Saturation Mapping of a Gene-Rich Recombination Hot Spot Region in Wheat

Corresponding author: Bikram S. Gill, Department of Plant Pathology, 4307 Throckmorton Plant Sciences Ctr., Kansas State University, Manhattan, KS 66506., bsg{at}ksu.edu (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Physical mapping of wheat chromosomes has revealed small chromosome segments of high gene density and frequent recombination interspersed with relatively large regions of low gene density and infrequent recombination. We constructed a detailed genetic and physical map of one highly recombinant region on the long arm of chromosome 5B. This distally located region accounts for 4% of the physical size of the long arm and at least 30% of the recombination along the entire chromosome. Multiple crossovers occurred within this region, and the degree of recombination is at least 11-fold greater than the genomic average. Characteristics of the region such as gene order and frequency of recombination appear to be conserved throughout the evolution of the Triticeae. The region is more prone to chromosome breakage by gametocidal gene action than gene-poor regions, and evidence for genomic instability was implied by loss of gene collinearity for six loci among the homeologous regions. These data suggest that a unique level of chromatin organization exists within gene-rich recombination hot spots. The many agronomically important genes in this region should be accessible by positional cloning.

THE polyploid nature of wheat (Triticum aestivum L. emend. Thell., 2n = 6x = 42, AABBDD genomes) allows it to tolerate, and transmit through gametes, a certain degree of aneuploidy. Over 400 chromosome deletion lines covering the entire wheat genome are now available (ENDO and GILL 1996 ). The deletion stocks are powerful tools for constructing physical maps as they eliminate the requirement for intragenomic polymorphism and can be used to localize agronomically important genes to relatively small chromosomal regions. Physical maps have been constructed for each of the 21 chromosomes of wheat using molecular markers (GILL et al. 1993 , GILL et al. 1996A , GILL et al. 1996B ; HOHMANN et al. 1994 ; DELANEY et al. 1995A , DELANEY et al. 1995B ; MICKELSON-YOUNG et al. 1995 ). On the physical maps, most markers were tightly clustered in small-sized physical segments. These markers were identified primarily with cDNA probes and represent expressed genes. Furthermore, GILL et al. 1996A , GILL et al. 1996B compared physical maps with recombination-based maps and found that these gene-rich regions undergo recombination much more frequently than do gene-poor regions. Kilobase pair per centimorgan (cM) estimates ranged from 118 kb for gene-rich regions to 22,000 kb for gene-poor regions (GILL et al. 1996A ). Other estimates of a gene-rich region on the short arm of chromosome 1D indicate that 1 cM may constitute as little as 20 kb (W. SPIELMEYER, personal communication).

Physical distribution of recombination events is nonrandom in other plant species as well (RICK 1971 ). In tomato, recombination is commonly suppressed near the centromeres (TANKSLEY et al. 1992 ). An extensive study of chromosome 4 of Arabidopsis by SCHMIDT et al. 1995 identified regions in which the base pair to centimorgan ratios ranged from 30 to >550 kb/cM.

Recombination usually results in reciprocal exchange between two nonsister chromatids or gene conversion. THURIEAUX 1977 postulated that recombination is confined to coding regions because different eukaryotic organisms have essentially the same number of genes, and the number of map units per genome is relatively constant even though the physical sizes of the genomes vary. Meiotic recombination seems to occur preferentially at defined sites, termed hot spots, along chromosomes of various eukaryotic organisms (SHIROISHI et al. 1993 ; SMITH 1994 ; LICHTEN and GOLDMAN 1995 ). In maize, genes per se are recombination hot spots (CIVARDI et al. 1994 ; XU et al. 1995 ). Recombination frequencies at the maize loci a1 (BROWN and SUNDARESAN 1991 ; CIVARDI et al. 1994 ), adh1 (FREELING 1978 ), b (PATTERSON et al. 1995 ), wx (NELSON 1968 ), gl1 (SALAMINI and LORENZONI 1970 ), r (DOONER and KERMICLE 1986 ), and bz1 (DOONER 1986 ; DOONER and KERMICLE 1986 ) are about 2 orders of magnitude higher than the average rate of recombination per kilobase for the whole genome.

Meiotic recombination in the yeast Saccharomyces cerevisiae is initiated by the formation of meiosis-specific DNA double-strand breaks at hot spots (NICOLAS et al. 1989 ; SUN et al. 1989 ; CAO et al. 1990 ; ZENVIRTH et al. 1992 ; DE MASSEY and NICOLAS 1993 ; GOYON and LICHTEN 1993 ; NAG and PETES 1993 ; WU and LICHTEN 1994 ). The double-strand breaks occur preferentially at nucleosome-free regions that show hypersensitivity to nucleases (OHTA et al. 1994 ; WU and LICHTEN 1994 ; FAN and PETES 1996 ). Therefore, chromatin structure that allows DNA accessibility seems essential for hot spot activity. Much less is known about the mechanisms of recombination in plants, but at least some aspects of meiotic recombination in yeast can be extended to plants. In both, the mechanism for meiotic recombination seems to follow the double-strand break model (XU et al. 1995 ).

The objectives of this research were to (1) saturate a physically small gene-rich region of wheat chromosome 5B with molecular markers, (2) assess the degree of recombination that occurs within the region, (3) compare the collinearity of markers in the region among the physical maps of homeologous group 5 chromosomes, and (4) compare levels of recombination among homologous regions of wheat, barley, rice, and Aegilops tauschii chromosomes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant materials:
We used 36 lines of "Chinese Spring" (CS) with terminal chromosomal deletions in the long arms of group 5 chromosomes (ENDO and GILL 1996 ) for physical mapping. The number of deletion lines for each chromosome was 16, 12, and 8 for 5AL, 5BL, and 5DL, respectively. Of these deletion lines, 28 are either homozygous or hemizygous (monosomic) for the deletion chromosome, and 8 (5AL-16, 5AL-19, 5BL-3, 5BL-7, 5BL-15, 5DL-7, 5DL-8, and 5DL-10) are heterozygous for the deletion chromosome.

