Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea
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To perform comparative studies of CR (clubroot resistance) loci in Brassica oleracea and Brassica rapa and to develop marker-assisted selection in B. oleracea, we constructed a B. oleracea map, including specific markers linked to CR genes of B. rapa. We also analyzed CR-QTLs using the mean phenotypes of F3 progenies from the cross of a resistant double-haploid line (Anju) with a susceptible double-haploid line (GC). In the nine linkage groups obtained (O1-O9), the major QTL, pb-Bo(Anju)1, was derived from Anju with a maximum LOD score (13.7) in O2. The QTL (LOD 5.1) located in O5, pb-Bo(GC)1, was derived from the susceptible GC. Other QTLs with smaller effects were found in O2, O3, and O7. Based on common markers, it was possible to compare our finding CR-QTLs with the B. oleraceaCR loci reported by previous authors; pb-Bo(GC)1 may be identical to the CR-QTL reported previously or a different member contained in the same CR gene cluster. In total, the markers linked to seven B. rapaCR genes were mapped on the B. oleracea map. Based on the mapping position and markers of the CR genes, informative comparative studies of CR loci between B. oleracea and B. rapa were performed. Our map discloses specific primer sequences linked to CR genes and includes public SSR markers that will promote pyramiding CR genes in intra- and inter-specific crosses in Brassica crops. Five genes involved in glucosinolates biosynthesis were also mapped, and GSL-BoELONG and GSL-BoPro were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. The linkage drag associated with the CR-QTLs is briefly discussed.
Clubroot disease is caused by a soil-borne obligate biotroph, Plasmodiophora brassicae, and is one of the most devastating diseases in Brassica crops worldwide (Crute et al. 1980; Hirai 2006). The pathogen causes gall formation on roots in the shape of a club or spindle. Severely infected roots cannot take up sufficient amounts of nutrients and water, and such infected plants are stunted and wilt in direct sunlight, causing a reduction in yield of both vegetable and oleiferous Brassicas (Voorrips 1995). Agricultural practices such as crop rotation, application of calcium to raise the pH, good drainage and sanitation, as well as use of agrochemicals are insufficient to control clubroot disease (Dobson et al. 1983; Voorrips 1995; Niwa et al. 2008). In addition, it is difficult to control the disease because the pathogen survives in soil as resting spores for long periods (Wallenhammar 1996). Therefore, development of genetically resistant cultivars to minimize crop losses caused by clubroot disease is highly desired.
Various genetic loci encoding clubroot resistance (CR) were extensively screened in Brassica oleracea (Dixon and Robinson 1986; Crips et al. 1989; Dias et al. 1993; Manzanares-Dauleux et al. 2000). However, only a few resistant populations such as German cabbage landrace ‘Bindsachsener’ and ‘Böhmerwaldkohl’ are available for resistance breeding with B. oleracea (Crute et al. 1983; Crips et al. 1989; Voorrips 1995; Diederichsen et al. 2009). Rutabaga cv. ‘Wilhelmsburgar’ is a known source of resistance to the clubroot pathogen in Brassica napus. Chiang and Crete (1983) successfully transferred CR genes from B. napus to B. oleracea. Although cultivars resistant to clubroot disease are bred using such strategies, production of durable resistant B. oleracea cultivars has not been successful due to poor knowledge of the inheritance of resistance and complexity of the plant–pathogen interaction.
