An AFLP-Based Procedure for the Efficient Mapping of Mutations and DNA Probes in Barley

Corresponding author: F. Salamini, Max-Planck-Institut für Züchtungsforschung, Carl-von-Linne ´weg, 10, 50829 Köln, Germany., salamini{at}mpiz-koeln.mpg.de (E-mail).

Communicating editor: W. F. SHERIDAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A strategy based upon AFLP markers for high-efficiency mapping of morphological mutations and DNA probes to linkage groups in barley is presented. First, 511 AFLP markers were placed on the linkage map derived from the cross Proctor x Nudinka. Second, loci controlling phenotypic traits were assigned to linkage groups by AFLP analysis, using F₂ populations consisting of 30–50 mutant plants derived from crosses of the type "mutant x Proctor" and "mutant x Nudinka." To map DNA probes, 67 different wild-type barley lines were selected to generate F₂ populations by crossing with Proctor and Nudinka. F₂ plants that were polymorphic for a given RFLP fragment were classified into genotypic classes. Linkage of the RFLP polymorphism to 1 of the 511 AFLP loci was indicated by cosegregation. The use of the strategy is exemplified by the mapping of the mutation branched-5 to chromosome 2 and of the DNA probes Bkn2 and BM-7 to chromosomes 5 and 1, respectively. Map expansion and marker order in map regions with dense clustering of markers represented a particular problem. A discussion considering the effect of noncanonical recombinant products on these two parameters is provided.

MORE than 1000 molecular markers, predominantly RFLPs, are mapped onto barley chromosomes (GRANER et al. 1991 ; HEUN et al. 1991 ; KLEINHOFS et al. 1993 ; KASHA and KLEINHOFS 1994 ). Recently, the amplified fragment length polymorphism (AFLP) procedure (VOS et al. 1995 ) has provided a convenient and reliable tool with which to generate markers to further facilitate map construction (BECKER et al. 1995 ; QI et al. 1997 ; WAUGH et al. 1997 ). The AFLP method is a PCR-based technique that avoids the laborious steps involved in restriction fragment length polymorphism (RFLP) mapping. Like RFLPs, the majority of AFLP fragments define unique loci in the barley genome (VOS et al. 1995 ; QI and LINDHOUT 1997 ; WAUGH et al. 1997 ). Here we report the use of AFLP markers to efficiently map mutations and DNA probes to barley linkage groups. AFLP analysis has a very high diversity index (RUSSELL et al. 1997 ), resulting in a limited number of primer combinations required to screen a whole genome. In this respect, the method for integrating genetic and molecular maps presented in this article is novel. To implement this procedure it was necessary to (1) place a sufficient number of AFLP markers on a barley linkage map constructed from a cross of two specific barley lines, (2) obtain F₂ populations in which barley mutations segregated in crosses with the mapping parents, and (3) generate a set of F₂ populations segregating at specific RFLP loci that can be mapped on the basis of their linkage with AFLP polymorphisms.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material:
The 113 doubled haploid barley lines (DH lines) used for mapping originated from a cross between the lines Proctor and Nudinka (HEUN et al. 1991 ). Seeds were provided, together with the parental lines, by M. HEUN in 1991, and were maintained at the Max-Planck-Institut für Züchtungsforschung (MPIZ; Köln, Germany). The 67 barley lines used in crosses with Proctor and Nudinka for mapping of DNA probes were selected from a collection of 5842 accessions obtained from the plant germplasm bank in Braunschweig (Germany). Their origins, gene bank numbers, and MPIZ collection numbers are reported in Table 1. Crosses between each of these lines and the varieties Proctor and Nudinka were done at the MPIZ. Seed from F₂ progeny of individual F₁ plants was harvested separately and stored at 4°. F₃ seed was harvested from single F₂ plants from the cross v.h. elses (G397 in Table 1) x Nudinka for mapping of the Bkn2 gene, and from the cross v.h. isthmos (G392 in Table 1) x Nudinka to map the BM-7 gene.

A set of barley mutants (Table 2) was crossed with Proctor and Nudinka to generate F₂ populations. These were stored as such or grown in the field, where wild-type (WT) and mutant (M) plants were selected and stored as F₃ seed families. The segregating populations of the mutants listed in Table 2, together with the genetic materials reported in Table 1, are available to those interested in using our procedure.

The barley mutant branched-5 (brc-5) was isolated from the Braunschweig seed collection (see above). This line is also homozygous for the dominant allele K at the Hooded locus. The mutant was crossed to Nudinka and Proctor, to generate F₂ populations. The 45 F₂ M plants from the cross with Nudinka and the15 F₂ M plants from the cross with Proctor, used in mapping, were selected in the field and F₃ seed was harvested. DNA was extracted from a pool of 20 F₃ seeds for each F₂ plant.

DNA techniques:
Seeds of the barley lines were planted in the greenhouse and seedlings were harvested at the four-leaf stage for DNA extraction (SAGHAI-MAROOF et al. 1984 ; or the "QIAtip 100" protocol of QIAGEN, Hilden, Germany).