A mapping population consisting of 117 recombinant substitution lines (RSLs) was generated from the cross of CS with CS that had a pair of Triticum dicoccoides 5B chromosomes substituted for the native 5B chromosomes (GILL et al. 1996A ). Briefly, the F₁ plant was crossed as a male with a monotelosomic 5BL plant. Plants possessing 40 normal wheat chromosomes and a 5B chromosome (lacking the 5BL telosome) were selected and allowed to self. From the progeny of each plant, a 42-chromosome plant having the 5B recombinant chromosome in the disomic condition was selected and used for mapping.

RFLP analysis:
Leaf tissue (~5 g) was collected from 3- to 4-wk-old plants, frozen in liquid nitrogen, ground with a mortar and pestle, and transferred to 50-ml polypropylene tubes. Sodium bisulfite (3.8 g liter^-1) was added to the extraction buffer [0.5 M NaCl, 0.1 M Tris-HCl, pH 8.0, 50 mM EDTA, 0.84% (w/v) SDS], and the pH was adjusted to 8.0 with NaOH. Extraction buffer (10–15 ml) was heated to 65°, added to frozen tissue, and incubated at 65° for 30–45 min. A 24:1 solution of chloroform:isoamyl alcohol was added, mixed vigorously, and centrifuged at 8000 x g for 15 min. The upper phase was removed, and the DNA was precipitated with 1.5 volumes of cold 95% (v/v) ethanol, rinsed in 70% (v/v) ethanol, dried, dissolved in TE buffer, and quantified on a 0.9% agarose gel.

A total of 20 µg of DNA was digested with 40 units of endonuclease (EcoRI, EcoRV, DraI, HindIII, or XbaI) in the presence of the appropriate buffer in a final volume of 35 µl. After 16 hr at 37°, the reactions were stopped by adding 8 µl of gel loading buffer [7.6 M glycerol, 0.5x neutral electrophoresis buffer (NEB) (1x NEB: 0.1 M Tris, 1 mM EDTA, 12.5 mM sodium acetate·3H₂O, pH 8.1), 0.02 mM EDTA, 0.2% (w/v) SDS, and 6 g liter^-1 bromphenol blue]. The resulting mix was loaded on a 0.9% agarose gel made using 1x NEB and was run for 16 hr at 22 V in a horizontal gel apparatus. Gels were stained with ethidium bromide, rinsed in distilled water, and photographed.

DNA was transferred from gels to Hybond N⁺ membranes (Amersham, Arlington Heights, IL) according to manufacturer's instructions, except that a large sponge soaked in 0.4 M NaOH served as the base of the blot.

The prehybridization and hybridization solutions were as described in KAM-MORGAN et al. 1989 . Probes were labeled by the random hexamer method with [³²P]dCTP (FEINBERG and VOGELSTEIN 1983 ), purified through spun columns containing Sephadex G50, denatured by boiling for 2 min, added to the membranes, and allowed to hybridize for 18–22 hr. Membranes were washed at 65° for 20 min each in 2x SSC and 1x SSC followed by 1 hr in 0.5x SSC (1x SSC: 0.15 M NaCl plus 0.015 M sodium citrate). All washing solutions also contained 0.1% (w/v) SDS. Membranes were placed in plastic sheets and were exposed to X-ray film for 3–7 days.

Microsatellite analysis:
Three 5B microsatellite markers (Xgwm371, Xgwm408, and Xgwm499) were selected on the basis of the map positions determined by RODER et al. 1998B . PCR reactions were performed as described in RODER et al. 1998A . Amplified products were run on a 2.3% agarose gel made with 1x NEB at 57 V for 4 hr. Gels were stained with ethidium bromide, visualized under UV light, and photographed.

Clone selection and sources:
We used 135 RFLP clones that could hybridize to the physical segment of wheat chromosome 5B that is flanked by fraction breakpoints 0.75 and 0.79. The descriptions of the clone libraries and their sources are given in Table 1. Most of the clones had been localized previously to chromosome group 5 of wheat (XIE et al. 1993 ; OGIHARA et al. 1994 ; DEVOS et al. 1995 ; NELSON et al. 1995 ; FARIS et al. 1996 ; GILL et al. 1996A ; KOJIMA and OGIHARA 1998 ; LI et al. 1999 ).

We selected many candidate markers from barley 5H maps (GRANER et al. 1991 ; HEUN et al. 1991 ; KLEINHOFS et al. 1993 ) because the order and constitution of genes and markers on barley chromosome 5H is highly conserved with that of wheat group 5 chromosomes. Other candidate clones were selected from conserved regions of rice chromosomes 3 and 9 (CAUSSE et al. 1994 ), oat linkage group E (O'DONOUGHUE et al. 1992 ; RAYAPATI et al. 1994 ), and Ae. tauschii chromosome 5D (KAM-MORGAN et al. 1989 ; GILL et al. 1991 ; BOYKO et al. 1999 ; LI et al. 1999 ).

Mapping and calculations:
The computer program MAPMAKER (LANDER et al. 1987 ) V2.0 for Macintosh was used to calculate linkage distances using the Kosambi mapping function (KOSAMBI 1944 ) and a LOD of 3.00. There are several methods used to calculate crossover interference along a chromosome, and there are limitations to each of them (for review see OTT 1997 ). We estimated crossover interference by calculating the actual number of crossovers within each marker interval. We divided the chromosome map into four marker intervals using the most informative loci separated by 10- to 15-cM intervals, which is the intermarker distance optimal for detecting interference (OTT 1991 ). Each adjacent pair of intervals was tested for interference by calculating the coefficient of coincidence for the interval pair.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Physical mapping:
The previous chromosome group 5 long arm physical maps consisted of 155 markers of which 44, 54, and 57 loci were on 5AL, 5BL, and 5DL, respectively (GILL et al. 1996A ). Of the 135 low-copy RFLP clones used in this research, 93 (69%) hybridized to the long arms of group 5 chromosomes resulting in 85, 82, and 78 additional loci on 5AL, 5BL, and 5DL, respectively (Figure 1). The group 5 long arm physical maps now consist of 129 loci on 5AL, 139 loci (including three microsatellites) on 5BL, and 135 loci on 5DL, for a total of 403 loci.

