Fine mapping of the clubroot resistance gene, Crr3, in Brassica rapa

A linkage map of Chinese cabbage (Brassica rapa) was constructed to localize the clubroot resistance (CR) gene, Crr3. Quantitative trait loci analysis using an F₃ population revealed a sharp peak in the logarithm of odds score around the sequence-tagged site (STS) marker, OPC11-2S. Therefore, this region contained Crr3. Nucleotide sequences of OPC11-2S and its proximal markers showed homology to sequences in the top arm of Arabidopsis chromosome 3, suggesting a synteny between the two species. For fine mapping of Crr3, a number of STS markers were developed based on genomic information from Arabidopsis. We obtained polymorphisms in 23 Arabidopsis-derived STS markers, 11 of which were closely linked to Crr3. The precise position of Crr3 was determined using a population of 888 F₂ plants. Eighty plants showing recombination around Crr3 locus were selected and used for the mapping. A fine map of 4.74 cM was obtained, in which two markers (BrSTS-41 and BrSTS-44) and three markers (OPC11-2S, BrSTS-54 and BrSTS-61) were cosegregated. Marker genotypes of the 21 selected F₂ families and CR tests of their progenies strongly suggested that the Crr3 gene is located in a 0.35 cM segment between the two markers, BrSTS-33 and BrSTS-78.     

Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa

Clubroot disease is one of the major diseases affecting Brassicaceae crops, and a number of these crops grown commercially, such as Chinese cabbage (Brassica rapa L. ssp. pekinensis), are known to be highly susceptible to clubroot disease. To provide protection from this disease, plant breeders have introduced genes for resistance to clubroot from the European turnip into susceptible lines. The CRa gene confers specific resistance to the clubroot pathogen Plasmodiophora brassicae isolate M85. Fine mapping of the CRa locus using synteny to the Arabidopsis thaliana genome and partial genome sequences of B. rapa revealed a candidate gene encoding a TIR-NBS-LRR protein. Several structural differences in this candidate gene were found between susceptible and resistant lines, and CRa expression was observed only in the resistant line. Four mutant lines lacking clubroot resistance were obtained by the UV irradiation of pollen from a resistant line, and all of these mutant lines carried independent mutations in the candidate TIR-NBS-LRR gene. This genetic and molecular evidence strongly suggests that the identified gene is CRa. This is the first report on the molecular characterization of a clubroot Resistance gene in Brassicaceae and of the disease resistance gene in B. rapa.     

SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage ( Brassica rapa ssp. pekinensis)

Clubroot disease, caused by Plasmodiophora brassicae Wor., is highly damaging for Chinese cabbage. The CR (clubroot resistant) Shinki DH (doubled haploid) line of Chinese cabbage carries a single dominant gene, CRb, which confers resistance to the P. brassicae races 2, 4, and 8. An F₂ population derived from a cross between the CR Shinki DH line and a susceptible line, 94SK, was used to map the CRb gene. Inoculation of F₃ families with SSI (single-spore isolate) resulted in a 1:2:1 segregation ratio. Use of the AFLP technique combined with bulked segregant analysis allowed five co-dominant AFLP markers, and four and seven dominant AFLP markers linked in coupling and repulsion, respectively, to be identified. Six of the 16 AFLP markers showing low frequencies of recombination with the CRb locus among 138 F₂ lines were cloned. A reliable conversion procedure allowed five AFLP markers to be successfully converted into CAPS and SCAR markers. An F₂ population (143 plants) was analyzed with these markers and a previously identified SCAR marker, and a genetic map around CRb covering a total distance of 6.75 cM was constructed. One dominant marker, TCR09, was located 0.78 cM from CRb. The remaining markers (TCR05, TCR01, TCR10, TCR08, and TCR03) were located on the other side of CRb, and the nearest of these was TCR05, at a distance of 1.92 cM.Communicated by R. Hagemann     

Accumulation of quantitative trait loci conferring broad-spectrum clubroot resistance in Brassica oleracea

