Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea

Background

Cabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F₂ population of 4000 individuals derived from the same parental lines.

Results

We confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99–77 and the highly susceptible line 99–91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F₂ population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes’ sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.

Conclusions

This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.     

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.     

Genetic mapping of a fusarium wilt resistance gene in Brassica oleracea

Fusarium wilt caused by Fusarium oxysporum f. sp. conglutinans is one of the most important diseases of Brassica crops, resulting in severe reductions in yield and quality. To characterize the inheritance pattern of fusarium resistance, a cross between a susceptible broccoli and a resistant cabbage was subjected to segregation analysis. Results indicated that resistance was controlled by a single dominant allele. This gene was named Foc-Bo1 and mapped to linkage group seven (O7) by both the segregation test and quantitative trait locus (QTL) analysis. The QTL on O7 was detected with a logarithm of odds score (LOD) of 19.5, which was above the threshold value with genome-wide 1% significance level (2.01). A minor QTL was also detected on O4 with a LOD score of 2.06. Inoculation tests indicated that stable expression of fusarium resistance at high temperatures required Foc-Bo1 homozygosity. The association between Foc-Bo1 and the closest simple sequence repeat marker (KBrS003O1N10) was analyzed in three F3 populations. Based on these studies, KBrS003O1N10 represents an effective marker-assisted selection (MAS) tool for breeding fusarium wilt resistance into Brassica oleracea crops. To our knowledge, this is the first paper to map the fusarium-resistance gene in Brassica species and to validate the effectiveness of MAS in improving fusarium resistance in these important plants.     

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.     

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var.capitata

INTRODUCTIONPlantshavedevelopedseveralbi0chemicaldefensemechanismsinresp0nsetopath0gensandabioticstress.Fo1l0wingpathogenattack,plantsynthesizephenyl-propaniodpr0ductssuchaslignin,l0wm0l.wt.antimicrobia1comp0undsknownasphyt0alexins,andseveraldefense-relatedproteins.Amongthesepr0teinsare"pathogenesis-relatedproteins"includingthefungalcellwalldegradingenzymeschitinaseandP-1,3-glucanase[1].Endochitinasefromhigherplantscatalyzethehydr0lysis0fchitin,aP-1,4-linkedhomop0lymerofN-acetyl-D-glucos…     

Map-based cloning of the ERECT PANICLE 3 gene in rice

Panicle architecture in rice can have a strong influence on yield. Using N-methyl-N-nitrosourea mutagenesis, we isolated an erect panicle mutant, Hep, from Hwasunchalbyeo, a glutinous japonica rice cultivar. Genetic analysis revealed that the erect panicle phenotype was controlled by a single recessive mutation designated erect panicle 3 (ep3). Genetic mapping revealed that the ep3 mutation was located on the short arm of chromosome 2 in a 0.1 cM region delimited by the STS markers STS5803-5 and STS5803-7. The ep3 locus corresponded to 46.8 kb region and contained six candidate genes. Comparison of the DNA sequences of the candidate genes from wild-type and erect panicle plants revealed a single base-pair change in the second exon of LOC_Os02g15950, which is predicted to result in a nonsense mutation. LOC_Os02g15950 encodes a putative F-box protein containing 515 amino acids and is expressed throughout the plant during all growth stages. A line carrying a T-DNA insertion in LOC_ Os02g15950 was obtained and shown to have the same phenotype as the ep3 mutant, thus confirming the identification of LOC_Os02g15950 as the ERECT PANICLE 3 (EP3) gene. The ep3 mutation causes a significant increase in the number of small vascular bundles as well as the thickness of parenchyma in the peduncle, which results in the erect panicle phenotype.     

cDNA cloning and characterization of a new stress-responsive gene BoRS1 from Brassica oleracea var. acephala

A new stress-responsive gene BoRS1 (GenBank accession No. AY373021 ) was isolated from Brassica oleracea var. acephala by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoRS1 was 2076 bp and contained a 1851 bp open reading frame (ORF) encoding 617 amino acids. Sequence analysis indicated that the deduced BoRS1 shared some identities with LTI65 , RD29A, RD29B and COR78 from Arabidopsis thaliana . Southern blot analysis of genomic DNA indicated that other related genes existed and there were two copies of BoRS1 in the genome of B. oleracea . Northern blot analysis revealed that BoRS1 was up-regulated by cold, mannitol, NaCl and abscisic acid (ABA). Expressional fluctuation of time course with ABA implied a two-step induction process. Tissue-specific expression analysis indicated that BoRS1 was expressed in all the tested plant tissues including leaves, stems and roots. Our studies imply that BoRS1 is a new gene that is responsive to environmental stresses such as low temperature, salinity and osmotic stress.     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400?kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100?kb, spanned by nine overlapping cosmid clones.     

Map-based isolation of disease resistance genes from bread wheat: cloning in a supersize genome

The genome of bread wheat is hexaploid and contains 1.6 x 10 10 bp of DNA, of which more than 80% is repetitive sequences. Its size and complexity represent a challenge for the isolation of agronomically important genes, for which we frequently know only their position on the genetic map. Recently, new genomic resources and databases from genome projects have simplified the molecular analysis of the wheat genome. The first genes to be isolated from wheat by map-based cloning include three resistance genes against the fungal diseases powdery mildew and leaf rust. In this review, we will describe the approaches and resources that have contributed to this progress, and discuss genomic strategies that will simplify positional cloning in wheat in the near future.     