View larger version (47K): In this window In a new window Download PPT slide   Figure 1. Physical maps of the long arms of wheat group 5 chromosomes. Fraction breakpoints and the corresponding line numbers are indicated to the left of each chromosome. Boxes to the right of chromosomes indicate deletion intervals. Markers and the intervals in which they map are indicated to the right. The X symbol preceding the names of DNA markers according to the wheat genetics nomenclature is omitted to conserve space. Dark regions of the chromosomes represent prominent C-bands, and hatched regions represent lightly stained C-bands that are observed cytologically.

Of the 93 probes that detected group 5 loci, 57 (61%) detected loci on all three homeologous group 5 chromosomes (Table 2). A total of 7 probes was specific to chromosome 5A, but 3 of these mapped within the chromosome 4A translocation segment at the tip of 5AL (MICKELSON-YOUNG et al. 1995 ; NELSON et al. 1995 ). A total of 8 probes was specific to 5B, with 1 of these (Xbcd307) detecting two loci; no probes were specific to 5D. A total of 5 probes detected loci on 5A and 5B, but not 5D. A total of 8 probes hybridized to fragments on 5A and 5D, but not 5B, and another 8 probes detected loci on 5B and 5D, but not 5A. A total of 39 probes was specific to group 5 chromosomes and did not hybridize to any other chromosome.

The three microsatellite markers were specific to chromosome 5B. Two of these markers mapped within the 0.55–0.59 interval, and one mapped within the 0.75–0.79 interval.

Previously, GILL et al. 1996A identified 12 markers within the deletion interval 5BL 0.75–0.79. Of the 93 RFLP clones and three microsatellites mapping to group 5 chromosomes in this experiment, 64 probes and one microsatellite mapped within the targeted deletion interval. Combining these 65 markers with the 12 mapped by GILL et al. 1996A , this small deletion interval now consists of 77 markers (Figure 1 and Figure 2). We also identified 16 markers in addition to those previously mapped by GILL et al. 1996A within the interval flanked by fraction breakpoints 0.55 and 0.59, and 3 additional markers were mapped within the interval flanked by fraction breakpoints 0.59 and 0.75.

View larger version (42K): In this window In a new window Download PPT slide   Figure 2. The physical consensus map, recombination-based map, and physical map of the chromosomal region that corresponds to the physical segment on 5BL flanked by fraction breakpoints 0.75 and 0.79. On the recombination-based map, centimorgan distances are indicated on the left and marker names are indicated on the right. Recombination was calculated for markers mapping at a LOD >3.00, and markers mapping at a LOD <3.00 (XksuQ11 and Xabc155) are presented in their most likely intervals. Asterisks indicate skewed segregation of markers (*P < 0.05, **P < 0.01). The description for the physical map and the physical consensus map is the same as for Figure 1. The physical consensus map was constructed on the basis of comparisons of deletion breakpoints and marker orders within the regions of 5AL and 5DL that are homeologous to the targeted region on 5BL. Markers along the consensus physical map that showed discrepant locations among 5AL, 5BL, and 5DL are indicated in parentheses and in boldface type.

We constructed a consensus physical map of the 5BL 0.75–0.79 region using 58 markers that were also present on 5AL and 5DL (Figure 2). By comparing deletion breakpoints and the markers mapping within deletion intervals on the three homeologues, we constructed a physical map that consists of nine deletion intervals defined by 10 breakpoints. The order of markers across the three homeologous chromosomes agreed relatively well with 52 (90%) of the 58 markers showing a conserved order. Markers Xtag644 and Xbcd1734 were more proximal on 5A and 5B, but they mapped in different, more distal intervals on 5D. The location of Xcdo87 on 5A and 5D agreed with each other, but it mapped more proximal on 5B. Similarly, the locations of Xmwg900 and dhn2 agreed with each other on 5B and 5D, but they were more distal on 5A. Xabg473 had a more proximal location on 5A than 5D, but the location of Xabg473 on 5B could not be determined relative to 5A and 5D.

Genetic mapping:
We tested probes that hybridized within interval 5BL 0.75–0.79 on the physical map for polymorphism between the parents of the mapping population. The microsatellite marker and 41 of the 76 RFLP probes were polymorphic. The resulting map has a genetic length of 50 cM (Figure 2).

With one exception, the order of the markers on the recombination-based map was the same as that of the 5BL physical map and the consensus physical map. Markers in deletion interval 5BL 0.75–0.76 on the physical map were at the proximal end of the recombination-based and consensus physical maps, and markers in deletion interval 5BL 0.76–0.79 on the physical map were on the distal end of the recombination-based and consensus physical maps (Figure 2). The exception to the collinearity, Xmwg900, was placed at different locations on the 5AL, 5BL, and 5DL physical maps. The location of Xmwg900 in the 5B recombination-based map corresponds most closely to its location on the 5AL physical map.

Only two markers (XksuQ11 and Xabc155) mapped at a LOD <3.00, and two markers (Xcdo400 and Xbcd183) did not fit a 1:1 segregation ratio (Figure 2). Neighboring markers appeared to have slightly skewed segregation ratios as well but were not significant at P < 0.05. FARIS et al. 1998 reported a segregation distortion locus within the homeologous region of chromosome 5D in Ae. tauschii. It is likely that a 5B homeoallele of the distortion factor is the cause of the skewed ratios observed for these two markers in this population.