Studies have disagreed as to whether clubroot resistance in B. oleracea is a qualitative or a quantitative trait. In reports describing clubroot resistance as qualitative, clubroot resistance can be either dominant (Chiang and Crete 1983) or recessive (Chiang and Crete 1970; Yoshikawa 1983). When employing QTL (quantitative trait loci) analysis for genetic behavior of resistance to clubroot, on the other hand, authors usually conclude that clubroot resistance in B. oleracea progenies is quantitative and under polygenic control, illustrating the existence of one or two major QTLs and some QTLs with minor effects (Landry et al. 1992; Figdore et al. 1993; Grandclement and Thomas 1996; Voorrips et al. 1997; Moriguchi et al. 1999; Rocherieux et al. 2004; Nomura et al. 2005). Because none of these studies has disclosed specific primer sequences or sequences of restriction fragment length polymorphism (RFLP) markers linked to the CR genes, additional studies that disclose these data are necessary to develop marker-assisted selection for clubroot resistance in B. oleracea (Hirai 2006). In Brassica rapa, many studies have demonstrated oligogenic control of clubroot resistance (reviewed by Hirai 2006; Piao et al. 2009). In total, eight CR loci were mapped and allocated to five different B. rapa chromosomes. The authors reported the specific primer sequences of the DNA markers linked to the B. rapaCR genes so that marker-assisted selection is now available for clubroot resistance. It is also now possible to compare whether the published CR genes are mutually homologous. The genes Crr2, CRc, Crr4, and Crr1 map to R1, R2, R6, and R8 of B. rapa linkage groups, respectively. The genes CRa, CRb, CRk, and Crr3 map to R3. CRk and Crr3 map closely together in R3 but are not at the same locus because these two loci were derived from different CR origins and had been inoculated with different isolates and conditions (Sakamoto et al. 2008). Therefore, it is important to clarify whether the CR genes identified so far are homologous; intra- and inter-specific comparison of B. oleracea and B. rapa is a means to address this issue.
The aims of this study were (1) to identify PCR-based markers linked to CR genes in B. oleracea, (2) to report the specific marker sequences of the CR genes to develop marker-assisted selection to clubroot resistance in B. oleracea, and (3) to perform a comparative study of CR genes in B. rapa and B. oleracea by simultaneously mapping the DNA markers linked to the CR genes identified in this study and in previous reports.
Materials and methods
Double-haploid (DH) lines were obtained from the clubroot-resistant cabbage cultivar ‘Anju’ (Nippon Norin Seed Co., Japan) and the susceptible broccoli cultivar ‘Green Comet’ (GC) (Takii & Co., Ltd.) using microspore culture. A preliminary inoculation test using a Plasmodiophora brassicae isolate revealed that the Anju DH line (P01) and GC DH line (P04) were resistant and susceptible, respectively. The GC DH line (P04) was crossed as the female parent to the Anju DH line (P01). A single F1 plant was self-pollinated to produce F2 seeds. F2 plants were self-pollinated to produce F3 seeds.
Pathogen isolation and inocula preparation
The isolate of P. brassicae that was determined as race 4 using the inoculation method of Williams (1966) was provided by A. Kiso (Musashino Seed Co., Ltd.). The pathogen was propagated on turnips, and the clubs in infected roots were stored at −20°C until required. Inocula were prepared from slowly thawed clubs. The clubs were ground in distilled water using a mortar and pestle, and the homogenized tissue was squeezed through four-layered gauze. The squeezed fluid was gathered and centrifuged at 1000×g for 10 min at room temperature. The pellet, which contained resting spores, was suspended in sterile distilled water. The final resting spore concentration was adjusted to 107 spores/ml using a hemocytometer.
Test for clubroot resistance
Seeds were germinated in 8-cm-diameter plastic pots containing soil in a greenhouse. Twelve plants of each parent and 12 F1 plants were used for the inoculation test. For the F3 test, a subset of 94 F3 progeny obtained from randomly selected F2 plants was sown, and 12 plants per F3 progeny were used for phenotypic evaluation. One-week-old seedlings were inoculated by applying 1 ml of spore suspension at the bottom of the stem base of each seedling (pipette method). The inoculated plants were grown during spring months in a greenhouse at a maximum temperature of 25°C. The soil was kept moist throughout the test. Four plants in each F3 strain were grouped to make one replication, and each strain was tested in randomized complete block design with three replications. The plants were evaluated for clubroot infection 6 weeks after inoculation. The roots were thoroughly washed. The status of each root system was rated on a scale of 0–5, where 0 = no clubs, 1 = a few small clubs usually confined to lateral roots, 2 = moderate clubbing on lateral roots, 3 = larger clubs on lateral roots and slight swelling of main roots, 4 = larger clubs in main roots, and 5 = severe clubbing (no roots left, only one big gall). The disease severity index (DI) was calculated from the results as the mean value for the 12 F3 seedlings. The phenotype evaluation was carried out twice, once in 2006 and once in 2008, using the same F3 seed obtained from each F2 plant, and the mean grades of the two F3 progeny tests were calculated.