The original AFLP procedure as described by ZABEAU and VOS 1993 and VOS et al. 1995 was followed using the minor modifications of BECKER et al. 1995 . The selection of biotinylated fragments was avoided in the mapping experiments for BM-7 and Bkn2. Adapters and the MseI and EcoRI primers used were as follows: MseI adapters, GACGATGAGTCCTGAG and TACTCAGGACTCAT; MseI universal primer (MU), GATGAGTCCTGAGTA; MseI +1 primer (M01), MU+A; MseI +3 primers, M32, MU+AAC; M33, MU+AAG; M34, MU+AAT; M36, MU+ACC; M38, MU+ACT; M40, MU+AGC; M43, MU+ATA; M44, MU+ATC; M46, MU+ATT; EcoRI adapters, CTCGTAGACTGCGTACC and CATCTGACGCATGGTTAA; EcoRI universal primer (EU), GACTGCGTACCAATTC; EcoRI +1 adapters, EU+A; EcoRI +3 primers: E34, EU+AAT; E35, EU+ACA; E36, EU+ACC; E37, EU+ACG; E40, EU+AGC; E41, EU+AGG; E42, EU+AGT; and E43, EU+ATA. All sequences are given in the 5' to 3' direction. All PCR reactions were carried out in a UNO-Thermoblock (Biometra, Göttingen, Germany). Amplified fragments were separated on 4.5% polyacrylamide gels, at 58 W for 1 hr in 0.5x TBE. A dephosphorylated and -³³P-labeled 1-kb ladder (GIBCO BRL, Gaithersburg, MD) was used as size marker.

The RFLP analysis was performed essentially as described by GEBHARDT et al. 1989 . The restriction enzymes TaqI, MspI, MseI, RsaI, and AluI (Boehringer Mannheim, Mannheim, Germany) were used to digest the DNA samples. A total of 7 µg of DNA was loaded per lane on 4.5% polyacrylamide gels and run at 40 W for 6 hr, electroblotted onto Hybond-N filters at 20 A for 1 hr, and probed with [-³²P]dCTP randomly labeled probes (FEINBERG and VOGELSTEIN 1984 ).

The inverse sequence-tagged repeat (ISTR)-based technique was performed as described in ROHDE 1996 . Forward and backward primers, designed to reveal polymorphisms connected with copia-like elements, were labeled with -³³P and used in standard PCR reactions incorporating an annealing step at 45° for 30 sec. PCR products were separated on 4% polyacrylamide gels.

Scoring and mapping:
The E and M AFLP primers were combined in all 72 possible combinations [16 were used earlier by BECKER et al. 1995 ]. Each mapped AFLP fragment can be identified by the number of its primer combination and an additional digit that refers to the figure stored under "A visual catalog of AFLP bands polymorphic between the barley lines Proctor and Nudinka," at the Web site http://www.mpiz-koeln.mpg.de/salamini/salamini.html (for example, the AFLP marker e3432-7 corresponds in the figure to band 7 obtained with the primer combination E34-M32).

In the 113 DH lines, polymorphic bands were scored as 0 or 1 for absence or presence, respectively, and were tested against the expected 1:1 segregation ratio using a chi-squared test (P = 0.05). Only AFLP data segregating 1:1 were added to the datafile of BECKER et al. 1995 and analyzed using MAPMAKER (LANDER et al. 1987 ; UNIX version /EXP3.0b) and JoinMap (STAM 1993 ; PC/MS-DOS 1.4 version) programs. All the AFLP-mapped bands are reported in Table 3, with the corresponding subgroup assignment. Allelic state of AFLP bands in autoradiograms was controlled independently twice. Furthermore, singletons (or doubletons; see DISCUSSION) were identified by computer analysis, and the existence of the concerned polymorphisms was checked again in the autoradiograms.

Data analysis with MAPMAKER was performed with and without the ERROR DETECTION option. RFLP loci mapped in the original Proctor x Nudinka cross (HEUN et al. 1991 ) were chosen as backbone markers, by virtue of their order reliability supported by data from other mapping populations. The backbone RFLPs are indicated, in Figure 1, to the left of each chromosome, where the number in parentheses refers to the mapping population from which they are derived [their relative distances were recalculated from the Proctor x Nudinka RFLP/AFLP integrated map of BECKER et al. 1995 ]. The mapping populations are numbered in Figure 1 as follows: (1) Steptoe x Morex (KLEINHOFS et al. 1993 ); (2) Harrington x TR306 (TINKER et al. 1996 ); (3) Blenheim x E24/3 (THOMAS et al. 1995 ); (4) Franger x Rupee (DE SCENZO et al. 1994 ); (5) T. Prentice x V. Gold (KJAER et al. 1995 ); (6) Betzes x Golden Promise (LAURIE et al. 1993 ); (7) Captain x H. spontaneum (LAURIE et al. 1993 ); (8) Steffi x Atlas (SCHWEIZER et al. 1995 ); (9) Igri x Triumph (LAURIE et al. 1995 ); (10) H. spontaneum x SE16 (SHERMAN et al. 1995 ); (11) Dicktoo x Morex (HAYES and MESZAROS 1997 ); (12) Chebec x Harrington (LANGRIDGE et al. 1996A ); (13) Igri x Franka (GRANER et al. 1994 ); (14) Vada x H. spontaneum (GRANER et al. 1991 ); (15) Galleon x Haruna nijo (LANGRIDGE et al. 1996C ); (16) Proctor x Nudinka (LIU et al. 1993 ); (17) Proctor x Nudinka (RODER et al. 1993 ); (18) Bonus lax-a¹ x H. spontaneum (LAURIE et al. 1996 ); (19) Clipper x Sahara (LANGRIDGE et al. 1996B ); and (20) Integrated map (QI et al. 1997 ).