Much of the recombination within the targeted interval 5BL 0.75–0.79 occurred toward the distal end. Of the 50 cM on the genetic map, 22 cM is accounted for by the five most distal markers. We determined the number of crossovers that occurred in each member of the mapping population. Of the 117 RSLs, 45 had no crossovers in this region, 41 had a single crossover, 28 had double crossovers, and 3 had triple crossovers. In the RSLs with multiple crossovers, none of the crossover pairs flanked single marker loci, but one RSL had a double crossover within a distance of 3.6 cM where one crossover occurred between Xmwg516 and Xrz328/Xrz589/Xbcd881, and the second crossover occurred between Xwg908/Xcdo548 and Xmwg900.

Coefficient of coincidence values for the interval pairs 1, 2 and 3, 4 suggested positive crossover interference. Slight negative interference was observed between intervals 2 and 3 where the coefficient of coincidence was 1.05.

We compared our 5B genetic map with the corresponding region of chromosome 5H of barley, chromosome 5D of Ae. tauschii, durum chromosome 5B from "Langdon" (T. turgidum) x Langdon/T. dicoccoides 5B disomic substitution, chromosome 5B of wheat from W7984 (synthetic) x "Opata 85" and W7976 (synthetic) x "Kulm," and rice chromosome 3 (Figure 3). With the exception of the durum and rice maps, a higher degree of recombination was observed in all of these maps with respect to the CS x CS/T. dicoccoides 5B map.

View larger version (26K): In this window In a new window Download PPT slide   Figure 3. The comparison of homologous and homeologous regions of group 5 maps. The regions presented correspond to the physical segment of wheat chromosome 5B flanked by deletion breakpoints 0.75 and 0.79. The maps are barley chromosome 5H from Proctor x Nudinka (HEUN et al. 1991 ), wheat chromosome 5B from W7984 (synthetic) x Opata 85 (NELSON et al. 1995 ), wheat chromosome 5B from CS x CS/T. dicoccoides disomic 5B substitution (this experiment), durum chromosome 5B from Langdon x Langdon/T. dicoccoides disomic 5B substitution (K. M. HAEN, J. D. FARIS and B. S. GILL, unpublished observations), wheat chromosome 5B from W7976 (synthetic) x Kulm (FARIS et al. 1996 ), Ae. tauschii chromosome 5D from TA1691 x TA1704 (BOYKO et al. 1999 ), and rice chromosome 3 from BS125 (Oryza sativa)/WL02(Oryza longistaminata)//BS125 (CAUSSE et al. 1994 ).

The barley chromosome 5H map is 197 cM, and the region corresponding to the 0.75–0.79 deletion interval on wheat 5B is 74 cM. Therefore, this region accounts for 38% of the recombination on barley chromosome 5H in the cross "Proctor" x "Nudinka." The Ae. tauschii chromosome 5D map is 429 cM and the corresponding region is 94 cM, accounting for ~22% of the total genetic length.

The marker interval lengths on the corresponding regions of 5B maps developed from W7984 x Opata 85 and W7976 x Kulm were similar to each other. The region of the 5B map from W7984 x Opata 85 corresponding to the 0.75–0.79 deletion interval is ~60 cM, and the length of the entire 5B map from this cross is ~150 cM. Therefore, this region accounts for ~40% of the recombination along chromosome 5B in this population. Fewer markers were mapped in the W7976 x Kulm population, and the map is 36 cM. However, the genetic distance between markers Xmwg914 and Xbcd450 is ~30 cM in the 5B maps from both W7984 x Opata 85 and W7976 x Kulm, indicating a similar degree of recombination within this region between these two populations.

Recombination appeared to be suppressed in the Langdon x Langdon/T. dicoccoides 5B population. All of the markers on the map showed a high level (P < 0.005) of segregation distortion (data not shown). FARIS et al. 1998 reported a segregation distortion factor (QSd.ksu.3-5D) in the homeologous region of chromosome 5D in Ae. tauschii when the F₁ was used as the male parent in a backcross. It is likely that a homeoallele of the distortion factor is active in this cross and it may have an effect on the degree of recombination. Alternatively, the suppressed recombination may be due to a lack of homology between Langdon 5B and T. dicoccoides 5B.

Comparison with the genetic linkage maps of rice (CAUSSE et al. 1994 ) indicated a segment of rice chromosome 3 having some homology with wheat chromosome 5B and there were five markers in common. However, the order of the markers on the rice map differed from that of wheat, which was probably due to the more distant evolutionary relatedness of rice to species of the Triticeae.

With one exception, the order of markers along the CS x CS/T. dicoccoides 5B map is in complete agreement with the order of markers on the compared maps (excluding the rice map; Figure 3). There appears to be an inversion within a small segment at the proximal region of the CS x CS/T. dicoccoides 5B map involving several closely linked markers.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Physical mapping:
The first physical maps constructed using deletion lines indicated that certain regions of the chromosomes had high gene density (GILL et al. 1993 , GILL et al. 1996A , GILL et al. 1996B ; HOHMANN et al. 1994 ; DELANEY et al. 1995A , DELANEY et al. 1995B ; MICKELSON-YOUNG et al. 1995 ), and when compared to recombination-based maps, the gene-rich regions preferentially participated in recombination (GILL et al. 1996A , GILL et al. 1996B ). We attempted to saturate a gene-rich region with molecular markers and to assess the degree of recombination. We chose the region on the long arm of chromosome 5B flanked by fraction breakpoints 0.75 and 0.79 because it is small (~4% of the long arm), apparently gene rich (GILL et al. 1996A ), and carries agronomically important genes such as the Pyrenophora tritici-repentis toxin resistance gene tsn1 (FARIS et al. 1996 ).

