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.     

Natural variation in a calreticulin gene causes reduced resistance to Ca2+ deficiency‐induced tipburn in Chinese cabbage (Brassica rapa ssp. pekinensis)

Tipburn is an irreversible physiological disorder of Chinese cabbage that decreases crop value. Because of a strong environmental component, tipburn‐resistant cultivars are the only solution, although tipburn resistance genes are unknown in Chinese cabbage. We studied three populations of Chinese cabbage over four growing seasons under field conditions: (a) 194 diverse inbred lines, (b) a doubled haploid (DH100) population, and (c) an F₂ population. The 194 lines were genotyped using single nucleotide polymorphism markers, and genome‐wide‐association mapping showed that 24 gQTLs were significantly associated with tipburn disease index. Analysis of the DH100 and F₂ populations identified a shared tipburn‐associated locus, gqbTRA06, that was found to cover the region defined by one of the 24 gQTLs. Of 35 genes predicted in the 0.14‐Mb quantitative trait locus region, Bra018575 (calreticulin family protein, BrCRT2) showed higher expression levels during disease development. We cloned the two BrCRT2 alleles from tipburn‐resistant (BrCRT2^R) and tipburn‐susceptible (BrCRT2^S) lines and identified a 51‐bp deletion in BrCRT2^S. Overexpression of BrCRT2^R increased Ca²⁺ storage in the Arabidopsis crt2 mutant and also reduced cell death in leaf tips and margins under Ca²⁺‐depleted conditions. Our results suggest that BrCRT2 is a possible candidate gene for controlling tipburn in Chinese cabbage.     

Development of InDel markers linked to Fusarium wilt resistance in cabbage

Cabbage Fusarium wilt (CFW) is a destructive disease causing great losses to cabbage (Brassica oleracea L. var. capitata L.) production worldwide. At present, there are few reports concerning molecular marker research on cabbage resistance to CFW. In this study, 160 double haploid (DH) lines were obtained from the F1 population of a 99–77 (highly resistant to CFW) × 99–91 (highly susceptible to CFW) cross. Insertion–deletion (InDel) markers were designed according to the reference genome sequence of cabbage and the whole-genome re-sequencing data of the two parents. A genetic map of chromosome C06 including seven InDel markers was constructed based on the DH population. Thus, FOC (resistance gene to Fusarium oxysporum f. sp. conglutinans) was located on chromosome C06 and two InDel markers out of the seven, M10 and A1, flanked the gene at 1.2 and 0.6 cM, respectively. Marker A1 revealed a significant consistency with the phenotype assay in the F2 population as well as in 40 inbred lines (96 and 82 %, respectively). This study lays the foundation for fine mapping and cloning of the FOC gene and for marker-assisted selection in cabbage resistance breeding.     

Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage (Brassica rapa L.)

The extreme resistance to Turnip mosaic virus observed in the Chinese cabbage (Brassica rapa) line, BP8407, is monogenic and recessive. Bulked segregant analysis was carried out to identify simple sequence repeat and Indel markers linked to this recessive resistance gene, termed recessive Turnip mosaic virus resistance 02 (retr02). Mapping of PCR-specific Indel markers on 239 individuals of a BP8407 × Ji Zao Chun F₂ population, located this resistance gene to a 0.9-cM interval between two Indel markers (BrID10694 and BrID101309) and in scaffold000060 or scaffold000104 on chromosome A04 of the B. rapa genome. Eleven eukaryotic initiation factor 4E (eIF4E) and 14 eukaryotic initiation factor 4G (eIF4G) genes are predicted in the B. rapa genome. A candidate gene, Bra035393 on scaffold000104, was predicted within the mapped resistance locus. The gene encodes the eIF(iso)4E protein. Bra035393 was sequenced in BP8407 and Ji Zao Chun. A polymorphism (A/G) was found in exon 3 between BP8407 and Ji Zao Chun. This gene was analysed in four resistant and three susceptible lines. A correlation was observed between the amino acid substitution (Gly/Asp) in the eIF(iso)4E protein and resistance/susceptibility. eIF(iso)4E has been shown previously to interact with the TuMV genome-linked protein, VPg.     