Detection of DNA polymorphism
DNA markers used in this study
Cited from Cheng et al. (2009)
Cited from Suwabe et al. (2006)
Cited from Saito et al. (2006)
Cited from Iniguez-Luy et al. (2008)
Cited from Okazaki et al. (2007)
Hayashida et al. (2008). Primers were designed from sequences collected from the NCBI nucleotide database.
Qiu et al. (2006). Primers were designed from sequences collected from the IMSORB nucleotide database.
Designed from terminal sequences of BAC clones released from the B. rapa genome project.
Designed from terminal sequences of BAC clones released from the B. rapa genome project.
Cited from Radoev et al. (2008)
Cited from Li et al. (2003)
Cited from Radoev et al. (2008)
Cited from Sakamoto et al. (2008)
Udall et al. (2005). Primers were designed from sequences collected from the NCBI nucleotide database.
Cited from Piao et al. (2004)
Annealing temperatures and extension times for PCR were determined according to the primer sequence and gene size. The PCR products were digested with one of four restriction enzymes (AfaI, AluI, MspI, or MboI). To identify the positions of the B. rapa CR genes in the B. oleracea map, we used the PCR-based markers reported by Suwabe et al. (2003, 2006), Piao et al. (2004), Saito et al. (2006), and Sakamoto et al. (2008). In addition, to identify the position of another CR gene, CRa, in B. rapa, a primer pair, HC352F2 and HC352R2, was designed as 5′-gctacaccaaaagattcgag-3′ and 5′- tgtccttcatagacaatgac-3′ based on the closest marker (HC352; Accession No. AB302983) to CRa. The amplified product was digested with AluI before polyacrylamide gel electrophoresis.
Electrophoresis was conducted using an 8–13% polyacrylamide gel (Kikuchi et al. 2003). The gel was stained with a Gelstar solution (0.1 μl/10 ml; Takara Biomedicals, Japan).
Construction of the map and QTL analysis
Linkage analysis of the markers was performed using the program Antmap 1.2 (Iwata and Ninomiya 2006). QTLs for clubroot resistance were analyzed using a composite interval mapping (CIM) analysis (Zeng 1994) with QTL Cartographer version 2.5 (Basten et al. 2002). A 1,000-permutation test was performed with QTL Cartographer to estimate the appropriate significance threshold of a logarithm of odds (LOD) score for analysis.
Synteny analysis of the QTL regions
To identify syntenic regions between the B. rapa and B. oleracea genomes, KBr markers designed with the BAC sequences released from BrGP were aligned with the JWF3p map published on the BrGP web site (http://www.brassica-rapa.org/BRGP/index.jsp). In addition, we compared our map with the B. rapa maps published by Suwabe et al. (2006) and Sakamoto et al. (2008). To identify homologous regions between the Arabidopsis thaliana genome and our map, the sequences harboring the markers were aligned with the A. thaliana genome sequence using BLASTn in DDBJ. Based on a threshold value of E < 10−10, we identified regions that were relatively conserved between the A. thaliana genome and our map. Groups of two or more markers showing homology and collinearity with A. thaliana were regarded as syntenic regions.