View larger version (148K): In this window In a new window Download PPT slide   Figure 1. Linkage maps of the seven barley chromosomes based on 113 DH lines derived from the cross Proctor x Nudinka. (A) Chromosome 1, (B) chromosome 2, (C) chromosome 3, (D) chromosome 4, (E) chromosome 5, (F) chromosome 6, (G) chromosome 7. For each chromosome, a backbone RFLP map is given on the left (see text for details). On the right, the backbone map is integrated with AFLP and ISTR loci. The RFLP/AFLP/ISTR map is completed by indications of linkage subgroups (1–68). Markers represented in large boldface type have been placed at LOD 3.00 in the framework of each chromosome; markers in a smaller boldface type have been placed in unique positions at LOD 2.00; markers in italics have been assigned to an interval; and markers with an asterisk have been placed with the TRY command of MAPMAKER. Numbers in parentheses correspond to references cited in MATERIALS AND METHODS, scoring and mapping section.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mapping of AFLP markers and ISTRs in the Proctor x Nudinka cross:
Proctor and Nudinka were analyzed with 72 AFLP primer combinations and each combination yielded on average 7.1 polymorphic AFLP markers. Of 6299 readable bands (87.5 per primer combination), 833 (14.0%) were polymorphic. The 116 AFLP markers mapped by BECKER et al. 1995 by using 113 DHs were considered together with the 395 new AFLP loci. In total, 511 AFLP markers and 32 ISTRs were added to the RFLP map of HEUN et al. 1991 . In total, 57,743 AFLP data points were produced, with 12% missing data. There were slightly more Nudinka than Proctor alleles (51% vs. 49%). In performing MAPMAKER analysis, the backbone markers assigned to linkage groups were not ordered. By means of the ASSIGN command (LOD 3.0 and 2.5), all other markers were placed. The LINKS and ATTACH commands were used to attribute markers to the most likely chromosome in a few cases. To order all markers on the assigned chromosomes, three-point data analysis was performed at LOD 3.0, with a maximum distance of 50 cM. The ORDER command was given twice for each chromosome using 100 as the minimum number of informative DH lines. When the program failed to find a starting order, this number was decreased to 50. The ORDER command was given also at LOD 2.0 to map markers that could not be placed at LOD 3.0. The TRY command was given to place all those markers for which the program was unable to find a location. Figure 1 shows the combined RFLP/AFLP/ISTR map (on the right-hand side) of each linkage group. Since the order of markers in dense clusters cannot be precisely established with a population of the size that we have used (see DISCUSSION), we divided the seven linkage groups into 68 subgroups, within which the most probable (although not definitive) order of markers is given in Figure 1. The order and the relative distance between the backbone markers were in good agreement with the data of BECKER et al. 1995 . Minor changes were observed in marker-dense regions, especially when flanked by gaps (map regions extending for long distances without intervening markers). Such changes concerned chromosome 2 (subgroup 19), chromosome 4 (in a region spanning the subgroups 36–39), and the telomeric region of chromosome 7. The inversion of marker order on chromosome 2 was also observed by SHERMAN et al. 1995 . The rearrangement on chromosome 4 affects a cluster containing many AFLP markers; an inverted order of RFLPs is reported here by LANGRIDGE et al. 1996A , LANGRIDGE et al. 1996B . The finding of AFLP markers beyond the putative telomeric marker XcsuBG141 (RODER et al. 1993 ) on chromosome 1 is in agreement with SHERMAN et al. 1995 . The backbone markers on chromosome 6 are in agreement with BECKER and HEUN 1995 . On chromosome 2, markers e4238-3 and e4133-1 are inverted as compared to BECKER et al. 1995 . Some gaps present on the BECKER et al. 1995 map have been filled: on chromosome 3 by the subgroup 26 markers; on chromosome 3 by ISTR9 between subgroups 26 and 27; on chromosome 1 by subgroup 3 markers; on chromosome 6 by ISTR34.

The RFLP/AFLP/ISTR data were also analyzed using the ERROR DETECTION option of MAPMAKER. This option considers the probability at each locus that its allelic configuration with respect to flanking markers arises in part from typing errors. Significant corrections in the total length of the map resulted, leading to a reduction from 2673 to 1597 cM (see DISCUSSION). Other changes were also observed: chromosome 1 was shortened by less then 10%, with markers e4040-2 and e4138-3 being inverted; chromosome 3 was shortened about 10-fold within each subgroup and 1.5-fold in the intervals between subgroups; chromosome 4 was 4-fold shortened mainly in the region spanning subgroups 36–38, resulting in a placement of markers XcnlWG181 and XcnlWG232 in agreement with the original Proctor x Nudinka map; chromosome 5 was shortened by 3-fold on average within subgroups, and by a factor of two in the intervals; chromosome 7 was shortened within subgroups 59 (10x), 60–63 (4x), 65 (3x), 66 (5x), and 67–68 (2x). In the latter case, a drastic rearrangement of marker order occurred. When the JoinMap program was used, the total length of the map resulted in 1264 cM.

Mapping mutant alleles of loci that control phenotypic traits to the AFLP map:
The brc-5 mutation is recessive and conditions the elongation of the rachilla, which is the second-order ramification axis of the barley ear. The elongated rachilla develops as an ear rachis, thus generating a ramified ear phenotype (Figure 2, A–C). The brc-5 mutation was mapped using 45 F₂ brc-5/brc-5 plants derived from the cross brc-5 x Nudinka, together with 5 WT F₂ plants. Primer combination E36M36 produced one AFLP band (e3636-2) linked to the brc-5 allele. Two out of 45 homozygous brc-5 F₂ plants were recombinants, which corresponds to a linkage of 2.5 cM ± 1 (P = 0.05). The primer combinations E40M32 and E43M38 revealed linkage of brc-5 with e4032-10 and e4338-2, two markers that map on chromosome 2 close to e3636-2. Linkage mapping, which considered 36 segregating bands obtained with 11 AFLP primer combinations, positioned the locus on chromosome 2 between markers e4338-2 and e3636-2, in a region spanning 8 cM. Figure 2E shows the segregation of AFLP band e3636-2 in 45 homozygous brc-5 F₂ plants, while Figure 2D depicts the region of chromosome 2 where the brc-5 locus maps. AFLP amplifications with primers E42M46, E41M40, E43M36, E35M46, E37M32, E41M34, and E41M44 were also carried out to confirm that associations between mutant phenotype and other segregating AFLP bands were not caused by linkage, but by distorted or chance segregation. Data derived from a small F₂ population of 15 brc-5 plants from the cross Proctor x brc5 confirmed the map location and allowed the scoring of markers that were previously uninformative in theNudinka cross, such as e4246-6 (repulsion; 2 recombinants), e3732-5 (repulsion; 1 recombinant), e4336-2 (repulsion; 0 recombinants), and e4140-8 (repulsion; 0 recombinants).

