Comparative Mapping of the Barley Ppd-H1 Photoperiod Response Gene Region, Which Lies Close to a Junction Between Two Rice Linkage Segments

Corresponding author: David A. Laurie, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom., david.laurie{at}bbsrc.ac.uk (E-mail)

Communicating editor: B. S. GILL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions. We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments. RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs. Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley. The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome. Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region. The implications of the results for comparative trait mapping in junction regions are discussed.

COMPARATIVE mapping of cereals using restriction fragment length polymorphism (RFLP) markers has shown considerable colinearity of marker order (recently reviewed by GALE and DEVOS 1998 ; DEVOS and GALE 2000 ; PATERSON et al. 2000 ). This has enabled the genomes of barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) to be described in terms of chromosome sections of rice (Oryza sativa; AHN and TANKSLEY 1993 ; KURATA et al. 1994 ; MOORE et al. 1995 ; VAN DEYNZE et al. 1995 ). Here we use the term "rice linkage segment" (RLS) following the terminology of MOORE et al. 1995 . The group 2 chromosomes of barley and wheat, which are highly colinear, can be represented as the insertion of rice chromosome 7 (RLS7) into rice chromosome 4 (RLS4a and RLS4b). Although this overall structure seems clear, much less is known about the organization of the junction regions between linkage segments apart from the observation that telomere and centromere regions are frequently involved.

The Ppd-H1 photoperiod response gene plays a major role in regulating flowering time in barley, and its position on chromosome 2H suggests that it is homeologous to the wheat (T. aestivum) Ppd gene series (LAURIE et al. 1995 ; BORNER et al. 1998 ). Previous RFLP mapping has shown that the Ppd genes of barley and wheat probably lie in the vicinity of the RLS7/RLS4a junction (VAN DEYNZE et al. 1995 ). More recently, LAURIE 1997 showed that Ppd-H1 is located between cDNA markers that map to the most distal part of rice chromosome 7L and that are therefore expected to be immediately adjacent to the junction. This region of rice chromosome 7 contains the Hd2 heading date gene shown by YAMAMOTO et al. 1998 to be tightly linked to four markers (Xrgc728, Xrgr411, Xrgs1979, and Xrgs2267), which are among those analyzed in the present work.

We are interested in isolating Ppd-H1 and although this could presumably be achieved by a direct map-based approach, as used for barley disease resistance genes (BUSCHGES et al. 1997 ; SHIRASU et al. 1999 ), we were also interested in exploring comparative approaches that utilize the small genome size of rice and in investigating the relationship between the Ppd-H1 and Hd2 regions. The primary aim was to determine if colinearity was disrupted by the proximity of the linkage segment junction. A secondary objective was to analyze RFLP probes from the distal region of rice chromosome 7 that detected two or more loci in rice and barley. We investigated whether the region as a whole might be duplicated and, if so, whether duplicated segments were associated with flowering time variation. RFLP markers were also mapped in maize to identify candidate regions that might contain counterparts of Hd2 or Ppd-H1. The majority of RFLP markers that were used were wholly or partially sequenced and this information was used to determine if the Hd2/Ppd-H1 region had a recognizable counterpart in Arabidopsis. If so, this might provide candidate genes for Ppd-H1 and Hd2.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material:
Wheat (Triticum aestivum L.) "Chinese Spring"/barley "Betzes" addition lines (ISLAM et al. 1981 ) were used to assign clones to barley chromosomes. The following populations were used for genetic mapping: rice: "Nipponbare" x "Kasalath" F₂ (KURATA et al. 1994 ; HARUSHIMA et al. 1998 ) and "IR20" x "63-83" F₂ (QUARRIE et al. 1997 ); barley: "Igri" x "Triumph" doubled haploid lines (LAURIE et al. 1995 ) and "Captain" x H. spontaneum F₂ (WANG et al. 1992 ; LAURIE et al. 1993 ); and maize: "Tx232" x "Cm37" and "Tx303" x "CO159" recombinant inbred lines (RIL; BURR and BURR 1991 ; http://burr.bio.bnl.gov/acemaz.html and http://www.agron.missouri.edu/images/).

