| Edited by Toru Terachi. Shuhei Nasuda: Corresponding author. E-mail: nasushu@kais.kyoto-u.ac.jp |
Genes inherited in selfish manners are known in a wide variety of eukaryotic organisms including fungi, animals and plants. Such selfish genes are transmitted to offspring more frequently than the expected Mendelian ratio and therefore are often referred to as segregation distorters. Segregation distorters are genetic elements that exhibit meiotic drives, and potentially evolutional forces (Sandler et al., 1959). Segregation distortion is arisen from a variety of mechanisms including unequal chromosome segregations during meiosis (de Villena and Sapienza, 2001) and abortion of gametes or zygotes (Lyttle, 1991).
In plants, the genes acting as segregation distorters have been known in species belonging to families Solanaceae and Gramineae including tobacco (Cameron and Moav, 1957), tomato (Rick, 1966), maize (Maguire, 1963), rice (Sano, 1990) and wheat (Loegering and Sears, 1963). These genes cause gamete abortions by allelic interaction when plants are heterozygous for the alleles. On the other hand, no gamete abortion is induced when plants are homozygous for the alleles. The molecular mechanism of gamete abortion induced by those genes is largely unknown. More recently, advances in genome mapping by DNA markers revealed that distortion of segregation is quite common in almost every plant species studied, although the levels of distortion are not strong enough to be recognized as major genes (e.g., Ritter et al., 1991; Brummer et al., 1993; Murigneux et al., 1993; Nodari et al., 1993).
In common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD), selfish genetic elements were identified in the process of recurrent backcross to produce alloplasmic lines (Tsunewaki, 1980, 1993; Endo, 1990) or alien chromosome addition lines (Endo and Katayama, 1978; Miller et al., 1982; Kota and Dvorak, 1988; Friebe et al., 1993, 1999). The alien chromosome with this genetic element was retained through generations in spite of recurrent backcrossing (Endo and Tsunewaki, 1975). This selfish chromosome is termed ‘gametocidal (Gc)’ (Endo, 1979) or ‘cuckoo’ chromosome (Miller, 1983). When Gc genes are heterozygous or hemizygous, gametes lacking the Gc genes become sterile (Endo, 1978). As a result, Gc chromosomes are preferentially transmited to offspring. This phenomenon is caused by chromosome breakage in gametes lacking the Gc gene (Finch et al., 1984; Tsujimoto et al., 1990; Nasuda et al., 1998). According to the dual function model for Gc action suggested by Endo (1990), Gc genes consist of two factors, one of which confers ‘breaking’ function and another ‘protecting’ function. This model is supported by the findings of the inhibitor of a Gc gene (Tsujimoto and Tsunewaki, 1985) and the isolation of a mutant that only lost ‘breaking’ function (Friebe et al., 2003). However, the molecular mechanism of Gc action has not been unraveled yet.
Gc genes are widely distributed in the genus Aegilops, closest relatives of the genus Triticum, and so far found in three different genomes C, S, and M. The homoeologous groups carrying the Gc genes are 2, 3, and 4. Functional analyses of doubly hemizygous plants revealed that Gc genes are divided into at least three groups, Gc1, Gc2, and Gc3 (Tsujimoto, 1995; for detail, see recent reviews by Tsujimoto (2005), and Endo (2007)). The Gc gene from chromosome 3C of Ae. triuncialis (Gc3-C1) executes its function during post meiotic cell divisions in gametogenesis on both male (Tsujimoto and Tsunewaki, 1985) and female (Tsujimoto et al., 2001) sides. Endo (1978) reported different levels of male and female sterility in the genetic backgrounds of common wheat cultivars; semi-sterile in ‘Jones Fife’ (JF) and ‘Chinese Spring’ (CS), and in contrast, fertile in ‘Norin 26’ (N26). A genetic factor in N26 suppresses the Gc function of Gc3-C1, which was given the gene-symbol ‘Igc1’ (Tsujimoto and Tsunewaki, 1985). Igc1 inhibits only the Gc action by Gc3-C1 and does not have the suppression effect on other Gc genes (Tsujimoto, 2005). Igc1 does not completely protect chromosomes of gametes from breaking action of Gc3-C1, the gametes can survive with chromosome deletions (Tsujimoto and Tsunewaki, 1985). Chromosomal location of Igc1 on chromosome 3B of N26 (Tsujimoto and Tsunewaki, 1985) suggested that Gc3-C1 and the Igc1 are homoeologous genes and Igc1 may be a modified Gc gene that have the ‘protecting’ function but not the ‘breaking’ function (Tsujimoto, 2005). This relationship between Gc3-C1 and Igc1 is analogous to that between Gc2 and its mutant allele Gc2mut (Friebe et al., 2003). The Gc2mut allele does not exert chromosome breaking function but fully protects chromosomes from breaking.