View larger version (83K): In this window In a new window Download PPT slide   Figure 2. Assignment of the brc-5 locus to barley chromosome 2. (A) Phenotype of WT and (B) brc-5 plants. (C) Scanning electron microscope (SEM) image of the ear primordia of a brc-5 plant (1.5 cm = 500 µm). The rachilla (the axis of the spikelet) is elongated, giving the ear a ramified habitus. An ectopic ear is indicated by the arrow. SEM was performed according to BOWMAN et al. 1989 . (D) Region of chromosome 2, subgroup 17, where the brc-5 locus was mapped. (E) AFLP mapping of the brc-5 locus. N, Nudinka; P, Proctor; b, brc-5; m, missing datum. The other lanes refer to the 45 F2 M plants from the brc-5 x Nudinka cross. The AFLP band e3636-2, present in Nudinka and absent in brc-5, is present only in the F2 M plants 1 and 17.

Mapping DNA probes on the Nudinka x Proctor AFLP map:
The incidence of RFLPs in 67 barley stocks was assayed using genomic and cDNA probes. Genomic probes, in part obtained from A. GRANER (Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany), revealed various levels of polymorphism. Probe MWG58 was polymorphic when tested on TaqI or AluI digests. The MWG611-AluI probe-enzyme combination allowed the detection of polymorphisms in 20% of the barley stocks; when the combination Bkn3 promoter probe and AluI was tested, 15% of lines were polymorphic. MWG634, tested on MspI- and RsaI-digested DNAs, revealed different allelic states in 6 and 25% of genotypes, respectively. The degree of polymorphism detected was lower when cDNA probes were used, particularly when cDNAs for barley homeobox genes were tested (2.5% of the lines resulted polymorphic for cBkn3 when tested on AluI-digests). In some cases, no polymorphism was observed for these genes, even when using genomic probes. For MADS-box genes and Adh cDNAs, the level of polymorphism (between 4 and 20%) was relatively high, similar to the results reported for the hordein genes (KANAZIN et al. 1993 ), a barley embryo desiccation-induced gene, and the thiamin gene (PECCHIONI et al. 1993 ). The AFLP-based mapping procedure for DNA probes was tested with a homeobox (Bkn-2) and a MADS box-containing (BM-7) genes.

The Bkn2 gene contains a homeodomain and codes for a putative transcriptional activator. In Southern analysis with the enzyme RsaI, a 1.5-kb genomic PstI/SalI fragment revealed a polymorphism between the barley line vulgare hybernum and Nudinka. The polymorphism consisted of a 320-bp fragment in vulgare hybernum that was absent in Nudinka. Sixty F₂ plants were classified on the basis of their RFLP pattern and fingerprinted with the AFLP primer combinations E37M38, E40M38, E42M32, E37M33, E41M34, E42M44, E42M36, E35M46, E40M44, E35M40, E43M43, and E36M36. The primer combination E40M44 amplified a band, e4044-1, linked in coupling to the presence of the 320-bp RFLP fragment. Primers E43M43 and E42M36, which amplify bands linked to e4044-1, were also tested on the same 60 F₂ plants. The Bkn2 gene was mapped to chromosome 5 on linkage subgroup 47, close to markers e4044-1, e4236-7, e4343-9, and e4343-4. The mapping of Bkn2 was thus possible by testing 14 AFLP primer combinations, allowing the detection of about 98 polymorphisms (7 per primer combination). A similar approach carried out with RFLP markers would have been much more demanding.

The second probe mapped was the MADS box-containing gene BM-7. A cDNA clone of 600 bp revealed RFLP between Nudinka and v.h. isthmos (Figure 3A). The analysis of 45 F₂ plants derived from a cross between these two lines revealed three genotypic groups. Group 1 was homozygous for the 450-bp Nudinka fragment, group 2 was homozygous for the 410-bp v.h. isthmos fragment, and group 3 was heterozygous (Figure 3B). DNA from each of these F₂ plants was analyzed using the AFLP primer combinations E43M38, E36M36, E40M32, E40M40, E40M36, E42M43, E40M38, E35M46, E37M34, and E37M40. The primer combination E40M36 amplified a fragment (e4036-2) missing in the 11 plants homozygous for the 450-bp RFLP fragment of Nudinka (Figure 3B and Figure C), suggesting a close linkage for the two markers. The data obtained allowed the gene BM-7 to be placed on chromosome 1 in subgroup 7 (Figure 3D). The gene mapped near nudum (n), a locus mapping approximately 3 cM from the multiovary (TAZHIN 1980 ), which is a putative mutant for a MADS box-like gene resulting in transformation of stamens into female organs (MENA et al. 1986 ). The BM-7 DNA sequence is available at the Web site cited in MATERIALS AND METHODS.

