the sex locus to 24. These markers can be utilized for fine mapping of the sex gene in asparagus, which will pave the way for a map-based cloning approach. Received: 31 May 1999 / Accepted: 22 June 1999     

Further characterization of AFLP® data as a tool in genetic diversity assessments among maize (Zea mays L.) inbred lines

AFLP® markers generated by CNG methylation sensitive (PstI/MseI) and CNG methylation insensitive (EcoRI/MseI) enzyme combinations and AFLP markers collected from hypomethylated (PstI/MseI) and hypermethylated (^m PstI/MseI) regions were compared for their polymorphism information content, sampling variance and patterns of genetic diversity in a representative sample of 33 inbred lines of maize (Zea mays L.). We demonstrate that the mean polymorphism information content generated by sets of PstI/MseI and ^m PstI/MseI markers (0.38) is significantly higher than by sets ofEcoRI/MseI markers (0.33). Also the sampling variance highlighted the distinctive nature of the ^(m) PstI/MseI markers: to achieve a mean standard deviation of 5% in the estimation of genetic distance among the 33 inbreds, the PstI/MseI and ^m PstI/MseI marker sets (135 and 129 markers, respectively) are clearly smaller than the EcoRI/MseI marker set (173 markers). A further minimizing of the sampling variance of AFLP data in the estimation of genetic similarities was obtained by reducing marker information redundancy by selecting markers evenly distributed over each chromosome: a set of only 106 AFLP markers, sampled conditionally on their genetic map position, was required for a mean standard deviation of 5% in the estimation of genetic distance among the 33 inbreds.     

A study of modified forms of the Lr19 translocation of common wheat

 Following the induction of allosyndetic pairing between the Thinopyrum-derived Lr19 translocation in ‘Indis’ wheat and homoeologous wheat chromatin, eight suspected recombinants for the Lr19 region were recovered. These selections were characterised for marker loci that were previously used to construct a physical map of the Lr19 segment. At the same time near-isogenic lines were developed for some of the selected segments and tested for seedling leaf-rust resistance in order to confirm the presence of Lr19. It appeared that three of the four white-endosperm selections do not possess Lr19 and only one, 88M22-149, is a true Lr19 recombinant. The resistance gene in the three non-Lr19 selections resides on chromosome 6B, appears to derive from ‘Indis’, and was selected unintentionally during backcrossing. The pedigree of ‘Indis’ is suspect and it is believed that the Lr19 translocation in ‘Indis’ is in reality the Th. ponticum-derived (T4) segment rather than being of Th. distichum origin as was believed earlier. The white-endosperm recombinant, 88M22-149, retained the complete Lr19 resistance and was apparently re-located to chromosome arm 7BL in a double-crossover event. 88M22-149 has lost the Sd1 gene and often shows strong self-elimination in translocation heterozygotes. This effect may result from additional gametocidal loci or from an altered chromosome structure following re-location of the segment. 88M22-149 in fact contains a duplicated region involving the Wsp-B1 locus. Three selections had partially white endosperms and expressed Lr19 and other Thinopyrum marker alleles. Polymorphisms for the available markers confirmed that the translocated segment in at least one of them had been shortened through recombination with chromosome arm 7DL. Further markers need to be studied in order to determine whether the translocation in the remaining two partially white recombinants had also undergone recombination with wheat. The eighth selection has yellow endosperm and appears to self-eliminate in certain translocation heterozygotes. No evidence of recombination could be found with the markers used. If the latter selections are in fact recombinants they may prove useful in attempts to unravel the complex segregation distortion mechanism. Received: 8 August 1996 / Accepted: 10 January 1997     

Two genetic linkage maps of tetraploid roses

A tetraploid F₂ progeny segregating for resistance to black spot, growth habit, and absence of prickles on the stem and petioles was used to construct genetic linkage maps of rose. The F₁ of the progeny, 90–69, was created by crossing a black spot-resistant amphidiploid, 86–7, with a susceptible tetraploid, 82–1134. The F₁ was open-pollinated to obtain 115 seedlings. AFLP and SSR markers were used to eliminate seedlings produced through cross-fertilization. The remaining progeny set of 52 F₂ plants was used to study the inheritance of 675 AFLPs, one isozyme, three morphological and six SSR markers. AFLP markers were developed with three combinations of restriction enzymes, EcoRI/MseI, KpnI/MseI and PstI/MseI. Most of the markers appear to be in simplex or single-dose and segregated 3:1 in the progeny. One linkage map was constructed for each parent using only the single-dose markers. The map of 86–7 consists of 171 markers assigned to 15 linkage groups and covering more than 902 cM of the genome. The map of 82–1134 consists of 167 markers assigned to 14 linkage groups and covering more than 682 cM of the genome. In the AFLP analysis, EcoRI/MseI generated nearly twice as many markers per run than PstI/MseI. Markers developed with three restriction enzyme combinations showed a mixed distribution throughout the maps. A gene controlling the prickles on the petiole was located at the end of linkage group 7 on the map of 86–7. A gene for malate dehydrogenase locus 2 was located in the middle of linkage group 4 on the map of 86–7. These first-generation maps provide initial tools for marker- assisted selection and gene introgression for the improvement of modern tetraploid roses. Received: 20 June 2000 / Accepted: 13 January 2001     

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.     

A High-density Linkage Map of Lotus japonicus Based on AFLP and SSR Markers

A collection of 94 F₆ individuals derived from crosses between Lotus japonicus, Gifu B-129 (G) and Miyakojima MG-20 (M) were used for mapping. By using the HEGS running system, 427 EcoRI/MseI primer pairs were selected to generate a total of 2053 markers, consisting of 739 G-associated dominant markers, 674 M-associated dominant markers, 640 co-dominant markers, 95 SSR markers and 2 dCAPS markers. Excluding heavily distorted markers, 1588 were mapped to six chromosomes of the L. japonicus genome based on the 97 reference markers. This linkage map consisted of 1023 unique markers (excluding duplicated markers) and covered a total of 508.5 cM of the genome with an average chromosome length of 84.7 cM and interval distance of 0.50 cM. Fifteen quantitative traits loci for eight morphological traits were also mapped. This linkage map will provide a useful framework for physical map construction in L. japonicus in the near future.Key words: Lotus japonicus, AFLP, SSR, linkage map, HEGS (high efficiency genome scanning)