We identified 248 loci on the long arms of group 5 chromosomes in addition to the 155 identified by GILL et al. 1996A . Deletion mapping in wheat requires polymorphism only between genomes, but it is possible that some group 5 loci were not identified due to the lack of intergenomic polymorphism. Hybridizing the probe to DNA digested with alternate restriction enzymes probably would remedy this problem. Also, some probes detected more than one fragment mapping in the same deletion interval. In these cases, we cannot determine if each fragment corresponds to a separate locus, or if the presence of multiple bands is due to a restriction site(s) within a single locus. Therefore, it is possible that we detected more loci than we are reporting here. The use of alternate restriction enzymes, the identification of additional deletion lines, or sequencing could solve this problem.

The collinearity of wheat homeologous chromosomes allowed us to use markers that map to all three homeologous group 5 chromosomes to derive a consensus map of the targeted region (Figure 2). The physical collinearity of these markers among the three group 5 chromosomes is generally conserved. The locations of six markers were not consistent across the three chromosomes. If the deletion stocks were characterized incorrectly, we would expect to observe a group of markers in disagreement. But the discrepancies observed here consist of mainly single markers scattered across the region. It appears that these inconsistencies resulted from small rearrangements and lack of microcollinearity among the three homeologues; however, it is possible that these probes detect multiple loci on the individual chromosomes, but were not detected with the restriction enzymes used. Higher-resolution mapping and eventually sequencing of this region will be required to provide definitive answers.

Genetic mapping:
The linkage map corresponding to the 5BL 0.75–0.79 deletion interval has a genetic length of 50 cM (Figure 2). Our results agree with those of LUKASZEWSKI and CURTIS 1993 who determined that most of the recombination on long arms of B-genome chromosomes occurred within the distal 20–30% of the arm. Our data indicated that 31 (26%) of the 117 RSLs had more than one crossover in the targeted region, which lies in the distal 25% of the chromosome and accounts for only ~4% of the long arm.

NELSON et al. 1995 and XIE et al. 1993 constructed genetic maps of chromosome 5B that were ~150 cM in length. Therefore, the physical region marked by deletion breakpoints 0.75 and 0.79 accounts for as much as one-third of the recombination that occurs on chromosome 5B. This evidence is consistent with previous reports of uneven distribution of recombination in wheat (DVORAK and CHEN 1984 ; CURTIS and LUKASZEWSKI 1991 ; WERNER et al. 1992 ; GILL et al. 1993 , GILL et al. 1996A , GILL et al. 1996B ; KOTA et al. 1993 ). Uneven distribution of recombination has also been observed in other plants (RICK 1971 ; GANAL et al. 1989 ) and animals (STEINMETZ et al. 1987 ; BOLLAG et al. 1989 ), and there is much evidence to support the notion that recombination hot spots occur within or near genes (reviewed by SCHNABLE et al. 1998 ). Intragenic recombination frequencies may be influenced by various factors including transposon insertions (DOONER 1986 ; XU et al. 1995 ), trans-acting factors (TIMMERMANS et al. 1997 ), and the presence of small base pair heterologies between allelic combinations (BORTS and HABER 1989 ; DOONER and MARTINEZ-FEREZ 1997 ). In yeast, specific short DNA sequences required for recombination hot spot activity have been identified (reviewed by SMITH 1994 ). But the activity of these hot spot sequences seems to depend on binding-specific transcription factors and/or chromatin structure that allows hypersensitivity to nucleases (FOX et al. 1997 ; MIZUNO et al. 1997 ).

It seems logical that in wheat, a specific higher-order chromatin structure that allows DNA accessibility to trans-acting factors and other recombination machinery is required for a recombination hot spot. Gene-rich regions are expected to be highly decondensed to allow accessibility to transcription machinery, while heterochromatic regions and long stretches of highly repetitive sequences are highly condensed and, therefore, less accessible to recombination factors.

The chromosome deletion lines were produced by introducing a gametocidal (Gc) chromosome into wheat by interspecific hybridization and backcrossing with related Aegilops species. Plants monosomic for the Gc chromosome produce two types of gametes. Only those gametes possessing the Gc chromosome are normal. Gametes lacking the Gc chromosome undergo structural chromosome aberrations and, in most cases, are nonfunctional. However, if the damage caused by the chromosome breakage is not sufficient to kill the gamete, it may still function and be transmitted to the offspring. The gene-rich regions not only undergo frequent recombination, but most of the deletion breakpoints occurred within these regions as well (GILL et al. 1996A ). Although the mechanism of the Gc gene is not yet understood (NASUDA et al. 1998 ), it seems plausible that regions accessible to recombination factors may also be prone to Gc gene action.

Typically, it has been assumed that homologous synapsis precedes and restricts crossing over to sequences in similar positions on homologous chromosomes. Small regions of heterology between allelic combinations may suppress recombination (BORTS and HABER 1989 ; DOONER and MARTINEZ-FEREZ 1997 ). Recombination in the targeted region of chromosome 5B in the interspecific crosses between CS and T. dicoccoides and between Langdon durum and T. dicoccoides appears to be suppressed compared with recombination in the crosses that utilized synthetic parents. Less heterology along chromosome 5B is expected in the synthetic crosses than in the crosses that utilize T. dicoccoides chromosome 5B. In addition, an inversion was observed in the proximal region of the CS x CS/T. dicoccoides 5B map compared with the others. If T. dicoccoides carries the inversion, then the F₁ was heterozygous for the inversion, which would reduce the frequency of recombination within the inverted segment. Therefore, reduced recombination in the CS x CS/T. dicoccoides population compared with the synthetic wheat x cultivar populations is expected.

The paradigm of a 1:1 relationship between chiasmata and genetic crossovers has long been accepted, but has been challenged recently (reviewed by SYBENGA 1996 ). The increased use of molecular markers has inflated map lengths compared with those generated from chiasma counts (NILSSON et al. 1993 ; NILSSON 1994 ). One of the reasons for map inflation has been attributed to experimental error in classifying marker data. In our case, this is an unlikely source of error as we used only unambiguous data.