Clone libraries:
Sources of rice RFLP probes were described in KURATA et al. 1994 and HARUSHIMA et al. 1998 . Other RFLP probes were from John Innes Centre libraries (PSR and PSB clones) or from Cornell University libraries (BCD, CDO, and WG clones). Construction and analysis of the rice Nipponbare yeast artificial chromosome (YAC) library and methods for YAC DNA preparation and isolation of YAC end clones by PCR were as described in UMEHARA et al. 1995 . Nipponbare YAC sizes were from the Rice Genome Program website (http://bank.dna.affrc.go.jp/). Southern hybridization to genomic DNA of rice and to digested YAC or bacterial artificial chromosome (BAC) inserts used the Amersham enhanced chemiluminescence direct labeling method (KURATA et al. 1994 ) or ³²P labeling as described by LAURIE et al. 1993 . An IR20 rice YAC library (John Innes Centre, Norwich, UK) was also used. The Nipponbare and IR20 YAC libraries each represented about seven genome equivalents. Subcloning from rice YAC DNA was by ligation of a partial Sau3A digest into Bluescript vector (Stratagene, La Jolla, CA).

Rice BAC clones were from "IR-BB21" (WANG et al. 1995 ), "Lemont," and "Teqing" libraries (ZHANG et al. 1996 ), representing 10 genome equivalents in total. Plasmid DNA from BAC clones was prepared by alkaline lysis and end clones were isolated by the method of Mozo (http://www/mpimp-golm.mpg.de/101/mpi_mp_map/bac.html) except that the primers used to amplify the T7 end were 5'-CCTCTTCGCTATTACGCCAG-3' and 5'-GCCCTTCCCAACAGTTGCG-3'. A Nipponbare cosmid library (Rice Genome Program, Tsukuba, Japan) and an IR20 phage library (John Innes Centre) were also used.

Nomenclature:
In the text and figures, loci detected by Southern blot hybridization are italicized and prefixed by X and a three-letter lab designator (e.g., Xrgc213 and Xpsr109) and the corresponding clones by capital letters (e.g., C213 and PSR109) following the nomenclature of MCINTOSH et al. 1998 .

Database analyses:
Sequences of rice clones and putative functions were obtained from the Japanese Ministry of Agriculture, Forestry and Fisheries DNA Bank website (http://bank.dna.affrc.go.jp/). Further sequence analyses were carried out using BLAST programs provided at the United States National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/). Sequence database accession numbers and putative functions for clones used in this article are given in Table 1.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Comparative mapping of the Ppd-H1 region of barley with rice and maize:
Previously, rice clones C213 and C924 were shown to detect loci distal and proximal to the barley Ppd-H1 locus, respectively (LAURIE 1997 ). These flanking markers formed the starting point for the current analysis. Markers from the Xrgc924–Xrgc213 interval in rice were tested in barley but the only probes suitable for comparative mapping were R411 and a closely linked YAC end clone (Y2938R, Table 1). Both were located between Ppd-H1 and Xrgc924 and although the order of the mapped markers was conserved between rice and barley, the genetic length of the Xrgc213–Xrgc924 interval was considerably greater in the latter (1.2 cM vs. 13.5 cM). However, Xrgr411 and Xrgy2938R cosegregated in both species (Fig 1).

View larger version (40K): In this window In a new window Download PPT slide   Figure 1. Comparative maps of the Ppd-H1 region of barley chromosome 2HS in relation to rice chromosome 7 and maize chromosomes 2 and 7. For clarity, barley 2HS is shown inverted. Markers from the Ppd-H1 region are shown in boldface type. Maps with marker positions indicated by short solid lines are from (a) rice: Nipponbare x Kasalath (HARUSHIMA et al. 1998 plus additional data from this article); (b) barley: Igri x Triumph (LAURIE et al. 1995 plus additional data from this article); (c) maize: Cm37 x Tx232 and Co159 x Tx303 (http://www.agron.missouri.edu/images/ plus additional data from this article). Loci mapped in other crosses (e.g., Xrgc74c on maize 2L) are not connected to chromosomes and are positioned approximately using common markers. Additional markers in rice are from CAUSSE et al. 1994 , in barley from LAURIE et al. 1993 plus additional data from this article, and in maize from DAVIS et al. 1999 . Figures in brackets after a probe name indicate other chromosomes of rice (R) or maize (M) to which the probe maps. Open ovals show the approximate positions of the centromeres.