Construction of linkage maps
QTL analysis for clubroot resistance
Summary of QTLs detected for clubroot resistance against P. brassicae
Marker position (cM)
QTL analysis was carried out separately for each of the 2006 and 2008 tests and the average of the 2 years. Five QTLs for clubroot resistance were detected in O2 (two regions), O3, O5, and O7. Among them, the largest QTL located in O2 was detected in both years and in the combined data of the 2 years. The scores for the minor effect QTLs varied between the 2 years; the QTLs located in O3 and O7 exceeded the significance threshold score in 2006, and the minor effect QTLs in O2 and O5 were significant in 2008. These minor QTLs were significant for the combined data of the 2 years, except for the QTL located in O7 that had a score of 3.1, comparable to the threshold score (3.5). The largest QTL effect (LOD of 13.7) for clubroot resistance was detected between the loci TFL and pW176 on O2 and was closely linked to marker KBrH059L13R (Fig. 1; Table 2). This QTL explained 47% of the total phenotypic variation. This CR locus was named pb-Bo(Anju)1. The QTL located in O5 came from the susceptible broccoli parent (Table 2), and therefore this CR locus was named pb-Bo(GC)1. Despite high susceptibility of the susceptible parent GC to P. brassicae, this CR locus accounted for only 9% of the variation, suggesting that there may be epistatic genes that interact with Bo(GC)1 in other regions of the genome. Other small-effect QTLs found in O2, O3, and O7, which came from the resistant parent, accounted for 16% of the variation. These CR loci on O2, O3, and O7 were named pb-Bo(Anju)2, pb-Bo(Anju)3, and pb-Bo(Anju)4, respectively.
Phenotypic DI at the KBrH059L13R marker that was closely linked to the major QTL indicated that higher resistance was associated with the homozygous Anju genotype versus the homozygous GC genotype, with the heterozygotes having varying resistance levels (Fig. 2).
Mapping of molecular markers closely linked to B. rapaCR genes
Quantitative resistance to P. brassicae
In the phenotypic evaluation, the F1 plants obtained from the cross of susceptible GC and resistant Anju were partially resistant to P. brassicae. The F3 progenies revealed typical continuous distributions for clubroot resistance. This type of continuous trait is controlled not only by multiple genes but also by a few individual genes that reveal continuous distribution in their progeny due to environmental effects and experimental error when measuring the phenotype. In our study, the QTL analysis that was performed using the appropriate significance threshold successfully detected several significant QTLs, indicating that Anju resistance was controlled by a polygenic system. The fact that no plants exhibited transgressive segregation beyond the range between the two parents suggests that a large number of QTLs did not contribute to clubroot resistance in the progeny, and every QTL for clubroot resistance/susceptibility converged into either of the parental genotypes, a resistant homozygote (Anju type), or a susceptible homozygote (GC type). Alternatively, the DI used in this study, which was not a continuous variable, may be not suitable for detecting transgressive segregation in the progeny.
In the QTL analysis, we detected one major locus (pb-Bo(Anju)1 on O2 that accounted for 47% of the variation) and some QTLs with minor effects on O2, O3, O5, and O7. The QTL with the largest effect exhibited good stability in the 2006 and 2008 tests. The other QTLs showed annual variation, which may be due to an environmental effect or inoculation conditions. The minor effect QTLs were significant for at least one of the two inoculation tests, and three of the four minor QTLs were significant using the combined data of the two tests. A candidate QTL (LOD of 3.1), detected in the distal end of O7, was comparable to the threshold score (3.5). Therefore, although the minor QTL effects are thought to be located on O2, O3, O5, and O7, it will be essential to isolate the individual QTLs through recombination and selection in the progeny to reassess the effect of individual QTLs.