Map-based cloning identifies velvet A as a critical component of virulence in Fusarium pseudograminearum during infection of wheat heads

Although better known as a pathogen of wheat stem bases, Fusarium pseudograminearum also causes Fusarium head blight. A natural isolate of F. pseudograminearum was identified that showed severely reduced virulence towards wheat heads and a map-based cloning approach was undertaken to identify the genetic basis of this phenotype. Using a population of 95 individuals, a single locus on chromosome 1 was shown to be responsible for the low virulence. Fine mapping narrowed the region to just five possible SNPs of which one was in the F. pseudograminearum homologue of velvet A. Knockout mutants of velvet A, which were non-pathogenic towards wheat, confirmed that velvet A regulates virulence in this pathogen. The mutation in velvet A was only found in a single field isolate and the origin of the mutation is unknown.     

Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.     

Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea.

In cruciferous plants, self-pollination is prevented by the action of genes situated at the self-incompatibility locus or S-locus. The self-incompatibility reaction is associated with expression of stigma glycoproteins encoded by the S-locus glycoprotein (SLG) gene. Only a few cases of self-compatible plants derived from self-incompatible lines in the crucifer Brassica have been reported. In these cases, self-compatibility was generally ascribed to the action of single genes unlinked to the S-locus. In contrast, we report here a line of Brassica oleracea var acephala with a self-compatible phenotype linked to the S-locus. By means of both biochemical and immunochemical analyses, we showed that this self-compatible (Sc) line nonetheless possesses stigmatic SLGs (SLG-Sc) that are expressed with a similar spatial and temporal pattern to that described for the SLGs of self-incompatible Brassica plants. Moreover, the SLG-Sc products segregate with the self-compatibility phenotype in F2 progeny, suggesting that changes at the S-locus may be responsible for the occurrence of the self-compatibility character. A cDNA clone encoding the SLG-Sc product was isolated, and the deduced amino acid sequence showed this glycoprotein to be highly homologous to the pollen recessive S2 allele glycoprotein. Hence, self-compatibility in this Brassica Sc line correlates with the expression of a pollen recessive-like S allele in the stigma.     

Map-based cloning of leaf rust resistance gene Lr21 from the large and polyploid genome of bread wheat

We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a gene-rich region at the distal end of chromosome arm 1DS of bread wheat (Triticum aestivum L.). Molecular cloning of Lr21 was facilitated by diploid/polyploid shuttle mapping strategy. Cloning of Lr21 was confirmed by genetic transformation and by a stably inherited resistance phenotype in transgenic plants. Lr21 spans 4318 bp and encodes a 1080-amino-acid protein containing a conserved nucleotide-binding site (NBS) domain, 13 imperfect leucine-rich repeats (LRRs), and a unique 151-amino-acid sequence missing from known NBS-LRR proteins at the N terminus. Fine-structure genetic analysis at the Lr21 locus detected a noncrossover (recombination without exchange of flanking markers) within a 1415-bp region resulting from either a gene conversion tract of at least 191 bp or a double crossover. The successful map-based cloning approach as demonstrated here now opens the door for cloning of many crop-specific agronomic traits located in the gene-rich regions of bread wheat.     

Functional analysis of a Brassica oleracea SLR1 gene promoter.

The Brassica oleracea S-locus-related gene 1 (SLR1) is expressed in the papillar cells of Brassica stigmas from a few days before anthesis. We have previously shown that a 1500-bp fragment of the SLR1 gene promoter is sufficient to direct high-level, temporally regulated expression of the beta-glucuronidase reporter gene in the pistils of transgenic tobacco. We have carried out a deletion analysis of the SLR1 promoter and found that elements required for pistil expression are located between -258 and -327 bp (relative to the translation start site). Furthermore, specific binding of pistil nuclear factors to sequences within this region was demonstrated by gel retardation analysis. Sequences between -1350 and -1500 were found to be required for high-level expression.     

Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa

The use of clubroot resistance (CR) genes is an effective and economical approach for controlling Plasmodiophora brassicae, the causal agent of clubroot disease in Chinese cabbage (Brassica rapa) and other Brassica crops. In a previous study, we identified and mapped the CRb locus on chromosome A03 of B. rapa in the doubled-haploid (DH) line ‘CR Shinki DH line’ of Chinese cabbage. In this study, CRb, a dominant gene conferring resistance to pathotype 4 of P. brassicae, was finely mapped in combination with bulked segregant analysis and bioinformatics analysis (BIA). Using 1,486 highly susceptible individuals and 2,896 individuals from two separate F₂ populations of ‘702-5’ (B. rapa ssp. chinensis) ×  ‘CR Shinki DH line,’ the CRb locus was narrowed to a region of approximately 0.14 cM between two flanking markers, TCR79 and TCR108. The sequences of seven newly developed markers linked to CRb were landed on bacterial artificial chromosome (BAC) of the reference B. rapa ‘Chiifu-401-42’ by BIA, and a physical map consisting of three BAC clones was constructed. The CRb locus was defined as an interval of approximately 83.5 kb on a BAC clone (KBrB085J21). The target interval contained one Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) gene, one NBS–LRR gene, and several putative regulatory genes in the B. rapa genome. The CRb gene was tightly linked to two other CR genes, CRa and CRb ^Kato . These results provide useful information for isolation of the CRb gene and tightly linked molecular markers for breeding CR in B. rapa.