Maize is generally considered to have undergone tetraploidization at some stage in its evolution (HELENTJARIS et al. 1988 ; MOORE et al. 1995 ; GAUT and DOEBLEY 1997 ) and sequences from the relevant region of rice chromosome 7 were therefore expected to be located on two maize chromosomes. R411, Y2938R, and C924 detected closely linked loci on the long arm of maize chromosome 7 where they were tightly linked to Xnpi45b. This region shared a number of duplicated loci with maize chromosome 2L, including a second locus detected by R411 (Fig 1). Y2938R detected two strongly hybridizing bands in maize but only one, on 7L, was polymorphic. These results were consistent with previous observations on the relationship between maize and rice chromosomes by AHN and TANKSLEY 1993 . C213, however, did not map in the same region. C213 provides the most distal marker on rice chromosome 7 (HARUSHIMA et al. 1998 ) and the closest RFLP marker distal to Ppd-H1 in barley (Fig 1). In maize it detected a single band, which mapped to the proximal region of chromosome 6L. C924 detected a locus at the same 6L location but no other markers suggested a link to maize chromosome arms 2L or 7L.

The barley cDNA BCD221 detected 6–12 bands of varying intensity on Southern blots of barley, depending on the restriction enzyme used. BCD221 and two rice clones (R518 and C74) gave identical hybridization patterns. Partial sequences from these clones identified them as elongation factor 1 (EF-1), corresponding to a full-length barley gene described by NIELSEN et al. 1997 who mapped EF-1 sequences on barley chromosomes 2HS, 4H, 5HL, and 6H. The 2HS copy of BCD221 was located between Ppd-H1 and Xrgr411a (Fig 1), making it of particular interest for comparative mapping. In rice, three bands of similar intensity were detected with BCD221 or R518. One band was polymorphic in the Nipponbare x Kasalath and IR20 x 63-83 crosses and in both cases a locus on rice chromosome 3 was detected (HARUSHIMA et al. 1998 and QUARRIE et al. 1997 , respectively). To determine the location of the nonpolymorphic bands, BCD221 was used to screen the IR20 rice YAC library. Two clones were detected, both of which showed the same three bands that were detected on genomic Southern blots when digested with the appropriate restriction enzyme. This showed that all copies of the sequence in rice were at the chromosome 3 location, that barley had more genomic copies of BCD221 than did rice, and that the 2HS locus had no counterpart on rice chromosome 7. Thus, the gene content of the Xpsr924-Ppd-H1-Xrgc213 region of barley cannot be fully determined by analysis of rice chromosome 7.

BCD221 detected 12 strongly hybridizing bands in maize, depending on the enzyme used, and at least six loci have been mapped (CHAO et al. 1994 ; DAVIS et al. 1999 ; this article) using BCD221 itself, C74 (a rice EF-1 clone), or umc116 (maize EF-1; KEITH et al. 1993 ). One copy was mapped on chromosome 2L in the UMC maize population (DAVIS et al. 1999 ), and this location was similar to that of Xrgr411b. However, the copy mapped on chromosome 7L in the RIL populations was not closely linked to Xrgr411a, Xrgc924a, or Xrgy2938R (Fig 1). This may mean that the 2L and 7L copies of BCD221 are duplicate loci and that they are not the equivalent of the barley 2HS locus, but this conclusion must be treated with caution because not all the copies of BCD221 could be mapped. At least three loci were detected on maize 6L by BCD221 but these were not tightly linked to Xrgc213 (Fig 1).