Genotyping at the KBrH059L13R marker that was closely linked to pb-Bo(Anju)1 showed that the homozygous Anju genotypes were highly resistant, whereas the homozygous GC genotypes were susceptible, and the heterozygotes tended to express partial resistance. This result suggests that pb-Bo(Anju)1 acts as a partially dominant gene in heterozygous plants or F1 plants and has notably established stable expression in homozygous plants. It is well known that major QTL effects that account for more than 50% of the phenotypic variation contribute to B. oleracea clubroot resistance (Landry et al. 1992; Voorrips et al. 1997; Rocherieux et al. 2004). The intermediate value of DI to clubroot resistance in F1 plants is in agreement with the report of Figdore et al. (1993), who reported that the DI of the F1 plant derived from the cross of susceptible cauliflower and resistance broccoli was intermediate, and the F2 population studied revealed the polygenic control of clubroot resistance that involves one major QTL effect and some minor QTL effects. The stable expression of resistance in homozygotes at the pb-Bo(Anju)1 locus indicates that the CR genes cumulatively act in a dose-dependent manner, which is consistent with the results reported by Suwabe et al. (2003) and Nomura et al. (2005). Previous studies identified B. oleracea clubroot resistance genes in either a dominant (Hansen 1989; Landry et al. 1992; Figdore et al. 1993; Laurens and Thomas 1993) or recessive manner (Yoshikawa 1983; Voorrips and Visser 1991). The expression levels of the CR genes that are relevant to the dominant-recessive relationship are thought to be determined by the specific CR gene itself, differential races of P. brassicae, diverse genetic backgrounds of plants, environmental conditions, etc. Which factor contributes the most to the control of CR gene expression remains to be determined. Furthermore, studies are needed to understand the interaction of resistance genes with pathogenicity genes.
Linkage drag between the CR genes and glucosinolate pathway genes
The largest QTL, pb-Bo(Anju)1, was located on the distal end of O2. The QTL Bo(GC)1 was located on the central region of O5. In those regions, GSL-BoELONG and GSL-BoPro, which control the chain elongation of aliphatic glucosinolates, were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. This observation indicates that this linkage drag can lead to a correlation between specific glucosinolates and resistance to P. brassicae. In fact, Chong et al. (1985) found that the level of goitrin, a sulfur-containing metabolite that modulates thyroid hormone production, was higher in selected clubroot-resistant cabbages than in commercial cultivars. Similarly, Chiang et al. (1989) found a correlation between a low level of thiocyanate and clubroot disease in broccoli. In our study, using simultaneous mapping of the CR genes and the glucosinolate pathway genes, we clarified the reason why these groups found correlations between specific glucosinolates and resistance to P. brassicae. In general, glucosinolates and their breakdown products are thought to play a role in disease resistance against insects and fungal pathogens (Glen et al. 1990; Menard et al. 1999). However, no evidence has been reported showing that glucosinolates and their hydrolysis products are protective against P. brassicae (Chong et al. 1985; Chiang et al. 1989). Likewise, when we mapped the glucosinolate pathway genes to the B. oleracea map, no QTLs were detected around the positions of GSL-BoALK, GSL-BoOH, or GSL-BoCYP79F1, and the positions of GSL-BoELONG and GSL-BoPro were outside the confidence intervals of the QTLs detected around the two genes. Thus our data indicate that glucosinolate biosynthesis genes are not CR genes.
QTLs of clubroot resistance in B. oleracea
Recent studies on QTL analysis of CR genes have revealed that clubroot resistance in B. oleracea is characterized by oligogenic inheritance (Landry et al. 1992; Figdore et al. 1993; Grandclement and Thomas 1996; Voorrips et al. 1997; Moriguchi et al. 1999; Rocherieux et al. 2004; Nomura et al. 2005). Landry et al. (1992) identified two QTLs, CR2a, and CR2b, in the progeny derived from the cross between resistant Rutabaga and susceptible CrGC No85. Voorrips et al. (1997) identified two CR loci, pb-3 and pb-4, and a few minor CR QTLs in a population of DH lines of F1 plants obtained between cabbage landrace ‘Bindsachsener’ and broccoli ‘Greenia’. Nomura et al. (2005) identified three QTLs for clubroot resistance in a population from a cross between cabbage and the Kale line ‘K269’. Rocherieux et al. (2004) reported differential QTLs from different isolates of P. brassicae and found that one QTL (pb-Bo1) acts as a major resistance gene against the three isolates. In our study, we identified five QTLs, pb-Bo(Anju)1, pb-Bo(Anju)2, pb- pb-Bo(Anju)3, pb-Bo(Anju)4, and pb-Bo(GC)1, from the genetic and phenotypic analysis of a cross of resistant cabbage and susceptible broccoli.