Another source of map inflation can be attributed to the Kosambi mapping function (KOSAMBI 1944 ), which does not consider variation in interference or crossover localization and places markers in the most plausible positions on the basis of statistical, not biological, probability. It seems unlikely that the marker order of our map would be incorrect due to the fact that we used a LOD of 3.00, a fairly stringent parameter. If the marker order were incorrect, then the degree of recombination within the targeted interval should have been much greater than what we found, and we would have observed several single-allele exchanges that would resemble gene conversion-type events.

There are also cytological explanations for chiasma counts not agreeing with the number of crossovers. The difficulty arises when it is impossible to distinguish between a single chiasma and two closely apposed chiasmata (SYBENGA 1975 ). Therefore, if a double crossover occurs within a submicroscopic region, multiple chiasmata would not be resolved. In our case, 28 RSLs had double crossovers and 3 had triple crossovers in a very small physical segment of the chromosome. Multiple chiasmata probably could not be resolved microscopically, and chiasma counts would underestimate recombination.

It is also possible that recombination may occur without the formation of chiasmata. Observations of recombination in interspecific hybrids of plants indicate that, occasionally, recombination occurs in the absence of chiasmata (GILL et al. 1995 ). Similar exchanges of short interstitial segments that are not recovered as chiasmata have been found in interspecific rice hybrids (JENA et al. 1992 ; ISHII et al. 1994 ).

Genetic distance is defined on the basis of the assumption that recombination occurs randomly along the chromosome, but the occurrence of one crossover is thought to inhibit the formation of another nearby. This phenomenon is referred to as positive crossover interference and has been observed widely in many organisms. Interference has been thought to result from some steric chromosomal feature such as stiffness (HALDANE 1919 ), but little is known about the causes of interference. Negative interference occurs when one crossover is more likely to be associated with the formation of another nearby. Negative interference is seldom reported in plant and animal species but is observed frequently in some species of yeast and fungi (OLSON et al. 1978 ; KOHLI and BAHLER 1994 ). LUKASZEWSKI and CURTIS 1993 studied interference along B-genome chromosomes and found coefficients of coincidence ranging from 0 to 1.08 with an average of 0.19. Our data for the small segment of wheat chromosome 5B ranges from positive (coefficient of coincidence = 0.46) to no, or slightly negative (coefficient of coincidence = 1.05), interference. Significant deviation from 1 is evidence for the existence of interference. Our values do not significantly deviate from 1 probably because of the small population size. Under optimal interval lengths of ~15 cM, >800 fully informative meioses are required to detect Kosambi-level interference with a power of 80% at a significance level of 0.05 (OTT 1991 ).

Collinearity of markers along the genetic maps of chromosome 5B of wheat, 5H of barley, and 5D of Ae. tauschii is highly conserved. However, collinearity of markers along these maps was not well conserved with a homologous region of rice chromosome 3 (Figure 3). Comparative mapping studies between rice and other cereals have indicated sets of linked genes on rice chromosomes, known as linkage blocks, that contain homology to cereal chromosomes (AHN et al. 1993 ; MOORE et al. 1995A , MOORE et al. 1995B ; VAN DEYNZE et al. 1995 ). A segment of rice chromosome 3 was found to have homology to a segment of the long arm of group 5 chromosomes of the Triticeae. Although linkage blocks of chromosome regions may show homology between wheat and rice, in-depth studies of specific genomic regions have indicated that the level of microcollinearity between wheat, barley, and rice is limited (FOOTE et al. 1997 ; KILIAN et al. 1997 ; MOORE et al. 1997 ; GALLEGO et al. 1998 ).

KUNZEL et al. 2000 constructed physical maps of barley chromosomes using microdissected translocation chromosomes. They found, just as in wheat, that recombination is confined to a few small regions that have high gene density. The barley region corresponding to the 5BL 0.75–0.79 interval was very small in physical size, but it accounted for most of the recombination on chromosome 5H. They estimated that recombination frequency was 4.4 Mb cM^-1 at the whole genome level, but was reduced to <1.0 Mb cM^-1 in recombination hot spots.

The entire map of the wheat genome constructed in the W7984 x Opata 85 population consists of ~3700 cM, and the wheat haploid genome consists of 1.6 x 10¹⁰ bp. This translates into a recombination frequency of 4.4 Mb cM^-1 for the whole genome. We do not know the exact physical size of the chromosome region flanked by fraction breakpoints 0.75 and 0.79, but cytological experiments indicate that it consists of ~4% of the long arm. If we assume that each chromosome is of equal size and that the long arm of chromosome 5B accounts for two-thirds of the chromosome, then the segment would consist of ~20 Mb. We now have 77 markers for this region, or at least 1 marker per 260 kb. As more markers are identified, this ratio will become smaller. The recombination-based map of this region is ~50 cM, so the recombination frequency is ~400 kb cM^-1, an 11-fold increase in recombination compared to the genomic average.

Our data indicate that recombination is somewhat suppressed in the CS x CS/T. dicoccoides population compared to the W7984 x Opata 85 and W7976 x Kulm populations. For example, the distances between the markers Xmwg914 and Xcdo584 are 22 and 50 cM for CS x CS/T. dicoccoides and W7984 x Opata 85, respectively. This result could mean that recombination in the CS x CS/T. dicoccoides population is only 44% of that in the W7984 x Opata 85 population. If this estimate of the degree of recombination suppression in the CS x CS/T. dicoccoides population is accurate, then the recombination frequency is <200 kb cM^-1.

Due to the large genome of wheat, molecular manipulations of the genome and attempts to perform techniques such as map-based cloning have been avoided. But our study suggests that the gene-rich regions in the wheat genome may be as amenable to molecular manipulations as are the smaller genomes of plants such as rice.

	ACKNOWLEDGMENTS

This research was supported in part by a U.S. Department of Agriculture special grant to the Wheat Genetics Resource Center. This paper is contribution number 00-24-J from the Kansas Agricultural Experiment Station (Manhattan, KS).