Three markers distal to Xrgc213 are included on the barley map (Fig 1). PSR108 did not give a clear signal in rice but detected several bands in maize, one of which was mapped to chromosome 6L where it was linked to two Xbcd221 loci. PSR666 and PSR109 detected loci in the centromeric region of maize chromosome 2L but were not polymorphic in rice. Nevertheless, these results were consistent with previous work showing that the homology of wheat and barley group 2 chromosomes to rice chromosome 7 ends close to the Ppd-H1 region (VAN DEYNZE et al. 1995 ; LAURIE 1997 ), that more distal regions of 2HS are homeologous to rice 4 (MOORE et al. 1995 ; VAN DEYNZE et al. 1995 ), and that this part of rice 4 is homeologous to the centromere region and short arm of maize 2 (AHN and TANKSLEY 1993 ).

Development of a YAC and BAC contig of rice chromosome 7:
To investigate the relationship between rice chromosome 7 and the Ppd-H1 region of barley chromosome 2H, we used R411 as the starting point for a contig of rice YAC and BAC clones (Fig 2). RFLP probes were hybridized to large insert libraries and clone order was verified by hybridizing additional probes and by isolating and hybridizing end clones. The contig contained three sequences mapped proximal to Ppd-H1 in barley (C924, R411, and Y2938R) and C213. Cloned amplified fragment length polymorphism fragments closely linked to Ppd-H1 (DECOUSSET et al. 2000 ) were also tested but they hybridized poorly to rice DNA and were not useful for comparative analysis.

View larger version (45K): In this window In a new window Download PPT slide   Figure 2. Genetic and physical maps of the distal region of rice chromosome 7 and of a segment of rice chromosome 3. YAC and BAC clones show the positions of RFLP probes (solid square), genetically mapped YAC/BAC end fragments (open circle), and their hybridization to other YAC/BAC clones (solid circle); YAC/BAC end fragments that could not be genetically mapped are shown as open ovals and their hybridization to other YAC/BAC clones by solid ovals; one rice microsatellite is shown (solid diamond). For clarity, the YACs are not drawn to scale. Brackets show probes that could not be ordered from genetic or physical mapping data.

Duplicate loci in rice revealed by RFLP mapping and analysis of the rice chromosome 7 contig:
RFLP mapping in the rice Nipponbare x Kasalath cross showed that the rice cDNA clones R411, S1636, and S941 each detected two loci, one on chromosome 7 and one on chromosome 3. R411 and S1636 gave indistinguishable hybridization patterns on Southern blots, with strong hybridization to bands of the chromosome 7 locus and weak hybridization to bands of the chromosome 3 locus. S941 detected the same bands but with a reversed pattern of hybridization strengths. This, together with expressed sequence tag (EST) sequence comparisons, suggested that R411 and S1636 were cloned transcripts of a gene on chromosome 7 while S941 derived from a closely related gene on chromosome 3 (predicted coding regions and 3' untranslated regions of R411 and S941 had 86 and 40% nucleotide identity, respectively). Hybridization of R411 to restriction digested YAC DNA showed that the band mapped to chromosome 7 detected two YACs (Y1273 and Y2938) while the band mapped to chromosome 3 detected five YACs previously assigned to chromosome 3 by hybridization to other mapped bands (Fig 2).

Hybridization of C586 to YAC DNA showed that the band mapped on chromosome 7 in the Nipponbare x Kasalath cross was present in one of the YACs detected by R411 (Y2938) and that an additional nonpolymorphic band was present in four chromosome 3 YACs previously assigned to the region immediately adjacent to the Xrgr411b locus (Fig 2). The remaining RFLP markers from the region of chromosome 7 shown in Fig 2 did not detect bands in the chromosome 3 YACs.

Two chromosome 3 cDNAs mapped in the Nipponbare x Kasalath cross (R3156 and S1828) detected bands when hybridized to DNA of the chromosome 7 YACs. R3156 hybridized to Y3607 (chromosome 3) and to Y4504 and Y2938 (chromosome 7). For S1828, the mapped band was present on the chromosome 3 YACs Y4932, Y3608, Y4421, and Y5002. An additional faint band was found on the chromosome 7 YACs Y1273 and Y2938 and BAC 14L15. The nonpolymorphic band of R3156 and of S1828 assigned to chromosome 7 and the band of C586 assigned to chromosome 3 were detected on at least two YACs, making it unlikely that the results were due to chimerism.