View larger version (72K): In this window In a new window Download PPT slide   Figure 3. Assignment of the BM-7 gene to barley chromosome 1. (A) Southern analysis of 38 WT barley lines (only some of those listed in Table 1). Only the variety v.h. isthmos (v) revealed a polymorphism between Nudinka (N) when RsaI-digested DNAs were hybridized with the BM-7 probe. (B) Southern blot of F2 plants from the cross Nudinka x v.h. isthmos probed with BM-7. v, v.h. isthmos; N, Nudinka; m, missing datum. Arrows in A and B indicate v.h. isthmos-specific bands. (C) AFLP analysis of the 45 F2 plants with the primer combination E40M36. Genotype no. 1 was missing in the AFLP analysis. Note that all plants missing the AFLP band e4036 (arrow) are homozygous for the 450-bp RFLP Nudinka fragment, indicating a close linkage between the RFLP and AFLP loci. (D) Chromosome 1 linkage map in the region where the BM-7 gene was mapped.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Knowledge of the precise position of mutant loci on molecular maps can lead to their association with specific genes, when these are also precisely mapped. This approach was followed by MULLER et al. 1995 to associate the barley-Hooded phenotype with a mutation in the homeobox-encoding gene Knox3. This strategy requires a dense linkage map. To increase the number of mapped loci, 511 AFLP markers were placed on the Proctor x Nudinka map. As the genetic background of existing barley mutants was different from those of Proctor and Nudinka, F₂ populations from crosses with each of these two parental lines were generated. It was expected that a monomorphic AFLP allele identified in a "mutant x Proctor" cross would have been polymorphic in the "mutant x Nudinka" cross. These F₂ populations were used for AFLP mapping experiments, where linkage of an AFLP fragment to the mutant locus was revealed by significant deviations from the expected Mendelian ratio of 3:1. When the AFLP fragment was present in the wild type (coupling configuration), its presence in 75% of the F₂ plants homozygous for the mutant allele indicated independent segregation, while a frequency of 0% indicated tight linkage. On the other hand, the incidence of F₂ mutant plants having the AFLP marker in repulsion configuration varied from 75% for the absence of linkage to 100% for complete linkage. The estimate of linkage in repulsion was thus less secure than that of the coupling configuration. For this reason, in scoring AFLP markers in F₂ populations, more reliance was placed on bands linked in repulsion to a mutation.

AFLP bands closely linked to a given mutation can be identified in the figure reported at the Web site http://www.mpiz-koeln.mpg.de/salamini/salamini.htm/. Their positions on the linkage map can be found by consulting Table 3. The use of this table allowed the identification of further primer combinations capable of generating other polymorphisms at linked AFLP loci. In the best case of mutant mapping so far encountered, data from a few AFLP gels were sufficient to enable a single experienced scientist to map the mutation brc-5 on chromosome 2 at a distance of 2.3 cM from each of the nearest flanking markers. This was possible because several AFLP markers were scored in each gel, thus leading to more rapid mapping of mutations than described, for example, in Arabidopsis for the RFLP-based method by FABRI and SCHAFFNER 1994 . Several other PCR methods for rapid mutation mapping in Arabidopsis are reported by WILLIAMS et al. 1993 and KONIECZY and AUSUBEL (1993). However, these methods are only extensions of the bulk segregant analysis procedure described by MICHELMORE et al. 1991 . This method is useful to enrich for PCR markers in the vicinity of a given genetic locus but does not assign the locus to a specific linkage group. Our efforts will now concentrate on the production of F₂ populations from crosses with mutant lines not yet listed in Table 2.

The mapping of DNA probes required, in addition to AFLP analysis, an RFLP step. Once an RFLP was found between Proctor or Nudinka and 1 of the 67 barley lines chosen as representative of the genetic variability present within the species, the corresponding F₂ population was selected. F₂ plants were classified according to their allelic state at the RFLP locus and AFLP analysis was carried out on the same materials. The combined RFLP and AFLP data allowed the detection of linkage between the RFLP and AFLP loci, as shown for genes Bkn2 and BM-7.

The method proposed avoids some of the problems encountered when mapping DNA probes to barley chromosomes. Barley has a low degree of DNA polymorphism (GRANER et al. 1990 ; HEUN et al. 1991 ). Based on Southern data, the RFLP probes of HEUN et al. 1991 , as well as RFLP probes mapped in other crosses, revealed polymorphisms in only a limited number of genetic stocks (LAURIE et al. 1992 ; PECCHIONI et al. 1993 ). It follows that in crosses involving the lines Proctor and Nudinka, a considerable fraction of the RFLP loci revealed by random probes are monomorphic. Using the RFLP technique, nevertheless, one has a sufficient probability of finding at least 1 polymorphic line among the 67 listed in Table 1. In crosses between this line and Proctor and Nudinka, F₂ plants can be classified genotypically by using the RFLP probe. The AFLP analysis performed on the same F₂ plants exploits the very-high-diversity index of these markers (RUSSELL et al. 1997 ). The combination of the two marker techniques, in conclusion, is capable of overcoming the cited drawbacks. Thus, mapping of almost any DNA probe can be achieved using a single restriction enzyme for Southern analysis.