Manuscript received July 27, 1999; Accepted for publication October 4, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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				E. van der Knaap, A. Sanyal, S. A. Jackson, and S. D. Tanksley High-Resolution Fine Mapping and Fluorescence in Situ Hybridization Analysis of sun, a Locus Controlling Tomato Fruit Shape, Reveals a Region of the Tomato Genome Prone to DNA Rearrangements Genetics, December 1, 2004; 168(4): 2127 - 2140. [Abstract] [Full Text] [PDF]

				J. H. Peng, H. Zadeh, G. R. Lazo, J. P. Gustafson, S. Chao, O. D. Anderson, L. L. Qi, B. Echalier, B. S. Gill, M. Dilbirligi, et al. Chromosome Bin Map of Expressed Sequence Tags in Homoeologous Group 1 of Hexaploid Wheat and Homoeology With Rice and Arabidopsis Genetics, October 1, 2004; 168(2): 609 - 623. [Abstract] [Full Text] [PDF]

				E. J. Conley, V. Nduati, J. L. Gonzalez-Hernandez, A. Mesfin, M. Trudeau-Spanjers, S. Chao, G. R. Lazo, D. D. Hummel, O. D. Anderson, L. L. Qi, et al. A 2600-Locus Chromosome Bin Map of Wheat Homoeologous Group 2 Reveals Interstitial Gene-Rich Islands and Colinearity With Rice Genetics, October 1, 2004; 168(2): 625 - 637. [Abstract] [Full Text] [PDF]

				A. M. Linkiewicz, L. L. Qi, B. S. Gill, A. Ratnasiri, B. Echalier, S. Chao, G. R. Lazo, D. D. Hummel, O. D. Anderson, E. D. Akhunov, et al. A 2500-Locus Bin Map of Wheat Homoeologous Group 5 Provides Insights on Gene Distribution and Colinearity With Rice Genetics, October 1, 2004; 168(2): 665 - 676. [Abstract] [Full Text] [PDF]

				H. S. Randhawa, M. Dilbirligi, D. Sidhu, M. Erayman, D. Sandhu, S. Bondareva, S. Chao, G. R. Lazo, O. D. Anderson, Miftahudin, et al. Deletion Mapping of Homoeologous Group 6-Specific Wheat Expressed Sequence Tags Genetics, October 1, 2004; 168(2): 677 - 686. [Abstract] [Full Text] [PDF]

				K. G. Hossain, V. Kalavacharla, G. R. Lazo, J. Hegstad, M. J. Wentz, P. M. A. Kianian, K. Simons, S. Gehlhar, J. L. Rust, R. R. Syamala, et al. A Chromosome Bin Map of 2148 Expressed Sequence Tag Loci of Wheat Homoeologous Group 7 Genetics, October 1, 2004; 168(2): 687 - 699. [Abstract] [Full Text] [PDF]

				L. L. Qi, B. Echalier, S. Chao, G. R. Lazo, G. E. Butler, O. D. Anderson, E. D. Akhunov, J. Dvorak, A. M. Linkiewicz, A. Ratnasiri, et al. A Chromosome Bin Map of 16,000 Expressed Sequence Tag Loci and Distribution of Genes Among the Three Genomes of Polyploid Wheat Genetics, October 1, 2004; 168(2): 701 - 712. [Abstract] [Full Text] [PDF]

				B. S. Gill, R. Appels, A.-M. Botha-Oberholster, C. R. Buell, J. L. Bennetzen, B. Chalhoub, F. Chumley, J. Dvorak, M. Iwanaga, B. Keller, et al. A Workshop Report on Wheat Genome Sequencing: International Genome Research on Wheat Consortium Genetics, October 1, 2004; 168(2): 1087 - 1096. [Abstract] [Full Text] [PDF]

				K. G. Hossain, O. Riera-Lizarazu, V. Kalavacharla, M. I. Vales, S. S. Maan, and S. F. Kianian Radiation Hybrid Mapping of the Species Cytoplasm-Specific (scsae) Gene in Wheat Genetics, September 1, 2004; 168(1): 415 - 423. [Abstract] [Full Text] [PDF]

				M. Erayman, D. Sandhu, D. Sidhu, M. Dilbirligi, P. S. Baenziger, and K. S. Gill Demarcating the gene-rich regions of the wheat genome Nucleic Acids Res., July 7, 2004; 32(12): 3546 - 3565. [Abstract] [Full Text] [PDF]

				D. R. See, M. Giroux, and B. S. Gill Effect of Multiple Copies of Puroindoline Genes on Grain Softness Crop Sci., July 1, 2004; 44(4): 1248 - 1253. [Abstract] [Full Text] [PDF]

				K. M. Haen, H. Lu, T. L. Friesen, and J. D. Faris Genomic Targeting and High-Resolution Mapping of the Tsn1 Gene in Wheat Crop Sci., May 1, 2004; 44(3): 951 - 962. [Abstract] [Full Text] [PDF]

				M. E. Sorrells, M. La Rota, C. E. Bermudez-Kandianis, R. A. Greene, R. Kantety, J. D. Munkvold, Miftahudin, A. Mahmoud, X. Ma, P. J. Gustafson, et al. Comparative DNA Sequence Analysis of Wheat and Rice Genomes Genome Res., August 1, 2003; 13(8): 1818 - 1827. [Abstract] [Full Text] [PDF]

				S. Brunner, B. Keller, and C. Feuillet A Large Rearrangement Involving Genes and Low-Copy DNA Interrupts the Microcollinearity Between Rice and Barley at the Rph7 Locus Genetics, June 1, 2003; 164(2): 673 - 683. [Abstract] [Full Text] [PDF]

				J. D. Faris, J. P. Fellers, S. A. Brooks, and B. S. Gill A Bacterial Artificial Chromosome Contig Spanning the Major Domestication Locus Q in Wheat and Identification of a Candidate Gene Genetics, May 1, 2003; 164(1): 311 - 321. [Abstract] [Full Text] [PDF]