The genetic map locations and patterns of hybridization to YACs and BACs showed that the four duplicated sequences were in the same order on both chromosomes, suggesting that a chromosome segment was duplicated during the evolution of the rice genome. However, there was good evidence that the respective regions were no longer duplicated in their entirety. Mapping and cross-hybridization of end clones from the chromosome 7 YACs showed that they formed a contig spanning the Xrgr2576S to Xrgs1979 interval (Fig 2). Therefore, the failure of additional chromosome 3 probes from the Xrgr3156–Xrgr411b interval to detect sequences on chromosome 7 was not due to gaps in the chromosome 7 contig. Hybridizations using chromosome 3 RFLP markers and YAC end clones showed that the chromosome 3 YACs did not form a single contig. However, this was not the reason for the failure of the chromosome 7 probes L831, C924, and S2267 to detect chromosome 3 sequences because for these probes all bands detected by Southern hybridization using genomic DNA could be accounted for by hybridization to the chromosome 7 YACs.

The four duplicated clones were located within a region of no more than 400 kb on chromosome 7 because they hybridized to a single YAC of this size. The size of the equivalent chromosome 3 region remains to be determined but it must be >810 kb, which is the combined size of the nonoverlapping YACs Y2565 and Y5002. This may be a considerable underestimate because these YACs do not contain the sequences detected by R3156 and R411/S941. The genetic distance between the duplicated loci was 0.9 cM on chromosome 7 compared to 3.1 cM on chromosome 3, showing that the chromosome 3 region was physically and genetically larger.

Comparative mapping using probes detecting duplicate sequences in rice:
Probes detecting loci on rice chromosomes 3 and 7 (Fig 2) were used to analyze barley and maize. In barley, R411 detected a second locus on barley chromosome 4H (Fig 3) in addition to the 2HS locus (Fig 1) . In maize, hybridization of R411 to the parents of the two maize RIL populations detected three strongly hybridizing bands, all of which could be mapped. Two were the 2L and 7L copies (Fig 1) and the third mapped on 1S close to the PhyB1 locus, a region previously shown to be colinear with rice chromosome 3 (AHN and TANKSLEY 1993 ) and consistent with the location of the rice Xrgr411b locus (Fig 3). No copy of R411 was present on maize chromosome 9 although the long arm shares other duplicated loci with 1S. R411 detected a weakly hybridizing band in one of the RIL populations that was mapped to maize chromosome 3, a region not known to be related to any of the others where copies of R411 were detected.

View larger version (36K): In this window In a new window Download PPT slide   Figure 3. Comparative maps of rice chromosome 3, barley chromosome 4H, and maize chromosomes 1 and 9. Markers from the rice 3 and rice 7 regions in Fig 2 are shown in boldface type. Data sources are the same as in Fig 1.

R3156 detected one strongly hybridizing band in maize and several weaker bands. The strongly hybridizing band gave a polymorphism that cosegregated with Xrgr411c on chromosome 1S in both RIL populations (Fig 3). A second polymorphism was mapped to chromosome 7L where it cosegregated with Xrgr411a (Fig 1). Thus, R3156 behaved in a similar way to R411. In barley, R3156 detected one strongly hybridizing band and one to three weaker bands depending on the restriction enzyme used. The most strongly hybridizing band was assigned to 4H using wheat/barley addition lines and cosegregated with the 4H copy of R411 in the Captain x H. spontaneum cross (Fig 3). The weakly hybridizing bands were not polymorphic but at least one could be assigned to chromosome 2H using wheat/barley addition lines. C586 and S1828 gave poor hybridization with barley and maize DNA and were not suitable for comparative mapping.

Clones from the Xrgr3156–Xrgr411 intervals (Fig 2) that were not duplicated in rice were also tested. Of these only R216 was suitable for comparative mapping and polymorphic. R216 detected a single copy sequence in rice chromosome 3 (Fig 2) and a single band in barley that cosegregated with Xrgr411b on chromosome 4H (Fig 3). R216 detected two bands in maize, both of which were polymorphic in the Tx232 x Cm37 RIL population. One mapped on chromosome 1, cosegregating with Xrgr411c, and the other mapped on chromosome 9 close to PhyB2 (Fig 3). PHYB behaves similarly to R216. It is present only on maize chromosomes 1 and 9 and was present as a single copy sequence on barley chromosome 4H [determined by hybridization of a PHYB specific sequence (GenBank accession no. U08169) to wheat/barley addition lines]. BCD221 (EF-1) detected copies linked to Xrgr411a (2H) and Xrgr411b (4H) in barley (Fig 1 and Fig 3), but detected neither region in rice. In maize several copies were mapped on the relevant chromosomes but their relationship was less clear.