The second problem that has been encountered concerns map expansion and marker order in dense linkage maps. When 511 AFLP polymorphisms were added to the HEUN et al. 1991 map, a substantial increase in map length from 1096 to 2673 cM was observed. Typing errors are proposed to be, in part, responsible for map expansion (LINCOLN and LANDER 1992 ). SÄLL and NILSSON (1994) designate as "singletons" those cases of single markers that recombine in a chromosomal region where flanking markers have a parental allelic state. Singletons, in addition to originating from scoring errors, are also the products of double crossover events, which are increasingly detected when maps are enriched with more markers. For barley, a map density-dependent increase in detection of double crossovers contradicts the finding that the number of crossovers estimated from RFLP data of medium-density maps is already significantly higher than the number of chiasmata observed in cytological studies (NILSSON et al. 1993 ; SÄLL and NILSSON 1994). This suggests that meiotic products, which are assumed to derive from double crossover events, may have a different origin. For example, singletons could be the products of meiotic gene conversion as predicted by the double-strand break repair model of recombination in yeast (SZOSTAK et al. 1983 ), where 50% of products of the resolution of the Holliday junction retain parental flanking sequences. Data from maize (CIVARDI et al. 1994 ; XU et al. 1995 ; OKAGAKI and WEIL 1997 ) and barley (BUSCHGES et al. 1997 ) also support the occurrence of double-strand break repair in plants. Our data demonstrate that, regardless of their origin, singletons increase map length and influence gene order in dense maps. When we used the ERROR DETECTION option of MAPMAKER, a reduction in map length from 2673 to 1597 cM was observed and the marker order within linkage subgroups was also modified. Similar conclusions were drawn from the analysis of the same set of data with the JoinMap program, which also seems to eliminate the products of noncanonical recombination events. In the latter case, the contraction of the map length was even more drastic.

A known phenomenon related to dense linkage maps is the clustering of markers in specific chromosomal regions, as reported for barley (BECKER et al. 1995 ; POWELL et al. 1997 ; QI et al. 1997 ), wheat (HART 1994 ), tomato (TANKSLEY et al. 1992 ), rice (NANDI et al. 1997 ), and potato (VAN ECK et al. 1995 ). Although no unequivocal explanations for clustering have been found, the suggested hypotheses have considered centromeric suppression of recombination (TANKSLEY et al. 1992 ; FRARY et al. 1996 ), amplification of polymorphic centromeric repetitive sequences (QI et al. 1997 ), and preferential amplification of the AT-rich region by MseI-based primers, as possible mechanisms (ROUPPE VAN DER VOORT et al. 1997 ). It is interesting to note that some of the linkage gaps present in the RFLP Proctor x Nudinka map are still devoid of markers after AFLP analysis. As the linkage gaps present in different molecular maps of barley (for references see MATERIALS AND METHODS) are in part located in different chromosomal regions, it is tempting to speculate that they may in part correspond to regions of genetic similarity between the chromosomal DNAs of the two strains used to construct a given map. We are currently approaching the problem by developing dense AFLP maps in different mapping populations.

	FOOTNOTES

¹ These authors made equal contributions to this work and are listed in alphabetical order.

	ACKNOWLEDGMENTS

We thank Dr. UDDA LUNDQVIST, the Barley Genetic Stock Center (Colorado), the Braunschweig (Germany) germplasm collection for providing mutant genotypes and barley lines, and Prof. FRANCKOWIAK (North Dakota University) for the msg mutants. We also acknowledge S. EFFGEN and M. ACCERBI for their excellent technical assistance. C.P. received a European Community grant (contract no. BIO4CT965023).

Manuscript received December 26, 1997; Accepted for publication May 8, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BECKER, J. and M. HEUN, 1995  Mapping of digested and undigested random amplified microsatellite polymorphisms in barley. Genome 38:991-998[Medline].

BECKER, J., P. VOS, M. KUIPER, F. SALAMINI, and M. HEUN, 1995  Combined mapping of AFLP and RFLP markers in barley. Mol. Gen. Genet. 249:65-73[Medline].

BOWMAN, J. L., D. R. SMYTH, and E. M. MEYEROWITZ, 1989  Genes directing flower development in Arabidopsis.. Plant Cell 1:37-52[Abstract/Free Full Text].

BÜSCHGES, R., K. HOLLRICHER, R. PANSTRUNGA, R. SIMONS, and G. SIMONS et al., 1997  The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 88:695-705[Medline].

CIVARDI, L., Y. J. XIA, K. J. EDWARDS, P. S. SCHNABLE, and B. J. NIKOLAU, 1994  The relationship between genetic and physical distances in the cloned a1-sh2 interval of the Zea mays L. genome. Proc. Natl. Acad. Sci. USA 91:8268-8272[Abstract/Free Full Text].

DE SCENZO, R. A., R. P. WISE, and M. MAHADEVAPPA, 1994  High resolution mapping of Hor1/Mla/Hor2 region on chromosome 5S in barley. Mol. Plant-Microbe Interact. 7:657-666.

FABRI, C. O. and A. R. SCHÄFFNER, 1994  An Arabidopsis thaliana RFLP mapping set to localize mutations to chromosomal regions. Plant J. 5:149-156.

FEINBERG, A. P. and B. VOGELSTEIN, 1984  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137:266-267[Medline].

FRARY, A., G. G. PRESTING, and S. D. TANKSLEY, 1996  Molecular mapping of the centromeres of tomato chromosomes 7 and 9. Mol. Gen. Genet. 250:295-304[Medline].

GEBHARDT, C., E. RITTER, T. DEBENER, U. SCHACHTSCHABLE, and B. WALKEMEIER et al., 1989  RFLP analysis and linkage mapping in Solanum tuberosum.. Theor. Appl. Genet. 78:65-75.

GRANER, A., H. SIEDLER, A. JAHOOR, R. G. HERRMANN, and G. WENZEL, 1990  Assessment of the degree and type of restriction fragment length polymorphism in barley (Hordeum vulgare). Theor. Appl. Genet. 80:826-832.