Recently, agronomically important genes such as the leaf rust resistance genes Lr21 (Huang et al., 2003) and Lr10 (Feuillet et al., 2003), and the powdery mildew resistance gene Pm3b (Yahiaoui et al., 2004), have been identified by the map-based cloning approach in wheat. Among the Gc-related genes, Igc1 may be one of the most accessible genes by the map-based cloning approach for the following reasons. (1) It locates on wheat chromosome per se. Other Gc genes are on Aegilops chromosomes or on wheat-Aegilops translocated chromosomes. Therefore, we could expect fully normal segregation of the critical chromosome in a mapping population, a larger number of genetic markers, and genetic stocks for detailed mapping of Igc1. (2) It is located on chromosome 3B for which the most abundant information from genomics is available (Paux et al., 2008). Differential analyses of expressed genes by cDNA-AFLP and other methods is difficult to adopt to the Gc systems because the Gc action is only exerted in a hemizygous (or heterozygous) condition where normal and abnormal gametogenesis proceed simultaneously. The current situation of wheat genomics encouraged us to identify the Igc1 gene by a reverse genetic approach. We here report the results of mapping Igc1 by means of genetic and physical methods as a first step for map-based cloning. We will discuss the steps towards molecular identification of the Igc1 genes based on our results.
The genetic mapping population consisting of 267 individuals was developed by the following crosses; F1 hybrids were produced between Triticum aestivum cv. ‘Chinese Spring’ (CS) and ‘Norin 26’ (N26). N26 carries the dominant suppressor gene Igc1, and CS is homozygous for recessive allele igc1 (Tsujimoto and Tsunewaki, 1985). Then, F1 hybrids were backcrossed to the chromosome 3C disomic addition lines of CS (CS + 3C”, 2n = 42 + 2, 3C from Aegilops triuncialis). The line CS + 3C” has a gametocidal (Gc) gene Gc3-C1 (Endo, 1978), and is stored as strain ID ‘LPGKU2161’ in the seed stock of the National Bioresource Project-Wheat, Japan (http://www.shigen.nig.ac.jp/wheat/komugi/top/top.jsp). The deletion mapping population for chromosome 3B of Norin 26 was developed by the following crosses; 3C monosomic addition lines of N26 (N26 + 3C’, 2n = 42 + 1) were crossed by 3C(3B) disomic substitution line of CS (CS-3C”(3B”), 2n = 40 + 2).
To assign markers to chromosome 3B, we used two ditelosomic lines (Dt3BS and Dt3BL), eight chromosome 3B deletion lines and the nullisomic 3B - tetrasomic 3D line (N3BT3D) (Sears, 1954, 1966; Sears and Sears, 1978; Endo and Gill, 1996).
Plants were grown in the field located at the Department of Agriculture, Kyoto University, Kyoto, Japan in the years 2008 and 2009. Each line was planted in a pot. To determine the genotype of Igc1 in each F1 plant, seed fertility was scored as follows; ignoring uppermost and the next spikelets from the top and lowermost and the next spikelets from the bottom, the number of the seed in the first and second florets was counted and divided by total number of florets scored.
We extracted total genomic DNA by the cetyl trimethyl ammonium bromide (CTAB) method from the fresh leaves harvested four weeks after seeding.
A total of 203 molecular markers including 73 simple sequence repeat markers (SSRs, Somers et al., 2004; Torada et al., 2006), 28 PCR-based landmark unique gene markers (PLUGs, Ishikawa et al., 2007) located on the 3B chromosome of CS were selected for linkage analysis on the basis of their polymorphisms between CS and N26. A total of 102 insertion site-based polymorphism markers (ISBPs, Paux et al., 2006) were selected from each contig assigned to bins C-3BS1-0.33 (51 ISBP markers) and C-3BL2-0.22 (51 ISBP markers) for linkage and physical mapping.