Throughout the world, clubroot disease is one of the most damaging diseases affecting Brassica oleracea. To develop marker-assisted selection (MAS) that could assist the incorporation of durable clubroot resistance (CR) into cultivars, previous genetic analyses have identified several CR quantitative trait loci (CR–QTL). However, the independent and cumulative effects of each CR locus against various isolates have rarely been tested. Previously, we identified one major CR–QTL and four minor CR–QTL in the F2 plants from broccoli doubled haploid (DH) line × cabbage DH line of B. oleracea. In the present study, to clarify their effectiveness for controlling disease involving various isolates, inoculation testing was conducted in genotypes with various combinations of the CR genes, which were selected using the DNA markers closely associated with each CR–QTL. In exploring the overall disease incidence, it was apparent that a single involvement of the major CR gene located in the PbBo(Anju)1 locus, or accumulation of CR genes in the minor CR–QTL, is not enough to confer sufficient resistance. One major CR gene in the QTL PbBo(Anju)1 locus plus two to three minor CR genes conferred moderate resistance. The genotype in which all of the CR genes locating in the five QTL including PbBo(Anju)1 were accumulated showed the highest resistance, and it was broadly resistant against six isolates. Accumulation of several CR genes by MAS is necessary to conduct CR breeding in B. oleracea. Our developed DNA markers can be used efficiently to make selections of required loci for the acquisition of resistance, and use of these markers will be a powerful tool for CR breeding in B. oleracea.     

Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance

An SSR-based linkage map was constructed in Brassica rapa. It includes 113 SSR, 87 RFLP, and 62 RAPD markers. It consists of 10 linkage groups with a total distance of 1005.5 cM and an average distance of 3.7 cM. SSRs are distributed throughout the linkage groups at an average of 8.7 cM. Synteny between B. rapa and a model plant, Arabidopsis thaliana, was analyzed. A number of small genomic segments of A. thaliana were scattered throughout an entire B. rapa linkage map. This points out the complex genomic rearrangements during the course of evolution in Cruciferae. A 282.5-cM region in the B. rapa map was in synteny with A. thaliana. Of the three QTL (Crr1, Crr2, and Crr4) for clubroot resistance identified, synteny analysis revealed that two major QTL regions, Crr1 and Crr2, overlapped in a small region of Arabidopsis chromosome 4. This region belongs to one of the disease-resistance gene clusters (MRCs) in the A. thaliana genome. These results suggest that the resistance genes for clubroot originated from a member of the MRCs in a common ancestral genome and subsequently were distributed to the different regions they now inhabit in the process of evolution.     

Genetic mapping of a dominant gene conferring resistance to cassava mosaic disease

Cassava mosaic disease (CMD) is the most-important disease of cassava (Manihot esculenta) in Africa, and is a potential threat to Latin American (LA) cassava production. Although this viral disease is still unknown in LA, its vector - the whitefly - has recently been found. The disease is best controlled through host-plant resistance, which was first found in third backcross derivatives of an interspecific cross between cassava and Manihot glaziovii, and is thought to be polygenic. Recently, high levels of resistance were also found in several Nigerian cassava landraces. Classical genetic analysis and molecular genetic-mapping of the landraces showed that a major dominant gene confers this resistance. Bulk segregant analysis (BSA) was used to quickly identify a simple sequence repeat (SSR) marker linked to the CMD-resistance gene. The marker, SSRY28, is located on linkage group R of the male-parent-derived molecular genetic map. The gene, designated as CMD2, is flanked by the SSR and RFLP marker GY1 at 9 and 8 cM, respectively. To our knowledge, this is the first report of qualitative virus resistance in cassava, and of molecular markers that tag CMD resistance in cassava. We discuss the use of markers linked to CMD2 for marker-assisted breeding of CMD resistance in Latin America and for increasing the cost-effectiveness of resistance breeding in Africa.     

Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues.

Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.     

Fine mapping of the Ph-3 gene conferring resistance to late blight (Phytophthora infestans) in tomato

Late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is a devastating disease for tomato and potato crops. In the past decades, many late blight resistance (R) genes have been characterized in potato. In contrast, less work has been conducted on tomato. The Ph-3 gene from Solanum pimpinellifolium was introgressed into cultivated tomatoes and conferred broad-spectrum resistance to P. infestans. It was previously assigned to the long arm of chromosome 9. In this study, a high-resolution genetic map covering the Ph-3 locus was constructed using an F₂ population of a cross between Solanum lycopersicum CLN2037B (containing Ph-3) and S. lycopersicum LA4084. Ph-3 was mapped in a 0.5 cM interval between two markers, Indel_3 and P55. Eight putative genes were found in the corresponding 74 kb region of the tomato Heinz1706 reference genome. Four of these genes are resistance gene analogs (RGAs) with a typical nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 domain. Each RGA showed high homology to the late blight R gene Rpi-vnt1.1 from Solanum venturii. Transient gene silencing indicated that a member of this RGA family is required for Ph-3-mediated resistance to late blight in tomato. Furthermore, this RGA family was also found in the potato genome, but the number of the RGAs was higher than in tomato.     

Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea

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. oleracea CR 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. rapa CR 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.     

Molecular mapping and physical location of major gene conferring seedling resistance to Septoria tritici blotch in wheat

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola (anamorph: Septoria tritici), is one of the most important foliar diseases of wheat. We assessed three doubled-haploid (DH) populations derived from Chara (STB-susceptible)/WW2449 (STB-resistant), Whistler (STB-susceptible)/WW1842 (STB-resistant) and Krichauff (STB susceptible)/WW2451 (STB-resistant) for resistance to a single-pycnidium isolate 79.2.1A of M. graminicola at the seedling stage. STB resistance in each of the three DH populations was conditioned by a single major gene designated as StbWW2449, StbWW1842 and StbWW2451. Linkage analyses and physical mapping indicated that the StbWW loci were located on the short arm of chromosome 1B (IBS). Four simple sequence repeat (SSR) markers linked with STB resistance: Xwmc230, Xbarc119b, Xksum045 and Xbarc008 were located to the distal bin of 1BS.sat1BS-4 (FL: 0.52–1.00) in the 1BS physical map. Xwmc230, Xbarc119b and Xksum045 markers, mapped within 7 cM from StbWW were validated for their linkage and predicted the STB resistance with over 94% accuracy in the 79 advanced breeding lines having WW2449 as one of the parents. The marker interval Xwmc230/Xksum045-Xbarc119b also explained up to 38% of the phenotypic variance at the adult plant stage in all three DH mapping populations. These results have proven that SSR markers are useful in monitoring STB resistance both at seedling and adult plant stages and hence are suitable for routine marker-assisted selection in the wheat breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and mapping of a novel dominant resistance gene,TuRB07 to Turnip mosaic virus in Brassica rapa

Key message

A novel dominant resistance gene, TuRB07, was found to confer resistance to an isolate of TuMV strain C4 in B. rapa line VC1 and mapped on the top of chromosome A06.

Abstract

The inheritance of resistance to Turnip mosaic virus in Brassica rapa was investigated by crossing the resistant line, VC1 with the susceptible line, SR5, and genotyping and phenotyping diverse progenies derived from this cross. Both a doubled haploid population, VCS3M-DH, an F2 and two BC1 (F1 × VC1 and F1 × SR5) populations were created. Population tests revealed that the resistance to the TuMV C4 isolate in B. rapa is controlled by a single dominant gene. This resistance gene, TuRB07 was positioned on the top of linkage group A06 of the B. rapa genome through bulk segregation analysis and fine mapping recombinants in three doubled haploid- and one backcross population using microsatellite markers developed from BAC end sequences. Within the region between the two closely linked markers flanking TuRB07, H132A24-s1, and KS10960, in the Chiifu reference genome, two genes encoding nucleotide-binding site and leucine-rich repeat proteins with a coiled-coil motif (CC-NBS-LRR), Bra018862 and Bra018863 were identified as candidate resistance genes. The gene Bra018862 is truncated, but the gene Bra018863 has all the domains to function. Furthermore, the analysis of structural variation using resequencing data of VC1 and SR5 revealed that Bra018863 might be a functional gene because the gene has no structural variation in the resistant line VC1 when compared with Chiifu, whereas at the other NBS-LRR genes large deletions were identified in the resistant line. Allelic differences of Bra018863 were found between VC1 and SR5, supporting the notion that this gene is a putative candidate gene for the virus resistance.     

Fine physical mapping of the rice stripe resistance gene locus, Stvb-i

The Stvb-i gene confers stripe disease resistance to rice. For positional cloning, we constructed a physical map spanning 1.8-cM distance between flanking markers, consisting of 18 bacterial artificial chromosome (BAC) clones, around the Stvb-i locus on rice chromosome 11. The 18 clones were isolated by screening a BAC library derived from a japonica cultivar, Shimokita, with three Stvb-i-linked RFLP markers and DraI-digested DNAs of a yeast artificial chromosome (YAC) clone. The results of Southern hybridization and restriction enzyme analyses indicated that these BAC clones are contiguous and cover about a 700-kb region containing the Stvb-i allele. Utilizing end and internal fragments of the BAC insert DNAs, 33 molecular markers were generated within a small chromosomal region including the Stvb-i locus. Genotyping analysis with these markers for a resistant cultivar and four nearby recombinants selected from 120 F₂ individuals indicated that Stvb-i is contained within an approximately 286-kb region covered with two overlapping BAC clones. Received: 25 August 1999 / Accepted: 16 November 1999     

Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa

Brassica rapa is a member of the Brassicaceae family and includes vegetables and oil crops that are cultivated worldwide. The introduction of durable resistance against turnip mosaic virus (TuMV) into agronomically important cultivars has been a significant challenge for genetic and horticultural breeding studies of B. rapa. Based on our previous genome-wide analysis of DNA polymorphisms between the TuMV-resistant doubled haploid (DH) line VC40 and the TuMV-susceptible DH line SR5, we constructed a core genetic map of the VCS-13M DH population, which is composed of 83 individuals derived from microspore cultures of a F₁ cross between VC40 and SR5, by analyzing the segregation of 314 sequence-characterized genetic markers. The genetic markers correspond to 221 SNPs and 31 InDels of genes as well as 62 SSRs, covering 1,115.9 cM with an average distance of 3.6 cM between the adjacent marker loci. The alignment and orientation of the constructed map showed good agreement with the draft genome sequence of Chiifu, thus providing an efficient strategy to map genic sequences. Using the genetic map, a novel dominant TuMV resistance locus (TuMV-R) in the VCS-13M DH population was identified as a 0.34 Mb region in the short arm of chromosome A6 in which four CC–NBS–LRR resistance genes and two pathogenesis-related-1 genes reside. The genetic map developed in this study can play an important role in the genetic study of TuMV resistance and the molecular breeding of B. rapa.     

Resistance gene homologues in melon are linked to genetic loci conferring disease and pest resistance

Genomic and cDNA fragments with homology to known disease resistance genes (RGH fragments) were cloned from Cucumis melo using degenerate-primer PCR. Fifteen homologues of the NBS-LRR gene family have been isolated. The NBS-LRR homologues show high divergence and, based on the partial NBS-fragment sequences, appear to include members of the two major subfamilies that have been described in dicot plants, one that possesses a TIR-protein element and one that lacks such a domain. Genomic organization of these sequences was explored by DNA gel-blot analysis, and conservation among other Cucurbitaceae was assessed. Two mapping populations that segregate for several disease and pest resistance loci were used to map the RGH probes onto the melon genetic map. Several NBS-LRR related sequences mapped to the vicinity of genetic loci that control resistance to papaya ringspot virus, Fusarium oxysporum race 1, F. oxysporum race 2 and to the insect pest Aphis gossypii. The utility of such markers for breeding resistant melon cultivars and for cloning the respective R-genes is discussed.     

Genetic studies on resistance to clubroot in Brassica napus

The rate of softening of apples of the variety Spartan was reduced by addition of 6 or 8% CO₂ to a storage atmosphere of 2% O₂ at 1 -5 ^oC. This effect was observed in three seasons and in samples from five sources. Sensory assessments suggested that addition of carbon dioxide to the storage atmosphere had no adverse effect on eating quality. Storage in 2% O₂ at – 1 ^oC was as effective in maintaining flesh firmness as 8% CO₂+ 2%O₂ at 1–5 ^oC. Strategies for further reductions in firmness loss during storage of Spartan apples are discussed.     

Mapping of one major gene and of QTLs involved in resistance to clubroot in Brassica napus

Clubroot, caused by Plasmodiophora brassicae, is a damaging disease of Brassica napus. Genetic control and mapping of loci involved in high and partial quantitative resistance expressed against two single spore isolates (Pb137–522 and K92–16) were studied in the F₁ and DH progenies of the cross Darmor-bzh (resistant) x Yudal (susceptible). The high level of resistance expressed by Darmor-bzh to isolate Pb137–522 was found to be mainly due to a major gene, which we have named Pb-Bn1, located on linkage group (LG) DY4. Partial quantitative resistance showed by Darmor-bzh to the K92–16 isolate arose from the association of at least two additive QTLs detected on LGs DY4 and DY15; the QTL on DY4, explaining 19% of the variance, was mapped at the same position as the major gene Pb-Bn1. Epistatic interactions between nine regions with or without additive effects were detected. The total phenotypic variation accounted for by additive and epistatic QTLs ranged from 62% to 81% depending on the isolate. For one isolate, the relative effect due to additivity was similar to that due to epistasis. Received: 10 November 1999 / Accepted:18 February 2000