Identification of NBS-encoding genes linked to black rot resistance in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis>)

Heading cabbage is a nutritionally rich and economically important cruciferous vegetable. Black rot disease, caused by the bacterium Xanthomonas campestris pv. campestris, reduces both the yield and quality of the cabbage head. Nucleotide binding site (NBS)-encoding resistance (R) genes play a vital role in the plant immune response to various pathogens. In this study, we analyzed the expression and DNA sequence variation of 31 NBS-encoding genes in cabbage (Brassica oleracea var. capitata). These genes encoded TIR, NBS, LRR and RPW8 protein domains, all of which are known to be involved in disease resistance. RNA-seq revealed that these 31 genes were differentially expressed in leaf, root, silique, and stem tissues. Furthermore, qPCR analyses revealed that several of these genes were more highly expressed in resistant compared to susceptible cabbage lines, including Bol003711, Bol010135, Bol010559, Bol022784, Bol029866, Bol042121, Bol031422, Bol040045 and Bol042095. Further analysis of these genes promises to yield both practical benefits, such as molecular markers for marker-assisted breeding, and fundamental insights to the mechanisms of resistance to black rot in cabbage.     

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.     

Comprehensive Analysis of Wall-Associated Kinase Genes and Their Expression Under Abiotic and Biotic Stress in Chinese Cabbage (Brassica rapa ssp. pekinensis)

The wall-associated kinase (WAK) gene family, a subfamily of the receptor-like kinase (RLK) gene family, is associated with the cell wall in plants, and has vital functions in cell expansion, pathogen resistance, and heavy metal stress tolerance because of their roles of the extracellular environment sensors to trigger intracellular signals in Arabidopsis. In the present study, 96 Chinese cabbage (Brassica rapa ssp. pekinensis) BrWAK gene family members were identified from the B. rapa genome using a reiterative database search and manual confirmation. The protein domain characterization, gene structure analysis, and phylogenetic analysis of the BrWAKs classified them into three gene groups. Comparative genomic analysis between WAK genes from Chinese cabbage and Arabidopsis revealed that the BrWAK genes have undergone the gene expansion and deletion events during evolution. Furthermore, the conserved motifs in the kinase domains of the WAK proteins and eukaryotic protein kinase family proteins were compared and some non-RD kinase proteins among the BrWAKs were identified. Ultimately, expression analysis of BrWAK genes in six tissues and under various stress conditions revealed that some tissue-specific WAK genes might function in callus cell growth and reproduction process; Bra012273, Bra016426, Bra016427, and Bra025882 might be involved in downy mildew resistance and high humidity stress; Bra012273, Bra025882, and Bra025883 might be responded to drought and heat stress. Taken together, this research was identified and classified the WAK gene family in Chinese cabbage and provided valuable resources to explore the potential roles of BrWAK genes in plant development and stress responses.

     

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.     

Identification of a broad-spectrum recessive gene in Brassica rapa and molecular analysis of the eIF4E gene family to develop molecular markers

Two Chinese cabbage (Brassica rapa L. ssp. pekinensis) lines resistant to Turnip mosaic virus (TuMV) CHN5 were identified and found to have broad-spectrum resistance against three other TuMV strains (CHN2, 3, and 4). Genetic analysis indicated that this TuMV resistance is recessive, and a candidate gene approach was used to identify the resistance gene, which we named trs (TuMV resistance discovered at Seoul National University). Based on previous research in Arabidopsis showing that mutations in eIF(iso)4E determine TuMV resistance, the eIF(iso)4E gene was selected as a candidate for the trs gene in Brassica rapa. Three copies of eIF(iso)4E, Braiso4Ea, Braiso4Eb, and Braiso4Ec, were amplified, and polymorphisms between resistant and susceptible lines were analyzed. Sequence polymorphisms were found in Braiso4Ea and Braiso4Eb; in contrast, no sequence differences were found in Braiso4Ec between resistant and susceptible lines. A CAPS marker developed to test the linkage between Braiso4Eb and TuMV resistance displayed no linkage. A SCAR marker, trsSCAR, developed using allele-specific deletions and SNPs in Braiso4Ea, did co-segregate perfectly with trs in three F ₂ populations. However, the presence or absence of the Braiso4Ea sequence deletion was not consistent between resistant lines and susceptible lines, indicating that Braiso4Ea is not the actual resistance gene. Results from mapping analysis indicated that the trs is located at chromosome A04, between scaffold 000104 and scaffold 040552. This location demonstrated that trs may be another recessive resistance gene tightly linked to retr02 or another allele. The molecular markers developed in this study will be useful for breeding durable resistance.     