It is difficult to compare the map positions of CR loci identified so far, due to the lack of common DNA markers in the published B. oleracea linkage groups. Based on the few common markers linked to CR genes, however, Voorrips et al. (1997) revealed that CR2a (Landry et al. 1992) and pb-4 (Voorrips et al. 1997) are linked to the common marker 2NA8. However, those two CR genes are not likely to be identical because of their different origins; CR2a is derived from CR Rutabaga, and pb-4 is from CR cabbage landrace. Similarly, we attempted to compare the CR genes published in B. oleracea as follows: Landry et al. (1992), Rocherieux et al. (2004), and Moriguchi et al. (1999) identified CR2b, pb-Bo1, and QTL-LG1, respectively, which were mapped to the distal end of the largest linkage groups in their B. oleracea maps. Voorrips et al. (1997) also detected pb-3 at the distal end of the large linkage group. These data suggest that the largest linkage groups identified in these studies probably correspond to the largest linkage group (O3) in our map; thus, collectively the five B. oleraceaCR genes, CR2b, pb-3, pb-Bo1, QTL-LG1, and Pb-Bo(Anju)3, map to either end of O3. In fact, based on the marker 4NE11, which was mapped as a common RFLP marker to the distal ends of the largest linkage groups in Landry et al. (1992) and Voorrips et al. (1997), Voorrips et al. (1997) detected pb-3 at the marker 4NE11, whereas Landry et al. (1992) detected CR2b on the opposite sides of the marker 4NE11 in the largest linkage group. This mapping is consistent with our data that map the CR genes at both ends of the largest linkage group (O3) (Fig. 4).
Nomura et al. (2005) identified the major effect QTL (QTL-LG3) that is linked to the marker WG1G5 (equal to pW216) in linkage group 3. Using a specific primer to amplify the marker pW216 sequence that was collected from the NCBI database, we mapped this marker to O1 in our map. The QTLs found in our study were not detected in this region, indicating that the major CR locus reported by Nomura et al. (2005) is different from all the CR loci found in our study and may be lacking in our plant materials. Alternatively, this result suggests that some of the CR genes in B. oleracea are differentially expressed against the various P. brassicae races. The differential response of CR genes to the isolates was reported in B. rapa (Suwabe et al. 2006; Sakamoto et al. 2008) and in B. oleracea (Rocherieux et al. 2004). Nomura et al. (2005) also identified a minor QTL (QTL-LG9) that is linked to the marker WG6H1 (equal to pW237) in linkage group 9. The marker pW237 was mapped to the central region of O5 in our map where the CR locus pb-Bo(GC)1 was detected, suggesting that QTL-LG9 and pb-Bo(GC)1 may be the same locus. Alternatively, the two CR loci could be different members located in the same CR gene cluster.
Comparative analysis of CR genes between B. rapa and B. oleracea
Extensive QTL analyses of clubroot resistance in B. rapa were recently conducted using public SSR markers and CR genes and linked markers (Suwabe et al. 2003, 2006; Hirai et al. 2004; Piao et al. 2004; Matsumoto et al. 2005; Saito et al. 2006; Hayashida et al. 2008; Sakamoto et al. 2008). As a result, CR genes were found in B. rapa as follows: CRa (Matsumoto et al. 1998), CRb (Piao et al. 2004), Crr3 (Hirai et al. 2004), Crr1, Crr2, Crr4 (Suwabe et al. 2003, 2006), and CRc and CRk (Sakamoto et al. 2008). In total, eight CR loci were mapped and allocated to five different chromosomes (Fig. 4a). In our study, we mapped the B. rapaCR gene markers to the B. oleracea map so that the marker m6R that is closely linked to CRc was mapped to the central region of O2 where we detected a minor QTL, pb-Bo(Anju)2. In addition, the marker TCR05 that is linked to CRb was mapped to O7, where pb-Bo(Anju)4 was detected. Moreover, using several anchor markers available in both the B. oleracea and B. rapa genomes, we showed that the B. rapa chromosomal regions harboring B. rapaCR gene-specific markers are homologous to the corresponding region of B. oleracea (Fig. 3), indicating that the linkage of the CR genes versus the specific markers established in the B. rapa genome is maintained in B. oleracea to some extent. Therefore, these results raise the possibility that the pb-Bo(Anju)2 and the pb-Bo(Anju)4 loci in B. oleracea are homologous to CRc and CRb in B. rapa, respectively. However, it is difficult to conclude whether the QTLs that are linked to the same molecular markers involve just one gene or family members of clustered CR genes. Microsynteny analysis in those regions in B. rapa and B. oleracea is needed to identify the relationship between these CR loci.