GRANER, A., A. JAHOOR, J. SCHONDELMAIER, H. SIEDLER, and K. PILLER et al., 1991  Construction of an RFLP map of barley. Theor. Appl. Genet. 83:250-256.

GRANER, A., E. BAUER, A. KELLERMANN, S. KIRCHNER, and J. K. MURAYA et al., 1994  Progress of RFLP-map construction in winter barley. Barley Genet. Newslett. 23:53-59.

HART, G. E., 1994 RFLP maps of bread wheat, pp. 327–358 in DNA-Based Markers in Plants, edited by R. L. PHILLIPS and I. K. VASIL. Kluwer Academic Publishers, The Netherlands.

HAYES, P., and K. MESZAROS, 1997 Mapping in Dicktoo x Morex. http://wheat.pw.usda.gov.

HEUN, M., E. KENNEDY, J. A. ANDERSON, N. L. V. LAPITAN, and M. E. SORRELLS et al., 1991  Construction of a restriction fragment length polymorphism map for barley (Hordeum vulgare). Genome 34:437-447.

KANAZIN, V., E. ANAIEV, and T. BLAKE, 1993  Variability among members of the Hor-2 multigene family. Genome 36:397-403[Medline].

KASHA, K. J. and A. KLEINHOFS, 1994  Mapping of the barley cross Harrington x TR306. Barley Genet. Newslett. 23:65-69.

KJAER, B., J. JENSEN, and H. GIESE, 1995  Quantitative trait loci for heading date and straw characters in barley. Genome 38:1098-1104.

KLEINHOFS, A., A. KILIAN, M. A. SAGHAI-MAROOF, R. M. BIYASHEV, and P. HAYES et al., 1993  A molecular, isozyme and morphological map of barley (Hordeum vulgare) genome. Theor. Appl. Genet. 86:705-712.

KONIECZNY, A. and F. M. AUSUBEL, 1993  A procedure for mapping Arabidopsis mutations using co-dominant ecotype specific PCR-based markers. Plant J. 4:403-410[Medline].

LANDER, E. S., P. GREEN, J. ABRAHAMSON, A. BARLOW, and M. J. DALY et al., 1987  MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174-181[Medline].

LANGRIDGE, P., A. KARAKOUSIS, J. KRETSCHMER, S. MANNING and S. LOGUE, 1996a Mapping in Chebec x Harrington. Http://wheat.pw.usda.gov/.

LANGRIDGE, P., A. KARAKOUSIS, J. KRETSCHMER, S. MANNING, R. ISLAM et al., 1996b Mapping in Clipper x Sahara. http://wheat.pw.usda.gov/.

LANGRIDGE, P., A. KARAKOUSIS, J. KRETSCHMER, S. MANNING and S. LOGUE, 1996c Mapping in Galleon x Haruna nijo. http://wheat.pw.usda.gov/.

LAURIE, D. A., J. W. SNAPE and M. D. GALE, 1992 DNA marker techniques for genetic analysis in barley, pp. 115–132 in Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology, edited by P. R. SHEWRY. C.A.B. International, Oxon, U.K.

LAURIE, D. A., N. PRATCHETT, K. M. DEVOS, I. J. LEITCH, and M. GALE, 1993  The distribution of RFLP markers on chromosome 2(2H) of barley in relation to the physical and genetic location of 5S rDNA. Theor. Appl. Genet. 87:177-183.

LAURIE, D. A., N. PRATCHETT, J. H. BEZANT, and J. W. SNAPE, 1995  RFLP mapping of five major genes and eight quantitative trait loci controlling flowering time in a winter x spring barley (Hordeum vulgare L.) cross. Genome 38:575-585.

LAURIE, D. A., N. PRATCHETT, R. L. ALLEN, and S. S. HANTKE, 1996  RFLP mapping of the barley homeotic mutant lax-a.. Theor. Appl. Genet. 93:81-85.

LINCOLN, S. E. and E. S. LANDER, 1992  Systematic detection of errors in genetic linkage data. Genomics 14:604-610[Medline].

LIU, C. J., M. HEUN, and M. D. GALE, 1993  Intrachromosomal mapping of seven biochemical loci in barley, Hordeum vulgare.. Theor. Appl. Genet. 87:94-96.

MENA, M., B. A. AMBROSE, R. B. MEELEY, S. P. BRIGGS, and M. F. YANOFSKY et al., 1986  Diversification of C-function activity in maize flower development. Science 274:1537-1540[Abstract/Free Full Text].

MICHELMORE, R. W., I. PARAN, and R. V. KESSELI, 1991  Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88:9828-9832[Abstract/Free Full Text].

MÜLLER, K. J., N. ROMANO, O. GERSTNER, F. GARCIA-MAROTO, and C. POZZI et al., 1995  The barley Hooded mutation caused by a duplication in a homeobox gene intron. Nature 374:727-730[Medline].

NANDI, S., P. K. SUBUDHI, D. SENADHIRA, N. L. MANIGBAS, and S. SEN-MANDI et al., 1997  Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping. Mol. Gen. Genet. 255:1-8[Medline].

NILSSON, N. O., T. SÄLL, and B. O. BENGTSSON, 1993  Chiasma and recombination data in plants: are they compatible? Trends Genet. 9:344-348[Medline].

OKAGAKI, R. J. and F. W. WEIL, 1997  Analysis of recombination sites within the maize waxy locus. Genetics 147:815-821[Abstract].