Comparative mapping of Raphanus sativus genome using Brassica markers and quantitative trait loci analysis for the Fusarium wilt resistance trait

Fusarium wilt (FW), caused by the soil-borne fungal pathogen Fusarium oxysporum is a serious disease in cruciferous plants, including the radish (Raphanus sativus). To identify quantitative trait loci (QTL) or gene(s) conferring resistance to FW, we constructed a genetic map of R. sativus using an F₂ mapping population derived by crossing the inbred lines ‘835’ (susceptible) and ‘B2’ (resistant). A total of 220 markers distributed in 9 linkage groups (LGs) were mapped in the Raphanus genome, covering a distance of 1,041.5 cM with an average distance between adjacent markers of 4.7 cM. Comparative analysis of the R. sativus genome with that of Arabidopsis thaliana and Brassica rapa revealed 21 and 22 conserved syntenic regions, respectively. QTL mapping detected a total of 8 loci conferring FW resistance that were distributed on 4 LGs, namely, 2, 3, 6, and 7 of the Raphanus genome. Of the detected QTL, 3 QTLs (2 on LG 3 and 1 on LG 7) were constitutively detected throughout the 2-year experiment. QTL analysis of LG 3, flanked by ACMP0609 and cnu_mBRPGM0085, showed a comparatively higher logarithm of the odds (LOD) value and percentage of phenotypic variation. Synteny analysis using the linked markers to this QTL showed homology to A. thaliana chromosome 3, which contains disease-resistance gene clusters, suggesting conservation of resistance genes between them.     

Genetic mapping of rust resistance genes in confection sunflower line HA-R6 and oilseed line RHA 397

Few widely effective resistance sources to sunflower rust, incited by Puccinia helianthi Schwein., have been identified in confection sunflower (Helianthus annuus L.). The USDA inbred line HA-R6 is one of the few confection sunflower lines resistant to rust. A previous allelism test indicated that rust resistance genes in HA-R6 and RHA 397, an oilseed-type restorer line, are either allelic or closely linked; however, neither have been characterized nor molecularly mapped. The objectives of this study are (1) to locate the rust resistance genes in HA-R6 and RHA 397 on a molecular map, (2) to develop closely linked molecular markers for rust resistance diagnostics, and (3) to determine the resistance spectrum of two lines when compared with other rust-resistant lines. Two populations of 140 F_2:3 families each from the crosses of HA 89, as susceptible parent, with HA-R6 and RHA 397 were inoculated with race 336 of P. helianthi in the greenhouse. The resistance genes (R-genes) in HA-R6 and RHA 397 were molecularly mapped to the lower end of linkage group 13, which encompasses a large R-gene cluster, and were designated as R _13a and R _13b, respectively. In the initial maps, SSR (simple sequence repeat) and InDel (insertion and deletion) markers revealed 2.8 and 8.2 cM flanking regions for R _13a and R _13b, respectively, linked with a common marker set of four co-segregating markers, ORS191, ORS316, ORS581, and ZVG61, in the distal side and one marker ORS464 in the proximal side. To identify new markers closer to the genes, sunflower RGC (resistance gene candidate) markers linked to the downy mildew R-gene Pl ₈ and located at the same region as R _13a and R _13b were selected to screen the two F₂ populations. The RGC markers RGC15/16 and a newly developed marker SUN14 designed from a BAC contig anchored by RGC251 further narrowed down the region flanking R _13a and R _13b to 1.1 and 0.1 cM, respectively. Both R _13a and R _13b are highly effective against all rust races tested so far. Our newly developed molecular markers will facilitate breeding efforts to pyramid the R ₁₃ genes with other rust R-genes and accelerate the development of rust-resistant sunflower hybrids in both confection and oilseed sunflowers.     

Silencing of six susceptibility genes results in potato late blight resistance

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thaliana S-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.     