PCR reactions were carried out in 15 μl of 0.5 units of Taq polymerase, 5 pmol for primer, 1.5 mM for MgCl2, 0.2 mM for each dNTP and 25 ng for template DNA. SSR markers were amplified under the following cycling conditions; 95°C for 5 min, 40 cycles of 95°C for 30 sec, 60, 55 or 50°C (annealing temperature depended on the primer sets) for 30 sec and 72°C for 50 sec, and 72°C for 10 min. PLUG markers and ISBP markers were amplified under the following touchdown method; 95°C for 5 min, 10 cycles of 95°C for 30 sec, 65°C for 30 sec decreasing by 0.5°C per cycle and 72°C for 50 sec, and 30 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 50 sec, and 72°C for 10 min. PCR products of SSR markers and ISBP markers were visualized either on the 2% agarose gel stained by ethidium bromide, or by the capillary gel-electrophoretic apparatus HAD-GT12 (eGene Inc., Irvine, CA, USA, now available from QIAGEN as QIAxcel system). PCR products of PLUG markers were cleaved by restriction enzyme HaeIII or TaqI, and then separated on the 4% agarose gel followed by staining with ethidium bromide.
Segregation data was analyzed by using MAPMAKER 3.0 (Lander et al., 1987) for linkage mapping. Linkage groups and marker orders were determined setting LOD 3.0 threshold. Map distances in centimorgan (cM) were computed according to the Kosambi mapping function.
Genotypes of the Igc1 locus in each of 267 BC1F1 plants from the cross between (CSxN26)F1 and CS + 3C” were determined by seed fertility. Because of the dominant suppression effect of Igc1 on Gc3-C1, the Igc1 carriers were expected to be fertile (Tsujimoto and Tsunewaki, 1985). Differences in the seed-set rates were apparent from the spike morphology (Fig. 1) as well as actual counting of the fertile flowerets (Fig. 2). A typical bimodal distribution of plants with low (8.3 to 39.6%) and high (70.8 to 100.0%) fertilities was observed. The numbers of the low and high fertility plants were 128 and 139, respectively, which fit to the expected Mendelian segregation ratio of 1:1 (χ2 = 0.453, df = 1, p > 0.56).
 View Details | Fig. 1 Spike morphologies of F1 plants from the cross between (CSxN26)F1 and CS+3C”. (Upper) Fertile plant (Igc1/igc1). (Lower) Semi-sterile plant (igc1/igc1). |
 View Details | Fig. 2 Distribution of plants in the mapping population derived from the cross between (CSxN26)F1 and CS+3C” according to their seed setting rates. The genotype at the Igc1 locus was unambiguously determined because of the discrete bimodal distribution; plants with less than 40% fertility (open box) are scored as igc1/igc1, and plants with more than 70% fertility (filled box) are scored as Igc1/igc1. An arrowhead on the graph indicate the fertilities of parental lines cited from the previous studies; (CSxN26)F1: 94.9% (Murai and Tsunewaki, 1994), CS+3C”: 94% (Tsujimoto and Tsunewaki, 1985). |
We picked up 61 molecular markers, i.e., 33 SSR markers (Somers et al., 2004; Torada et al., 2006) and 28 PLUG markers (Ishikawa et al., 2007), from the genetic and deletion maps of chromosome 3B of CS, and screened them for polymorphism between CS and N26. Out of 61 molecular markers, 12 SSR markers were polymorphic, while no PLUG marker showed polymorphism. Using randomly selected nine BC1F1 progenies that had Igc1, the 12 polymorphic SSR markers were then prescreened for linkage to the Igc1 locus (we refer to this step as ‘bulk analysis’). Two SSR marker loci Xwmc78 and Xgwm566 co-segregated with the Igc1 locus among the nine lines tested. To find more loci linked to the Igc1 locus, we searched for additional 40 SSR markers adjacent to Xgwm566 in the GrainGenes 2.0 database (http://wheat.pw.usda.gov/GG2/index.shtml). After a polymorphism survey between CS and N26, 13 markers were subjected to ‘bulk analysis’ and seven markers showed potential linkage to Igc1. Segregation data of the nine SSR markers and the Igc1 loci in 267 BC1F1 indicated that two marker loci Xgwm285 and Xgwm376 were completely co-segregated with Igc1 (Fig. 3).