Two YAC end clones (Y1273L and Y2938R) from the rice chromosome 7 contig were suitable for comparative mapping. Y1273L detected several bands, and Y2938R one band, on Southern blots of rice genomic DNA. Both probes detected two loci in barley. For Y1273L, only one band was polymorphic and this mapped to the centromere region of chromosome 5H. For Y2938R, both bands could be mapped, detecting loci on 2HS and 7HS. Y1273L hybridized poorly to maize while Y2938R detected two bands in maize, one of which was polymorphic and shown to be on chromosome 7L (Fig 1). Thus, although R411 and Y2938R were closely linked, they showed different patterns of duplication in the three species tested.

These results suggest that a region spanning at least Xrgr411 to Xbcd221 was duplicated in the common ancestor of barley, rice, and maize and had evolved part of its present structure before these species diverged (Fig 4). For example, R216 would have been deleted from one linkage segment, or inserted into the other, before divergence. After the barley, rice, and maize lineages separated, BCD221 sequences were deleted from rice 3 and 7 but retained in barley 2H and 4H. The duplication of R411 was retained in rice, barley, and maize but one copy was subsequently deleted from maize 9, possibly after tetraploidization. If this model is correct, additional sequences such as Y2938R should have been within the original duplication. The absence of Y2938R from rice 3 and from barley 4H but its presence on 7HS suggests either additional deletion and duplication events or translocation of the 4H copy. It would be interesting to investigate the behavior of the duplicated sequences in other monocot lineages.

View larger version (19K): In this window In a new window Download PPT slide   Figure 4. Model for the evolution of the duplicated segments in barley, rice, and maize.

Duplicated sequences in Arabidopsis:
Candidate Arabidopsis homologs of sequences from the relevant regions of rice chromosomes 3 and 7 were identified by BLAST analyses using the predicted peptide sequences of the rice RFLP markers or, where available, the full-length sequence of the rice gene (Table 1). Where matches were found, the nucleotide sequence of the Arabidopsis gene was used to identify its genomic location. The full-length peptide corresponding to the R411 EST sequence was highly homologous to Arabidopsis 26S proteasome AAA-ATPase subunit RPT2 (predicted peptides were 93% identical and 96% similar). The R411 sequence, or its Arabidopsis equivalent, detected two highly conserved copies in the Arabidopsis genome, one on chromosome 2 (BAC T2G17) and the other on chromosome 4 (BAC F19B15). However, analysis of other sequences linked to R411 in rice (Fig 2), plus BCD221 (EF-1), showed no association with RTP2 sequences in Arabidopsis (Table 1).

The BAC sequences containing the R411 homologs were within the extensive duplication of Arabidopsis chromosomes 2 and 4 described by LIN et al. 1999 and MAYER et al. 1999 and might result from a separate duplication event. Therefore, more diverged Arabidopsis R411-like sequences were also analyzed (RPT1 and RTP3–RPT6, Table 1). The only significant clustering was the association of sequences homologous to C728, EF-1, RTP5, and R143 in a region of chromosome 1 spanning 600 kb. However, if marker order was conserved with the predicted ancestral region in cereals, R143 would be predicted to be between R411 and Ef-1, which was not observed. Thus, no clear equivalent to the Hd2 or Ppd-H1 regions that would provide candidate genes for further analysis was identified. Consequently, it was not possible to determine if the duplication observed in cereals had an equivalent in Arabidopsis. The lack of extensive fine-scale colinearity observed in this study is consistent with two recent comparisons of rice and Arabidopsis (DEVOS et al. 1999 ; VAN DODEWEERD et al. 1999 ).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Comparative mapping of the Ppd-H1 region:
Although the Ppd-H1 gene of barley lies close to a junction between linkage segments RLS7 and RLS4a, the order of markers derived from rice was not altered when they were mapped in barley. Duplicated sequences closely linked to Ppd-H1 complicated the comparison, but their analysis in barley, rice, and maize suggested that they were the remnants of an ancient duplication and were not generated by the formation of the 2HS chromosome. The results from rice 7 and rice 3 contrast with the well-conserved duplicated segments on chromosomes 11 and 12 (NAGAMURA et al. 1995 ; WU et al. 1998 ) and suggest that if a duplication occurred it is likely to have been considerably earlier in evolution. The results in rice, barley, and maize are consistent with the hypothesis that a duplication had occurred and had assumed at least part of its present form before these species diverged some 50–80 million years ago (WOLFE et al. 1989 ; BENNETZEN and FREELING 1997 ).