Detection and verification of quantitative trait loci for resistance to Fusarium ear rot in maize

Fusarium ear rot caused by Fusarium verticillioides is a prevalent disease in maize which can severely reduce grain yields and quality. Identification of stable quantitative trait loci (QTL) for resistance to Fusarium ear rot is a basic prerequisite for understanding the genetic mechanism of resistance and for the use of marker-assisted selection. In this study, two hundred and ten F _2:3 families were developed from a cross between resistant inbred line BT-1 and susceptible inbred line Xi502, and were genotyped with 178 simple sequence repeat markers. The resistance of each line was evaluated in two environments by artificial inoculation using the nail-punch method. The resistance QTL were detected using the composite interval mapping method. Three QTL were detected on chromosomes 4, 5 and 10. Of them, the QTL on chromosome 4 (bin 4.05/06) had the largest resistance to Fusarium ear rot, and could explain 17.95?% of the phenotypic variation. For further verification of the QTL effect, we developed near-isogenic lines (NILs) carrying the QTL region on chromosome 4 using parental line Xi502 as the recurrent parent. In the NIL background, this QTL can increase the resistance by 33.7?C35.2?% if the resistance region is homozygous, and by 17.8?C26.5?% if the resistance region contains the heterozygous allele. The stable and significant resistance effect of the QTL on chromosome 4 lays the foundation for further marker-assisted selection and map-based cloning in maize.     

Identification of Novel QTLs for Isolate-Specific Partial Resistance to Plasmodiophora brassicae in Brassica rapa

Plasmodiophora brassicae, the causal agent of clubroot disease of the Brassica crops, is widespread in the world. Quantitative trait loci (QTLs) for partial resistance to 4 different isolates of P. brassicae (Pb2, Pb4, Pb7, and Pb10) were investigated using a BC₁F₁ population from a cross between two subspecies of Brassica rapa, i.e. Chinese cabbage inbred line C59-1 as a susceptible recurrent parent and turnip inbred line ECD04 as a resistant donor parent. The BC₁F₂ families were assessed for resistance under controlled conditions. A linkage map constructed with simple sequence repeats (SSR), unigene-derived microsatellite (UGMS) markers, and specific markers linked to published clubroot resistance (CR) genes of B. rapa was used to perform QTL mapping. A total of 6 QTLs residing in 5 CR QTL regions of the B. rapa chromosomes A01, A03, and A08 were identified to account for 12.2 to 35.2% of the phenotypic variance. Two QTL regions were found to be novel except for 3 QTLs in the respective regions of previously identified Crr1, Crr2, and Crr3. QTL mapping results indicated that 1 QTL region was common for partial resistance to the 2 isolates of Pb2 and Pb7, whereas the others were specific for each isolate. Additionally, synteny analysis between B. rapa and Arabidopsis thaliana revealed that all CR QTL regions were aligned to a single conserved crucifer blocks (U, F, and R) on 3 Arabidopsis chromosomes where 2 CR QTLs were detected in A. thaliana. These results suggest that some common ancestral genomic regions were involved in the evolution of CR genes in B. rapa.     

Fine mapping and candidate gene screening of the downy mildew resistance gene <Emphasis Type="Italic">RPF1</Emphasis> in Spinach

Key message

A SLAF-BSA approach was used to locate the RPF1 locus. The three most likely candidate genes were identified which provide a basic for cloning the resistance gene at the RPF1 locus.

Abstract

Spinach downy mildew is a globally devastating oomycete disease. The use of downy mildew resistance genes constitutes the most effective approach for disease management. Hence, the objective of the present study was to fine map the first-reported resistance locus RPF1. The resistance allele at this resistance locus was effective against races 1–7, 9, 11, 13, and 15 of Peronospora farinosa f. sp. spinaciae (P. effusa). The approach fine mapped RPF1 using specific-locus amplified fragment sequencing (SLAF-Seq) technology combined with bulked segregant analysis. A 1.72 Mb region localized on chromosome 3 was found to contain RPF1 based on association analysis. After screening recombinants with the SLAF markers within the region, the region was narrowed down to 0.89 Mb. Within this region, 14 R genes were identified based on the annotation information. To identify the genes involved in resistance, resequencing of two resistant inbred lines (12S2 and 12S3) and three susceptible inbred lines (12S1, 12S4, and 10S2) was performed. The three most likely candidate genes were identified via amino acid sequence analysis and conserved domain analysis between resistant and susceptible inbred lines. These included Spo12729, encoding a receptor-like protein, and Spo12784 and Spo12903, encoding a nucleotide-binding site and leucine-rich repeat domains. Additionally, based on the sequence variation in the three genes between the resistant and susceptible lines, molecular markers were developed for marker-assisted selection. The results could be valuable in cloning the RPF1 alleles and improving our understanding of the interaction between the host and pathogen.