 View Details | Fig. 3 A genetic map of the DNA markers linked to the Igc1 locus (left) and chromosomal bin map of the chromosome 3B in common wheat (right). The locus names in center are connected to both genetic and physical maps by solid lines. The genetic distances in cM are given in leftmost. The fraction lengths of bins are given in rightmost. |
To investigate the physical location of the SSR markers linked to the Igc1 locus, the mapped nine markers were ‘bin mapped’ using ditelosomics 3BS and 3BL, and eight deletion lines for chromosome 3B. Two co-segregating loci were assigned to different chromosomal regions; Xgwm285 was assigned to bin C-3BS1-0.33 (i.e., the chromosomal region defined by the centromere and the breakpoint of the line 3BS-1, which correspond to the proximal 33% of the short arm), and Xgwm376 was assigned to bin C-3BL2-0.22 (i.e., the chromosomal region defined by the centromere and the breakpoint of the line 3BL-2, which correspond to the proximal 22% of the long arm) (Fig. 3). This result implies that the Igc1 locus is located in the pericentromeric region of 3B spanning the bins C-3BS1-0.33 and C-3BL2-0.22.
Furthermore, we employed 102 insertion site-based polymorphism (ISBP) markers, which are designed from the BAC-end sequences of the contigs assigned to each chromosomal bin of chromosome 3B (Paux et al., 2008). Out of 102 ISBP markers, 51 marker loci were assigned to bin C-3BS1-0.33 and the rest to bin C-3BL2-0.22. Only two ISBP markers (cfp1886 in bin C-3BS1-0.33 and cfp1144 in C-3BL2-0.22) were polymorphic between CS and N26. Consequently the two markers were mapped as follows; Xcfp1886 was complete linked to the Igc1 locus, and Xcfp1144 was mapped 8.2 cM apart from the Igc1 locus in spite of its reported position in bin C-3BL2-0.22 (Paux et al., 2008) (Fig. 3). Close investigation of electrophoretic patterns of the PCR products by cfp1144 primers indicated amplification of multiple fragments. Thus, we probably genetically mapped different loci (denoted as Xcfp1144n) than the original Xcfp1144 loci previously reported.
Physical proximity of the linked markers to Igc1 was uncertain because three marker loci co-segregated with Igc1 were assigned to two different bins on different arms. Therefore, deletion mapping was conducted to identify markers physically close to the Igc1 locus. We could recover 38 Gc-induced chromosome deletion lines that had been selected as the Igc1 carriers from the progeny of the cross between CS-3C”(3B) and N26 + 3C’. Testing the genotype of the nine SSR markers mapped in the present study, we identified four lines (N26del-1, N26del-21, N26del-35, and N26del-36) with deletion in chromosome 3B (Table 1). The deletion lines that have breakpoints in the critical chromosomal region (pericentromeric region spanning bins C-3BS1-0.33 and C-3BL2-0.22) were N26del-1, N26del-21, and N26del-36. Then, we examined the presence/absence of the 102 ISBP markers to estimate the size of the chromosome region missing in the lines N26del-1, N26del-21, and N26del-36. Confirming location of the ISBP markers on chromosome 3B by nullisomic 3B line, we could test 97 ISBP markers (Table 2 and Table 3). In N26del-1, all 50 ISBP markers in bin C-3BS1-0.33 were present, and seven out of 47 (14.9%) ISBP markers in bin C-3BL2-0.22 were present (Fig. 4). In N26del-21, 26 (52.0%) and 18 (38.3%) ISBP markers in bins C-3BS1-0.33 and C-3BL2-0.22, respectively, were present. In N26del-36, 44 out of 50 (88.0%) ISBP markers in bin C-3BS1-0.33 were present. In contrast, out of 47 ISBP markers in bin C-3BL2-0.22, only one marker cfp3119 (2.1%) was amplified in N26del-36 (Fig. 4). Loss of the majority of the long arm in the lines N26del-1, -21 and -36 were confirmed by absence of the additional two markers wmc1 and wmc527 that are located in bins 3BL1-0.31-0.38 and 3BL9-0.38-0.50, respectively. A conservative estimation for the location of the Igc1 locus would be within proximal 52.0% of the bin C-3BS1-0.33 on the short arm or within the proximal 2.1% of the bin C-3BL2-0.22.