The marker BSA3, which is closely linked to Crr2, and the marker BrSTS61, which is closely linked to Crr3, were mapped to O1 and O3, respectively. In the flanking regions of these markers, we did not detect any counterpart QTLs. On the other hand, we detected the major-effect pb-Bo(Anju)1, moderate-effect pb-Bo(GC)1, and minor-effect pb-Bo(Anju)3 on O2, O5, and O3, respectively, where none of the published CR loci derived from the B. rapa genome were included. These results raise the following possibilities: First, Crr2 and Crr3 are the original CR genes in the B. rapa genome, whereas pb-Bo(Anju)1, pb-Bo(GC)1, and pb-Bo(Anju)3 originally existed in B. oleracea. If so, interchange and pyramiding of CR genes between B. rapa and B. oleracea through interspecific hybridization will be useful for clubroot resistance breeding; success in this regard would support this hypothesis. Second, the CR genes responded differently to different isolates used in this study and in previous studies (Suwabe et al. 2003; Hirai et al. 2004). Third, the linkage of the CR genes versus the specific markers established in the B. rapa genome is not maintained in the corresponding region of the B. oleracea genome. To address these questions, cloning of CR genes and QTL analyses using various races are required to characterize the relationship between CR genes and races of P. brassicae.
Genetic origin and diversification of CR genes
R-genes (NBS-LRR) are clustered mainly in A. thaliana chromosomes 1, 4, and 5 (Jones 2001). This cluster of disease resistance genes is termed the major recognition complex. Studies have reported that the regions harboring the B. rapaCR genes overlap in major recognition complexes of the A. thaliana genome (Suwabe et al. 2006; Piao et al. 2009). In our study, we showed that the regions containing pb-Bo(Anju)1 and pb-Bo(Anju)3 correspond to the top of A. thaliana chromosome 5 (Fig. 1, Supplementary Table S3). pb-Bo(Anju)2 and pb-Bo(Anju)4 correspond to the middle of chromosome 4, and pb-Bo(Anju)4 corresponds to the distal end of chromosome 1. This observation demonstrates that the regions harboring the B. oleraceaCR genes correspond to the major recognition complexes of the A. thaliana genome, as in the case of the CR loci in B. rapa.
Fuchs and Sacristain (1996) identified a CR locus (RPB1) in A. thaliana chromosome 1. Jubault et al. (2008) mapped two CR loci, pb-At1, pb-At4, in chromosomes 1 and 4, respectively, and the other CR loci, pb-At5.1 and pb-At5.2, in chromosomes 5. In B. rapa, Crr1, Crr2, and CRb are syntenic with the central region of A. thaliana chromosome 4 (Suwabe et al. 2006; Piao et al. 2009). However, the region harboring Crr3 corresponds to A. thaliana chromosome 3 (Saito et al. 2006). These results indicate that the CR loci of crucifer crops, including B. oleracea, may express lineage commitment-related CR genes that originated from the common ancestor of crucifer plants. At present, however, as a result of the long-term host–parasite co-evolution, BrassicaCR genes have diversified against the various P. brassicae races (Suwabe et al. 2006; Sakamoto et al. 2008; Rocherieux et al. 2004). This study will promote a more comprehensive description of different CR genes of B. rapa, B. oleracea, as well as B. napus.
The authors sincerely thank Dr. K. Hatakeyama from the National Institute of Vegetable and Tea Science, Japan, for kindly providing DH lines.
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