If the model for the duplication of markers in the Ppd-H1 region shown in Fig 4 is correct, it is important to consider whether the duplication extended beyond BCD221 to include the closely linked Ppd-H1 gene in barley or Hd2 in rice. No markers distal to Ppd-H1 followed the same pattern of duplication and there is no evidence in the literature for a major flowering time gene in the relevant regions of barley 4H or any of the other Triticeae group 4 chromosomes. This suggests that Ppd-H1 was not duplicated, although deletion from the 4H location is possible. Similarly, the only major flowering time gene in the relevant regions of rice or maize is Hd2 on rice chromosome 7.

Strategies for isolating Ppd-H1:
Comparative analysis provided valuable additional markers for the Ppd-H1 region but there were significant complications in using rice to target the barley gene. Whether this is a general conclusion for rice-to-barley comparisons remains to be seen, but for the Ppd-H1 region it will be desirable to develop a physical map of the region in barley and to sequence a minimum region defined by flanking markers to identify genes that can be assayed for their effects on flowering. Despite the complications described above, rice genomic sequence is likely to provide significant benefits in the development of new markers for mapping and library screening. Rice genes identified from genomic sequencing could be used directly as probes or to identify homologous Triticeae ESTs. This is preferable to the use of random subclones of YAC DNA, which we found unrewarding.

As the relationship between the Ppd-H1 and Hd2 regions is clarified it will be possible to assess the relationship between the two genes in more detail. Recent work using larger mapping populations suggests that Hd2 is distal to Xrgc213 in rice (M. YANO, unpublished data), making it less likely to be a direct counterpart of Ppd-H1, but it will be interesting to see whether any equivalent of Ppd-H1 is present in rice or any equivalent of Hd2 is present in barley.

Analyzing the genomic sequences of Arabidopsis did not identify any region that could be equivalent to the Hd2 or Ppd-H1 regions of rice and barley, respectively, and therefore failed to provide candidate genes for further analysis. Thus, while homology at the gene level between Arabidopsis and cereals will be of considerable value, as shown by PENG et al. 1999 , map-based comparisons seem unlikely to be useful in this case.

More generally, further analysis of the genetic and physical maps of rice and of the rice genomic sequence will provide evidence of the extent to which duplication has occurred in the evolution of cereal genomes and will be useful for assessing the importance of segment duplication in the evolution of gene families. These data, in turn, will help determine if segment duplication has been significant in the evolution of agronomically important characters in crop species. Combined with expression data, this should provide insights into the selective advantage of duplication and the ways in which duplications evolve.

	FOOTNOTES

¹ Present address: Seminis Vegetable Seeds, Woodlands, CA 95695.

	ACKNOWLEDGMENTS

We thank Ian Bancroft, John Innes Centre, for provision of rice BAC clones. The physical mapping of rice chromosomes 3 and 7 using YAC clones was carried out by D.A.L. at the Rice Genome Program, Tsukuba, Japan, for which funding from the STAFF Visiting Research Fellowship Program is gratefully acknowledged. The work was also supported by a grant in aid to the John Innes Institute from the United Kingdom Biotechnology and Biological Sciences Research Council. G.H. and T.R. were visiting scientists, supported in part by Monsanto and Pioneer Hi-Bred International, at the John Innes Centre.

Manuscript received September 14, 2001; Accepted for publication March 18, 2002.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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