 View Details | Table 1 The presence or absence of SSR markers in deletion lines of chromosome 3B of N26 |
 View Details | Table 2 The presence or absence of markers of bin C-3BS1-0.33 in deletion lines of chromosome 3B of N26 |
 View Details | Table 3 The presence or absence of markers of bin C-3BL2-0.22 in deletion lines of chromosome 3B of N26 |
 View Details | Fig. 4 A schematic diagram showing the chromosomal region retained in the newly obtained deletion lines N26del-1, N26del-21, and N26del-36. Chromosomal region shown here is the bins C-3BS1-0.33 and C-3BL2-0.22 where the Igc1 locus resides. The circles represent the centromere of chromosome 3B of N26. Solid horizontal lines indicate the region retained, and dashed lines indicate the lost region. Numbers of markers present and its proportion to the normal 3B chromosome are given above and below the solid lines, respectively. Note that only a marker cfp3119 is present on the long arm of N26del-36. |
In plants and other species, recombination is not distributed evenly through the genome (see review by Mezard, 2006). In some plants with large genomes such as wheat (17 gigabases (Gb)), maize (2.5 Gb) and barley (5 Gb), gradual increase of recombination is found from the centromeres to telomeres (Lukaszewski and Curtis, 1993; Kunzel et al., 2000; Anderson et al., 2003; Saintenac et al., 2009). For chromosome 3B of wheat, which is eventually the longest (995 megabases (Mb)) chromosome in the wheat genome, Saintenac et al. (2009) reported that crossover frequency per physical distance (cM/Mb) within bins C-3BS1-0.33 and C-3BL2-0.22 were only 0.006 and 0.012, respectively in contrast to the highest value of 0.85 in bin 3BS8-0.78-0.87. Our results showed similar recombination suppression in pericentromeric region of chromosome 3B. Linkage analysis has shown that three loci Xgwm285, Xcfp1886, and Xgwm376 are cosegregating with the Igc1 locus (Fig. 4), of which Xgwm285 and Xcfp1886 are located on the short arm and Xgwm376 is on the long arm of 3B. This result implies that these three loci are still in substantial physical distances from the Igc1 locus although no genetic recombination was observed. The genetic distance of three markers to Igc1 might be smaller than 0.37 cM (assuming one recombination in 267 gametes), and thus the physical distance, if simply adopted the estimation by Saintenac et al. 2009, would be less than 62.4 Mb apart from Xgwm285 and Xcfp1886, and 31.2 Mb from Xgwm376. These values imply a sum of 93.6 Mb physical interval that carries the Igc1 locus, which is still about three fourth of the Arabidopsis genome (Bennett et al., 2003). This estimation seemed to be an underestimation from the cytological view point. Chromosome 3B is the largest chromosome in wheat and thus shares at least one 17th (ca. 995 Mb) of its genome. The pericentromeric region defined by bins C-3BS1-0.33 and C-3BL2-0.22 is estimated to be 266 Mb (Saintenac et al., 2009), roughly accounted for a quarter of the chromosome, which is more than 60% of whole rice genome (Matsumoto et al., 2005). Analysis of a large segregating population may not be a good approach for dissecting this large genomic region because recombination is highly suppressed.
Deletion mapping allowed us to use more markers than genetic mapping because deletion mapping does not need allelic polymorphism (Endo, 2007). Analysis of the Gc-induced chromosome deletion lines by using 113 markers (including genetically unmapped 100 ISBP markers) indicated that 26 loci on bin C-3BS1-0.33 and 18 loci on bin C-3BL2-0.22 were closer to the Igc1 locus than the Xgwm285 locus and the Xgwm376 locus, respectively. The locus Xgwm285 and Xgwm376 were cosegregating with the Igc1 locus in genetic mapping and physically mapped to the most proximal bin in the short arm and the long arm of chromosome 3B respectively. Because ISBP markers are evenly distributed along the chromosome 3B, this physical mapping with newly developed deletion lines suggested that the Igc1 locus locates on approximately 52.0% of bin C-3BS1-0.33 or 2.1% of bin C-3BL2-0.22 (Fig. 4). The genomic interval carrying the Igc1 gene was roughly estimated to be 76.4 Mb according to the physical size of the pericentromeric bins (Saintenac et al., 2009). Since the candidate chromosomal region in the short arm of 3B remains large, additional chromosome deletion lines with breakpoints in bin C-3BS1-0.33 are required to narrow the chromosomal region carrying Igc1. One of the methods to achieve the high resolution mapping in the recombination-depleted region of genome is the radiation hybrid (RH) mapping (Goss and Harris, 1975; Riera-Lizarazu et al., 2008). Kalavacharla et al. (2006) constructed a high-resolution physical map of chromosome 1D by irradiation treatment to wheat seed. This RH mapping approach allowed ordering molecular markers previously unordered within a chromosomal bin, and RH maps with the required resolution can be produced by altering the dosage of radiation. Therefore, the construction of high-resolution RH map might help us to identify the molecular marker locus that is in the close physical proximity of the Igc1 locus.