PECCHIONI, N., A. M. STANCA, V. TERZI, and L. CATTIVELLI, 1993  RFLP analysis of highly polymorphic loci in barley. Theor. Appl. Genet. 85:926-930.

POWELL, W., W. T. B. THOMAS, E. BAIRD, P. LAWRENCE, and A. BOOTH et al., 1997  Analysis of quantitative traits in barley by the use of amplified fragment length polymorphism. Heredity 79:48-59.

QI, X. and P. LINDHOUT, 1997  Development of AFLP markers in barley. Mol. Gen. Genet. 254:330-336[Medline].

QI, X., P. STAM, and P. LINDHOUT, 1997  Use of the locus specific AFLP markers to construct a high density molecular map in barley. Theor. Appl. Genet. 96:376-384.

RÖDER, M., N. L. V. LAPITAN, M. E. SORRELLS, and S. D. TANKSLEY, 1993  Genetic and physical mapping of the barley telomeres. Mol. Gen. Genet. 238:294-303[Medline].

ROHDE, W., 1996  Inverse sequence-tagged repeat (ISTR) analysis, a novel and universal PCR-based technique for genome analysis in the plant and animal kingdom. J. Genet. Breed. 50:249-261.

ROUPPE VAN DER VOORT, J. N. A. M., P. VAN ZANDVOORT, H. J. VAN ECK, R. T. FOLKERTSMA, and R. C. B. HUTTEN et al., 1997  Use of allele specificity of comigrating AFLP markers to align genetic maps from different potato genotypes. Mol. Gen. Genet. 255:438-447[Medline].

RUSSELL, J. R., J. D. FULLER, M. MACAULAY, B. G. HATZ, and A. JAHOOR et al., 1997  Direct comparison of levels of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs. Theor. Appl. Genet. 95:714-722.

SAGHAI-MAROOF, M. A., K. M. SOLIMAN, R. A. JORGENSEN, and R. W. ALLARD, 1984  Ribosomal DNA length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 91:5466-5470[Abstract/Free Full Text].

SÄLL, T. and N.-O. NILLSON, 1994  Crossover distribution in barley analysed through RFLP linkage data. Theor. Appl. Genet. 89:211-216.

SCHWEIZER, G. F., M. BAUMER, G. DANIEL, H. RUGEL, and M. S. RÖDER, 1995  RFLP markers linked to scald (Rhynchosporium secalis) reistance gene Rh2 in barley. Theor. Appl. Genet. 90:920-924.

SHERMAN, J. D., A. L. FENWICK, D. M. NAMUTH, and N. L. V. LAPITAN, 1995  A barley RFLP map: alignment of three barley maps and comparison to Gramineae species. Theor. Appl. Genet. 91:681-690.

SOGAARD, B. and P. WETTSTEIN-KNOWLES, 1987  Barley: genes and chromosomes. Carlsberg Res. Commun. 52:123-196.

STAM, P., 1993  Construction of integrated genetic linkage maps by means of a new computer package: JoinMap. Plant J. 3:739-744.

SZOSTAK, J. W., T. L. ORR-WEAVER, R. J. ROTHSTEIN, and F. W. STAHL, 1983  The double-strand-break repair model for recombination. Cell 33:25-35[Medline].

TANKSLEY, S. D., M. W. GANAL, J. P. PRINCE, M. C. DEVINCENTE, and M. W. BONIERBALE et al., 1992  High density molecular linkage maps of the tomato and potato genomes: biological inferences and practical applications. Genetics 132:1141-1160[Abstract].

TAZHIN, O. T., 1980  The linkage of the genes mo5 and n in barley. Barley Genet. Newslett. 10:69-72.

THOMAS, W. T. B., W. POWELL, R. WAUGH, K. J. CHALMERS, and U. M. BARUA et al., 1995  Detection of quantitative trait loci for agronomic, yield, grain and disease characters in spring barley (Hordeum vulgare L.). Theor. Appl. Genet. 91:1037-1047.

TINKER, N., D. E. MATHER, B. G. ROSSNAGELB, K. J. KASHA, and A. KLEINHOFS et al., 1996  Regions of the genome that affect agronomic performance in two-row barley. Crop. Sci. 36:1053-1062[Abstract/Free Full Text].

VAN ECK, H. J., J. R. VAN DER VOORT, J. DRAAISTRA, E. VAN ZANDVOORT, and H. VAN ENCKEVORT, 1995  The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring. Mol. Breeding 1:397-410.

VOS, P., R. HOGERS, M. BLEEKE, M. REIJANS, and T. VAN DE LEE et al., 1995  AFLP: a new concept for DNA fingerprinting. Nucleic Acids Res. 23:4407-4414[Abstract/Free Full Text].

WAUGH, R., N. BONAR, E. BAIRD, B. THOMAS, and A. GRANER et al., 1997  Homology of AFLP products in three mapping populations of barley. Mol. Gen. Genet. 255:311-321[Medline].

WILLIAMS, J. G. K., R. S. REITER, R. M. YOUNG, and P. A. SCOLNIK, 1993  Genetic mapping of mutations using phenotypic pools and mapped RAPD markers. Nucleic Acids Res. 21:2697-2702[Abstract/Free Full Text].

XU, X., A.-P. HSIA, L. ZHANG, B. J. NIKOLAU, and P. SCHNABLE, 1995  Meiotic recombination break points resolve at high rates at the 5' end of a maize coding sequence. Plant Cell 7:2151-2161[Abstract].

ZABEAU, M., and P. VOS, 1993 Selective restriction fragment amplification: a general method for DNA fingerprinting. European Patent Application n. 92402629.7, publication n. 0 534 858 A1.

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