				J. Peng, Y. Ronin, T. Fahima, M. S. Roder, Y. Li, E. Nevo, and A. Korol Domestication quantitative trait loci in Triticum dicoccoides, the progenitor of wheat PNAS, March 4, 2003; 100(5): 2489 - 2494. [Abstract] [Full Text] [PDF]

				R. Malik, G. L. Brown-Guedira, C. M. Smith, T. L. Harvey, and B. S. Gill Genetic Mapping of Wheat Curl Mite Resistance Genes Cmc3 and Cmc4 in Common Wheat Crop Sci., March 1, 2003; 43(2): 644 - 650. [Abstract] [Full Text] [PDF]

				L. Maleki, J. D. Faris, R. L. Bowden, B. S. Gill, and J. P. Fellers Physical and Genetic Mapping of Wheat Kinase Analogs and NBS-LRR Resistance Gene Analogs Crop Sci., March 1, 2003; 43(2): 660 - 670. [Abstract] [Full Text] [PDF]

				W. Ramakrishna, J. Emberton, M. Ogden, P. SanMiguel, and J. L. Bennetzen Structural Analysis of the Maize Rp1 Complex Reveals Numerous Sites and Unexpected Mechanisms of Local Rearrangement PLANT CELL, December 1, 2002; 14(12): 3213 - 3223. [Abstract] [Full Text] [PDF]

				W. Ramakrishna, J. Dubcovsky, Y.-J. Park, C. Busso, J. Emberton, P. SanMiguel, and J. L. Bennetzen Different Types and Rates of Genome Evolution Detected by Comparative Sequence Analysis of Orthologous Segments From Four Cereal Genomes Genetics, November 1, 2002; 162(3): 1389 - 1400. [Abstract] [Full Text] [PDF]

				D. Sandhu, D. Sidhu, and K. S. Gill Identification of Expressed Sequence Markers for a Major Gene-Rich Region of Wheat Chromosome Group 1 Using RNA Fingerprinting-Differential Display Crop Sci., July 1, 2002; 42(4): 1285 - 1290. [Abstract] [Full Text] [PDF]

				M. N. Islam-Faridi, K. L. Childs, P. E. Klein, G. Hodnett, M. A. Menz, R. R. Klein, W. L. Rooney, J. E. Mullet, D. M. Stelly, and H. J. Price A Molecular Cytogenetic Map of Sorghum Chromosome 1: Fluorescence in Situ Hybridization Analysis With Mapped Bacterial Artificial Chromosomes Genetics, May 1, 2002; 161(1): 345 - 353. [Abstract] [Full Text] [PDF]

				H. Yao, Q. Zhou, J. Li, H. Smith, M. Yandeau, B. J. Nikolau, and P. S. Schnable From the Cover: Molecular characterization of meiotic recombination across the 140-kb multigenic a1-sh2 interval of maize PNAS, April 30, 2002; 99(9): 6157 - 6162. [Abstract] [Full Text] [PDF]

				W. Li and B. S. Gill The Colinearity of the Sh2/A1 Orthologous Region in Rice, Sorghum and Maize Is Interrupted and Accompanied by Genome Expansion in the Triticeae Genetics, March 1, 2002; 160(3): 1153 - 1162. [Abstract] [Full Text] [PDF]

				D. Sandhu and K. S. Gill Gene-Containing Regions of Wheat and the Other Grass Genomes Plant Physiology, March 1, 2002; 128(3): 803 - 811. [Abstract] [Full Text] [PDF]

				J. Faris, A. Sirikhachornkit, R. Haselkorn, B. Gill, and P. Gornicki Chromosome Mapping and Phylogenetic Analysis of the Cytosolic Acetyl-CoA Carboxylase Loci in Wheat Mol. Biol. Evol., September 1, 2001; 18(9): 1720 - 1733. [Abstract] [Full Text] [PDF]

				D. Sandhu, J. A. Champoux, S. N. Bondareva, and K. S. Gill Identification and Physical Localization of Useful Genes and Markers to a Major Gene-Rich Region on Wheat Group 1S Chromosomes Genetics, April 1, 2001; 157(4): 1735 - 1747. [Abstract] [Full Text]

				Z. Cheng, G. G. Presting, C. R. Buell, R. A. Wing, and J. Jiang High-Resolution Pachytene Chromosome Mapping of Bacterial Artificial Chromosomes Anchored by Genetic Markers Reveals the Centromere Location and the Distribution of Genetic Recombination Along Chromosome 10 of Rice Genetics, April 1, 2001; 157(4): 1749 - 1757. [Abstract] [Full Text]

				N. Stein, C. Feuillet, T. Wicker, E. Schlagenhauf, and B. Keller Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.) PNAS, November 8, 2000; (2000) 230361597. [Abstract] [Full Text]

				J. Peng, A. B. Korol, T. Fahima, M. S. Röder, Y. I. Ronin, Y. C. Li, and E. Nevo Molecular Genetic Maps in Wild Emmer Wheat, Triticum dicoccoides: Genome-Wide Coverage, Massive Negative Interference, and Putative Quasi-Linkage Genome Res., October 1, 2000; 10(10): 1509 - 1531. [Abstract] [Full Text]

				H. Yao, Q. Zhou, J. Li, H. Smith, M. Yandeau, B. J. Nikolau, and P. S. Schnable From the Cover: Molecular characterization of meiotic recombination across the 140-kb multigenic a1-sh2 interval of maize PNAS, April 30, 2002; 99(9): 6157 - 6162. [Abstract] [Full Text] [PDF]

				N. Stein, C. Feuillet, T. Wicker, E. Schlagenhauf, and B. Keller Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.) PNAS, November 21, 2000; 97(24): 13436 - 13441. [Abstract] [Full Text] [PDF]