In a previous cytological study on the chromosome carrying Gc3-C1, the gene suppressed by Igc1, an isochromosome of the long arm of 3C of Aegilops triuncialis and a chromosome that lacks a short arm and most of the distal region of the long arm of 3C were produced (Endo, 2007). According to the fact that both structural mutants invoke Gc action, Gc3-C1 is predicted to be located in the proximal region of the long arm. Our mapping data indicated that Igc1 is located in the pericentromeric region of chromosome 3B, which is homoeologous to the region carrying Gc3-C1. Supposing Igc1 and Gc3-C1 are homoeologous genes, we hypothesized that Igc1 is located on the long arm. If this is the case, the Igc1 gene would be located in an extremely proximal region of the long arm because only one ISBP marker (cfp3119) in the long arm was retained in the deletion line N26del-36. Telosomic analysis using ditelosomic 3BS and 3BL of N26 should be conducted to identify whether Igc1 is located on the short arm or long arm of 3B.
The genes of the Segregation Distorter (SD) system of Drosophila melanogaster and t-haplotypes in mouse have been cloned (Powers and Ganetzky, 1991; Herrmann et al., 1999) and their biochemical function revealed (Bauer et al., 2007; McElroy et al., 2008). Although both distortion systems are caused by the interaction of linked genes ‘distorter’ and ‘responder’ and resulted in dysfunctions of male gametes, underlying systems are different. The responder of t-haplotype encodes a dominant-negative form of the protein kinase Smok1, but that of SD does not encode a protein. In plants, segregation distorter S1 in rice has been well studied (Koide et al., 2008). The S1 gene induces preferential abortion of both male (mTRD) and female gametes (fTRD) in interspecific hybrids. The degree of gamete dysfunction caused by S1 was altered depending on the genetic background in female gametes but always complete in male gametes, indicating that S1 is composed of two components for mTRD and fTRD and the presence of modifier(s) for fTRD. The S1 locus causing mTRD was narrowed down to a 40 kb region on chromosome 6, which includes eight open-reading frames. As segregation distorters are controlled by linked multi genes in other systems, the Gc factors may be a supergene composed of at least two genes tightly linked; a gene coding for the ‘breaking agent’ and another coding for the ‘protecting agent’. Pericentromeric localization, where recombination is strongly deflated, might help keep the structure of the supergene. Eventually, for the known Gc factors, we have repeated backcrossing for several decades, but we could not separate ‘breaking’ and ‘protecting’ factors by recombination. Synteny of orthologous genes between wheat and rice revealed high conservation and a one-to-one correspondence of centromeric regions between chromosome 3B of wheat and chromosome 1 of rice (Qi et al., 2009). For the centromeric region of chromosome 1 in rice, distortion of marker segregation has been found in the F2 population of indica-japonica hybrids (Harushima et al., 2001). Although factors causing segregation distortion reside in the syntenic chromosomal region, the mechanism and the causal factor for the segregation distortion may be different because chromosomal breakage in gametes of intraspecific hybrids in rice has not been reported so far. To find the differences, both genes in wheat and rice should be identified. In conclusion, linkage analysis using SSR markers, PLUG markers and ISBP markers showed that Xgwm285 and Xcfp1886 in bin C-3BS1-0.33 and Xgwm376 in bin C-3BL2-0.22 were cosegregated with the Igc1 locus, and physical analysis showed that the expected chromosomal region for the Igc1 locus is approximately 52.0% of bin C-3BS1-0.33 or 2.1% of bin C-3BL2-0.22. The physical mapping using deletion lines showed higher resolution in pericentromeric regions, where recombination is strongly suppressed. This study is the first step to fine mapping of the inhibitor of Gc factor in wheat. As the Igc1 locus was identified in the pericentromeric region in this study, our next effort will be extensive physical mapping by developing radiation-induced chromosomal deletion lines of N26, which is aimed to construct a high-resolution radiation hybrid map. Combination of recently developed genomic tools (ISBPs, Paux et al., 2006) for physical mapping of the chromosome 3B by INRA, France, and radiation hybrid panels of chromosome 3B of N26 should help us find molecular markers close enough to the Igc1 gene for future map-based cloning.
We thank Mr. Takehito Asami and Dr. Yutaka Okumoto, Laboratory of Plant Breeding, Graduate School of Agriculture, Kyoto University for help in genetic mapping. The polymorphism data of some SSR markers between CS and N26 were obtained from the National Bioresource Project (NBRP)-wheat (http://www.shigen.nig.ac.jp/wheat/komugi/strains/aboutNbrpMarker.jsp). This study was supported by Grant-in-Aid (B) (No. 20380006) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This publication is given the contribution number 601 from the Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Japan.
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